Chronic ethanol feeding causes depression of mitochondrial elongation factor Tu in the rat liver: implications for the mitochondrial ribosome.

PubMed

Weiser, Brian; Gonye, Gregory; Sykora, Peter; Crumm, Sara; Cahill, Alan

2011-05-01

Chronic ethanol feeding is known to negatively impact hepatic energy metabolism. Previous studies have indicated that the underlying lesion responsible for this may lie at the level of the mitoribosome. The aim of this study was to characterize the structure of the hepatic mitoribosome in alcoholic male rats and their isocalorically paired controls. Our experiments revealed that chronic ethanol feeding resulted in a significant depletion of both structural (death-associated protein 3) and functional [elongation factor thermo unstable (EF-Tu)] mitoribosomal proteins. In addition, significant increases were found in nucleotide elongation factor thermo stable (EF-Ts) and structural mitochondrial ribosomal protein L12 (MRPL12). The increase in MRPL12 was found to correlate with an increase in the levels of the 39S large mitoribosomal subunit. These changes were accompanied by decreased levels of nuclear- and mitochondrially encoded respiratory subunits, decreased amounts of intact respiratory complexes, decreased hepatic ATP levels, and depressed mitochondrial translation. Mathematical modeling of ethanol-mediated changes in EF-Tu and EF-Ts using prederived kinetic data predicted that the ethanol-mediated decrease in EF-Tu levels could completely account for the impaired mitochondrial protein synthesis. In conclusion, chronic ethanol feeding results in a depletion of mitochondrial EF-Tu levels within the liver that is mathematically predicted to be responsible for the impaired mitochondrial protein synthesis seen in alcoholic animals.

The mitochondrial ribosomal protein of the large subunit, Afo1p, determines cellular longevity through mitochondrial back-signaling via TOR1.

PubMed

Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Hager, Matthias; Bogengruber, Edith; Jarolim, Stefanie; Simon-Nobbe, Birgit; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael

2009-07-13

Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

URF6, Last Unidentified Reading Frame of Human mtDNA, Codes for an NADH Dehydrogenase Subunit

NASA Astrophysics Data System (ADS)

Chomyn, Anne; Cleeter, Michael W. J.; Ragan, C. Ian; Riley, Marcia; Doolittle, Russell F.; Attardi, Giuseppe

1986-10-01

The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.

Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

PubMed

Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

2016-01-01

In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Tripeptidyl peptidase I, the late infantile neuronal ceroid lipofuscinosis gene product, initiates the lysosomal degradation of subunit c of ATP synthase.

PubMed

Ezaki, J; Takeda-Ezaki, M; Kominami, E

2000-09-01

The specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of NCL (LINCL) is caused by a defect in the CLN2 gene product, tripeptidyl peptidase I (TPP-I). The data here show that TPP-I is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c. The inclusion of a specific inhibitor of TPP-I, Ala-Ala-Phe-chloromethylketone (AAF-CMK), in the culture medium of normal fibroblasts induced the lysosomal accumulation of subunit c. In an in vitro incubation experiment the addition of AAF-CMK to mitochondrial-lysosomal fractions from normal cells inhibited the proteolysis of subunit c, but not the b-subunit of ATP synthase. The use of two antibodies that recognize the aminoterminal and the middle portion of subunit c revealed that the subunit underwent aminoterminal proteolysis, when TPP-I, purified from rat spleen, was added to the mitochondrial fractions. The addition of both purified TPP-I and the soluble lysosomal fractions, which contain various proteinases, to the mitochondrial fractions resulted in rapid degradation of the entire molecule of subunit c, whereas the degradation of subunit c was markedly delayed through the specific inhibition of TPP-I in lysosomal extracts by AAF-CMK. The stable subunit c in the mitochondrial-lysosomal fractions from cells of a patient with LINCL was degraded on incubation with purified TPP-I. The presence of TPP-I led to the sequential cleavage of tripeptides from the N-terminus of the peptide corresponding to the amino terminal sequence of subunit c.

Mutation screening of 75 candidate genes in 152 complex I deficiency cases identifies pathogenic variants in 16 genes including NDUFB9.

PubMed

Haack, Tobias B; Madignier, Florence; Herzer, Martina; Lamantea, Eleonora; Danhauser, Katharina; Invernizzi, Federica; Koch, Johannes; Freitag, Martin; Drost, Rene; Hillier, Ingo; Haberberger, Birgit; Mayr, Johannes A; Ahting, Uwe; Tiranti, Valeria; Rötig, Agnes; Iuso, Arcangela; Horvath, Rita; Tesarova, Marketa; Baric, Ivo; Uziel, Graziella; Rolinski, Boris; Sperl, Wolfgang; Meitinger, Thomas; Zeviani, Massimo; Freisinger, Peter; Prokisch, Holger

2012-02-01

Mitochondrial complex I deficiency is the most common cause of mitochondrial disease in childhood. Identification of the molecular basis is difficult given the clinical and genetic heterogeneity. Most patients lack a molecular definition in routine diagnostics. A large-scale mutation screen of 75 candidate genes in 152 patients with complex I deficiency was performed by high-resolution melting curve analysis and Sanger sequencing. The causal role of a new disease allele was confirmed by functional complementation assays. The clinical phenotype of patients carrying mutations was documented using a standardised questionnaire. Causative mutations were detected in 16 genes, 15 of which had previously been associated with complex I deficiency: three mitochondrial DNA genes encoding complex I subunits, two mitochondrial tRNA genes and nuclear DNA genes encoding six complex I subunits and four assembly factors. For the first time, a causal mutation is described in NDUFB9, coding for a complex I subunit, resulting in reduction in NDUFB9 protein and both amount and activity of complex I. These features were rescued by expression of wild-type NDUFB9 in patient-derived fibroblasts. Mutant NDUFB9 is a new cause of complex I deficiency. A molecular diagnosis related to complex I deficiency was established in 18% of patients. However, most patients are likely to carry mutations in genes so far not associated with complex I function. The authors conclude that the high degree of genetic heterogeneity in complex I disorders warrants the implementation of unbiased genome-wide strategies for the complete molecular dissection of mitochondrial complex I deficiency.

Mutations in the Atp1p and Atp3p subunits of yeast ATP synthase differentially affect respiration and fermentation in Saccharomyces cerevisiae.

PubMed

Francis, Brian R; White, Karen H; Thorsness, Peter E

2007-04-01

ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

PubMed Central

Braun, H P; Emmermann, M; Kruft, V; Schmitz, U K

1992-01-01

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase. Images PMID:1324169

Evidence that the assembly of the yeast cytochrome bc1 complex involves the formation of a large core structure in the inner mitochondrial membrane.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-04-01

The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.

Evidence that assembly of the yeast cytochrome bc1 complex involves formation of a large core structure in the inner mitochondrial membrane

PubMed Central

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L.

2009-01-01

The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS-PAGE and immunodecoration, revealed the presence of the two catalytic subunits cytochrome b and cytochrome c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 complex, first because of its wide distribution in distinct yeast deletion strains and second for its characteristics of stability which resemble those of the intact homodimeric bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation*

PubMed Central

Letts, James A.; Degliesposti, Gianluca; Fiedorczuk, Karol; Skehel, Mark; Sazanov, Leonid A.

2016-01-01

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. PMID:27672209

Adaptation of the Mitochondrial Genome in Cephalopods: Enhancing Proton Translocation Channels and the Subunit Interactions

PubMed Central

Almeida, Daniela; Maldonado, Emanuel; Vasconcelos, Vitor; Antunes, Agostinho

2015-01-01

Mitochondrial protein-coding genes (mt genes) encode subunits forming complexes of crucial cellular pathways, including those involved in the vital process of oxidative phosphorylation (OXPHOS). Despite the vital role of the mitochondrial genome (mt genome) in the survival of organisms, little is known with respect to its adaptive implications within marine invertebrates. The molluscan Class Cephalopoda is represented by a marine group of species known to occupy contrasting environments ranging from the intertidal to the deep sea, having distinct metabolic requirements, varied body shapes and highly advanced visual and nervous systems that make them highly competitive and successful worldwide predators. Thus, cephalopods are valuable models for testing natural selection acting on their mitochondrial subunits (mt subunits). Here, we used concatenated mt genes from 17 fully sequenced mt genomes of diverse cephalopod species to generate a robust mitochondrial phylogeny for the Class Cephalopoda. We followed an integrative approach considering several branches of interest–covering cephalopods with distinct morphologies, metabolic rates and habitats–to identify sites under positive selection and localize them in the respective protein alignment and/or tridimensional structure of the mt subunits. Our results revealed significant adaptive variation in several mt subunits involved in the energy production pathway of cephalopods: ND5 and ND6 from Complex I, CYTB from Complex III, COX2 and COX3 from Complex IV, and in ATP8 from Complex V. Furthermore, we identified relevant sites involved in protein-interactions, lining proton translocation channels, as well as disease/deficiencies related sites in the aforementioned complexes. A particular case, revealed by this study, is the involvement of some positively selected sites, found in Octopoda lineage in lining proton translocation channels (site 74 from ND5) and in interactions between subunits (site 507 from ND5) of Complex I. PMID:26285039

Mitochondrial genes in the colourless alga Prototheca wickerhamii resemble plant genes in their exons but fungal genes in their introns.

PubMed Central

Wolff, G; Burger, G; Lang, B F; Kück, U

1993-01-01

The mitochondrial DNA from the colourless alga Prototheca wickerhamii contains two mosaic genes as was revealed from complete sequencing of the circular extranuclear genome. The genes for the large subunit of the ribosomal RNA (LSUrRNA) as well as for subunit I of the cytochrome oxidase (coxI) carry two and three intronic sequences respectively. On the basis of their canonical nucleotide sequences they can be classified as group I introns. Phylogenetic comparisons of the coxI protein sequences allow us to conclude that the P.wickerhamii mtDNA is much closer related to higher plant mtDNAs than to those of the chlorophyte alga C.reinhardtii. The comparison of the intron sequences revealed several unusual features: (1) The P.wickerhamii introns are structurally related to mitochondrial introns from various ascomycetous fungi. (2) Phylogenetic analyses indicate a close relationship between fungal and algal intronic sequences. (3) The P. wickerhamii introns are located at positions within the structural genes which can be considered as preferred intron insertion sites in homologous mitochondrial genes from fungi or liverwort. In all cases, the sequences adjacent to the insertion sites are very well conserved over large evolutionary distances. Our finding of highly similar introns in fungi and algae is consistent with the idea that introns have already been present in the bacterial ancestors of present day mitochondria and evolved concomitantly with the organelles. PMID:7680126

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Colombo, I.; Volta, M.

The electron-transfer flavoprotein (ETF), located in the mitochondrial matrix, is a nuclear-encoded enzyme delivering to the respiratory chain electrons by straight-chain acyl-CoA dehydrogenases and other dehydrogenases. ETF is composed of a 35-kDa [alpha]-subunit that is cleaved to a 32-kDa protein during mitochondrial import (ETFA) and a [beta]-subunit that reaches the mitochondrion unmodified (ETFB). The cDNA encoding both these subunits has been cloned and sequenced. 14 refs., 1 fig.

Ethanol Dose- and Time-dependently Increases α and β Subunits of Mitochondrial ATP Synthase of Cultured Neonatal Rat Cardiomyocytes.

PubMed

Mashimo, Keiko; Arthur, Peter G; Ohno, Youkichi

2015-01-01

Mitochondria are target subcellular organelles of ethanol. In this study, the effects of ethanol on protein composition was examined with 2-dimensional electrophoresis of protein extracts from cultured neonatal rat cardiomyocytes exposed to 100 mM ethanol for 24 hours. A putative β subunit of mitochondrial ATP synthase was increased, which was confirmed by Western blot. The cellular protein abundances in the α and β subunits of ATP synthase increased in dose (0, 10, 50, and 100 mM) - and time (0.5 hour and 24 hours) -dependent manners. The DNA microarray analysis of total RNA extract demonstrated that gene expression of the corresponding messenger RNAs of these subunit proteins did not significantly alter due to 24-hour ethanol exposure. Therefore, protein expression of these nuclear-encoded mitochondrial proteins may be regulated at the translational, rather than the transcriptional, level. Alternatively, degradation of these subunit proteins might be decreased. Additionally, cellular ATP content of cardiomyocytes scarcely decreased following 24-hour exposure to any examined concentrations of ethanol. Previous studies, together with this study, have demonstrated that protein abundance of the α subunit or β subunit or both subunits of ATP synthase after ethanol exposure or dysfunctional conditions might differ according to tissue: significant increases in heart but decreases in liver and brain. Thus, it is suggested that the abundance of subunit proteins of mitochondrial ATP synthase in the ethanol-exposed heart, being different from that in the liver and brain, should increase dose-dependently through either translational upregulation or decreased degradation or both to maintain ATP production, as the heart requires much more energy than other tissues for continuing sustained contractions.

The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum

PubMed Central

Feagin, Jean E.; Harrell, Maria Isabel; Lee, Jung C.; Coe, Kevin J.; Sands, Bryan H.; Cannone, Jamie J.; Tami, Germaine; Schnare, Murray N.; Gutell, Robin R.

2012-01-01

Background The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. Principal Findings The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. Significance All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered. PMID:22761677

Biogenesis of the yeast cytochrome bc1 complex.

PubMed

Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L

2009-01-01

The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.

Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

PubMed

Teng, Chang Ying; Dang, Yunkun; Danne, Jillian C; Waller, Ross F; Green, Beverley R

2013-01-01

Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

The mitochondrial genome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals two unsuspected trans-splicing events of group I introns.

PubMed

Pelin, Adrian; Pombert, Jean-François; Salvioli, Alessandra; Bonen, Linda; Bonfante, Paola; Corradi, Nicolas

2012-05-01

• Arbuscular mycorrhizal fungi (AMF) are ubiquitous organisms that benefit ecosystems through the establishment of an association with the roots of most plants: the mycorrhizal symbiosis. Despite their ecological importance, however, these fungi have been poorly studied at the genome level. • In this study, total DNA from the AMF Gigaspora margarita was subjected to a combination of 454 and Illumina sequencing, and the resulting reads were used to assemble its mitochondrial genome de novo. This genome was annotated and compared with those of other relatives to better comprehend the evolution of the AMF lineage. • The mitochondrial genome of G. margarita is unique in many ways, exhibiting a large size (97 kbp) and elevated GC content (45%). This genome also harbors molecular events that were previously unknown to occur in fungal mitochondrial genomes, including trans-splicing of group I introns from two different genes coding for the first subunit of the cytochrome oxidase and for the small subunit of the rRNA. • This study reports the second published genome from an AMF organelle, resulting in relevant DNA sequence information from this poorly studied fungal group, and providing new insights into the frequency, origin and evolution of trans-spliced group I introns found across the mitochondrial genomes of distantly related organisms. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

DMR1 (CCM1/YGR150C) of Saccharomyces cerevisiae encodes an RNA-binding protein from the pentatricopeptide repeat family required for the maintenance of the mitochondrial 15S ribosomal RNA.

PubMed

Puchta, Olga; Lubas, Michal; Lipinski, Kamil A; Piatkowski, Jakub; Malecki, Michal; Golik, Pawel

2010-04-01

Pentatricopeptide repeat (PPR) proteins form the largest known RNA-binding protein family and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly in mitochondria and chloroplasts, where they modulate organellar genome expression on the post-transcriptional level. The Saccharomyces cerevisiae DMR1 (CCM1, YGR150C) encodes a PPR protein that localizes to mitochondria. Deletion of DMR1 results in a complete and irreversible loss of respiratory capacity and loss of wild-type mtDNA by conversion to rho(-)/rho(0) petites, regardless of the presence of introns in mtDNA. The phenotype of the dmr1Delta mitochondria is characterized by fragmentation of the small subunit mitochondrial rRNA (15S rRNA), that can be reversed by wild-type Dmr1p. Other mitochondrial transcripts, including the large subunit mitochondrial rRNA (21S rRNA), are not affected by the lack of Dmr1p. The purified Dmr1 protein specifically binds to different regions of 15S rRNA in vitro, consistent with the deletion phenotype. Dmr1p is therefore the first yeast PPR protein, which has an rRNA target and is probably involved in the biogenesis of mitochondrial ribosomes and translation.

QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease

PubMed Central

Guarani, Virginia; Jardel, Claude; Chrétien, Dominique; Lombès, Anne; Bénit, Paule; Labasse, Clémence; Lacène, Emmanuelle; Bourillon, Agnès; Imbard, Apolline; Benoist, Jean-François; Dorboz, Imen; Gilleron, Mylène; Goetzman, Eric S; Gaignard, Pauline; Slama, Abdelhamid; Elmaleh-Bergès, Monique; Romero, Norma B; Rustin, Pierre; Ogier de Baulny, Hélène; Paulo, Joao A; Harper, J Wade; Schiff, Manuel

2016-01-01

Previously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients’ fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation. DOI: http://dx.doi.org/10.7554/eLife.17163.001 PMID:27623147

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology.

PubMed

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-05-21

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture.

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology

PubMed Central

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-01-01

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture. DOI: http://dx.doi.org/10.7554/eLife.06265.001 PMID:25997101

Mitochondrial genome of the freshwater jellyfish Craspedacusta sowerbyi and phylogenetics of Medusozoa.

PubMed

Zou, Hong; Zhang, Jin; Li, Wenxiang; Wu, Shangong; Wang, Guitang

2012-01-01

The 17,922 base pairs (bp) nucleotide sequence of the linear mitochondrial DNA (mtDNA) molecule of the freshwater jellyfish Craspedacusta sowerbyi (Hydrozoa, Trachylina, Limnomedusae) has been determined. This sequence exhibits surprisingly low A+T content (57.1%), containing genes for 13 energy pathway proteins, a small and a large subunit rRNAs, and methionine and tryptophan tRNAs. Mitochondrial ancestral medusozoan gene order (AMGO) was found in the C. sowerbyi, as those found in Cubaia aphrodite (Hydrozoa, Trachylina, Limnomedusae), discomedusan Scyphozoa and Staurozoa. The genes of C. sowerbyi mtDNA are arranged in two clusters with opposite transcriptional polarities, whereby transcription proceeds toward the ends of the DNA molecule. Identical inverted terminal repeats (ITRs) flank the ends of the mitochondrial DNA molecule, a characteristic typical of medusozoans. In addition, two open reading frames (ORFs) of 354 and 1611 bp in length were found downstream of the large subunit rRNA gene, similar to the two ORFs of ORF314 and polB discovered in the linear mtDNA of C. aphrodite, discomedusan Scyphozoa and Staurozoa. Phylogenetic analyses of C. sowerbyi and other cnidarians were carried out based on both nucleotide and inferred amino acid sequences of the 13 mitochondrial energy pathway genes. Our working hypothesis supports the monophyletic Medusozoa being a sister group to Octocorallia (Cnidaria, Anthozoa). Within Medusozoa, the phylogenetic analysis suggests that Staurozoa may be the earliest diverging class and the sister group of all other medusozoans. Cubozoa and coronate Scyphozoa form a clade that is the sister group of Hydrozoa plus discomedusan Scyphozoa. Hydrozoa is the sister group of discomedusan Scyphozoa. Semaeostomeae is a paraphyletic clade with Rhizostomeae, while Limnomedusae (Trachylina) is the sister group of hydroidolinans and may be the earliest diverging lineage among Hydrozoa.

Mitochondrial Genome of the Freshwater Jellyfish Craspedacusta sowerbyi and Phylogenetics of Medusozoa

PubMed Central

Zou, Hong; Zhang, Jin; Li, Wenxiang; Wu, Shangong; Wang, Guitang

2012-01-01

The 17,922 base pairs (bp) nucleotide sequence of the linear mitochondrial DNA (mtDNA) molecule of the freshwater jellyfish Craspedacusta sowerbyi (Hydrozoa,Trachylina, Limnomedusae) has been determined. This sequence exhibits surprisingly low A+T content (57.1%), containing genes for 13 energy pathway proteins, a small and a large subunit rRNAs, and methionine and tryptophan tRNAs. Mitochondrial ancestral medusozoan gene order (AMGO) was found in the C. sowerbyi, as those found in Cubaia aphrodite (Hydrozoa, Trachylina, Limnomedusae), discomedusan Scyphozoa and Staurozoa. The genes of C. sowerbyi mtDNA are arranged in two clusters with opposite transcriptional polarities, whereby transcription proceeds toward the ends of the DNA molecule. Identical inverted terminal repeats (ITRs) flank the ends of the mitochondrial DNA molecule, a characteristic typical of medusozoans. In addition, two open reading frames (ORFs) of 354 and 1611 bp in length were found downstream of the large subunit rRNA gene, similar to the two ORFs of ORF314 and polB discovered in the linear mtDNA of C. aphrodite, discomedusan Scyphozoa and Staurozoa. Phylogenetic analyses of C. sowerbyi and other cnidarians were carried out based on both nucleotide and inferred amino acid sequences of the 13 mitochondrial energy pathway genes. Our working hypothesis supports the monophyletic Medusozoa being a sister group to Octocorallia (Cnidaria, Anthozoa). Within Medusozoa, the phylogenetic analysis suggests that Staurozoa may be the earliest diverging class and the sister group of all other medusozoans. Cubozoa and coronate Scyphozoa form a clade that is the sister group of Hydrozoa plus discomedusan Scyphozoa. Hydrozoa is the sister group of discomedusan Scyphozoa. Semaeostomeae is a paraphyletic clade with Rhizostomeae, while Limnomedusae (Trachylina) is the sister group of hydroidolinans and may be the earliest diverging lineage among Hydrozoa. PMID:23240028

A PAC containing the human mitochondrial DNA polymerase gamma gene (POLG) maps to chromosome 15q25

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walker, R.L.; Meltzer, P.S.; Anziano, P.

The human mitochondrial DNA (mtDNA) is a closed circular, 16,569-bp double-stranded DNA, encoding 13 genes whose protein products are subunits of the oxidative phosphorylation system required for synthesis of most of the ATP consumed by eukaryotic cells. Point mutations of the mtDNA that cause multi-tissue, loss-of-energy syndromes, called mitochondrial encephalomyopathies (e.g., MERRF and MELAS), have been identified. In addition, large-scale deletions of the human mtDNA have been identified and are the molecular bases for the neonatal and adolescent onset loss-of-energy syndromes Pearson and Kearns-Sayer, respectively. 5 refs., 1 fig.

Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

PubMed

Duka, Tetyana; Collins, Zachary; Anderson, Sarah M; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

2017-07-01

The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of insulin on human skeletal muscle mitochondrial ATP production, protein synthesis, and mRNA transcripts

NASA Astrophysics Data System (ADS)

Stump, Craig S.; Short, Kevin R.; Bigelow, Maureen L.; Schimke, Jill M.; Sreekumaran Nair, K.

2003-06-01

Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels. cytochrome c oxidase | NADH dehydrogenase subunit IV | amino acids | citrate synthase

Purification of Ovine Respiratory Complex I Results in a Highly Active and Stable Preparation.

PubMed

Letts, James A; Degliesposti, Gianluca; Fiedorczuk, Karol; Skehel, Mark; Sazanov, Leonid A

2016-11-18

NADH-ubiquinone oxidoreductase (complex I) is the largest (∼1 MDa) and the least characterized complex of the mitochondrial electron transport chain. Because of the ease of sample availability, previous work has focused almost exclusively on bovine complex I. However, only medium resolution structural analyses of this complex have been reported. Working with other mammalian complex I homologues is a potential approach for overcoming these limitations. Due to the inherent difficulty of expressing large membrane protein complexes, screening of complex I homologues is limited to large mammals reared for human consumption. The high sequence identity among these available sources may preclude the benefits of screening. Here, we report the characterization of complex I purified from Ovis aries (ovine) heart mitochondria. All 44 unique subunits of the intact complex were identified by mass spectrometry. We identified differences in the subunit composition of subcomplexes of ovine complex I as compared with bovine, suggesting differential stability of inter-subunit interactions within the complex. Furthermore, the 42-kDa subunit, which is easily lost from the bovine enzyme, remains tightly bound to ovine complex I. Additionally, we developed a novel purification protocol for highly active and stable mitochondrial complex I using the branched-chain detergent lauryl maltose neopentyl glycol. Our data demonstrate that, although closely related, significant differences exist between the biochemical properties of complex I prepared from ovine and bovine mitochondria and that ovine complex I represents a suitable alternative target for further structural studies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Systematics of the Dioryctria abietella Species Group (Lepidoptera: Pyralidae) Based on Mitochondrial DNA Ann

Treesearch

G. Roux-Morabito; N.E. Gillette; A. Roques; L. Dormont; J. Stein; F.A.H. Sperling

2008-01-01

Coneworms of the genus Dioryctria Zeller include several serious pests of conifer seeds that are notoriously difficult to distinguish as species. We surveyed mitochondrial DNA variation within the abietella species group by sequencing 451 bp of cytochrome oxidase subunit 1 (COI) and 572 bp of cytochrome oxidase subunit 2 (COII...

The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

PubMed Central

Tetaud, Emmanuel; Godard, François; Giraud, Marie-France; Ackerman, Sharon H.; di Rago, Jean-Paul

2014-01-01

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F1 and the proton translocation channel in F0 for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F0-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F0 subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F0. PMID:24451261

Vacuolating cytotoxin A (VacA), a key toxin for Helicobacter pylori pathogenesis

PubMed Central

Palframan, Samuel L.; Kwok, Terry; Gabriel, Kipros

2012-01-01

More than 50% of the world's population is infected with Helicobacter pylori (H. pylori). Chronic infection with this Gram-negative pathogen is associated with the development of peptic ulcers and is linked to an increased risk of gastric cancer. H. pylori secretes many proteinaceous factors that are important for initial colonization and subsequent persistence in the host stomach. One of the major protein toxins secreted by H. pylori is the Vacuolating cytotoxin A (VacA). After secretion from the bacteria via a type V autotransport secretion system, the 88 kDa VacA toxin (comprised of the p33 and p55 subunits) binds to host cells and is internalized, causing severe “vacuolation” characterized by the accumulation of large vesicles that possess hallmarks of both late endosomes and early lysosomes. The development of “vacuoles” has been attributed to the formation of VacA anion-selective channels in membranes. Apart from its vacuolating effects, it has recently become clear that VacA also directly affects mitochondrial function. Earlier studies suggested that the p33 subunit, but not the p55 subunit of VacA, could enter mitochondria to modulate organelle function. This raised the possibility that a mechanism separate from pore formation may be responsible for the effects of VacA on mitochondria, as crystallography studies and structural modeling predict that both subunits are required for a physiologically stable pore. It has also been suggested that the mitochondrial effects observed are due to indirect effects on pro-apoptotic proteins and direct effects on mitochondrial morphology-related processes. Other studies have shown that both the p55 and p33 subunits can indeed be efficiently imported into mammalian-derived mitochondria raising the possibility that they could re-assemble to form a pore. Our review summarizes and consolidates the recent advances in VacA toxin research, with focus on the outstanding controversies in the field and the key remaining questions that need to be addressed. PMID:22919683

Pyruvate dehydrogenase complex (PDC) export from the mitochondrial matrix.

PubMed

Ng, Fanny; Tang, Bor Luen

2014-01-01

Studies on mitochondria protein import had revealed in detail molecular mechanisms of how peptides and proteins could be selectively targeted and translocated across membrane bound organelles. The opposite process of mitochondrial export, while known to occur in various aspects of cellular physiology and pathology, is less well understood. Two very recent reports have indicated that a large mitochondrial matrix protein complex, the pyruvate dehydrogenase complex (PDC) (or its component subunits), could be exported to the lysosomes and the nucleus, respectively. In the case of the latter, evidence was presented to suggest that the entire complex of 8-10 MDa could translocate in its entirety from the mitochondrial matrix to the nucleus upon mitogenic or stress stimuli. We discuss these findings in perspective to what is currently known about the processes of transport in and out of the mitochondrion.

Species boundaries of Gulf of Mexico vestimentiferans (Polychaeta, Siboglinidae) inferred from mitochondrial genes

NASA Astrophysics Data System (ADS)

Pia Miglietta, Maria; Hourdez, Stephane; Cowart, Dominique A.; Schaeffer, Stephen W.; Fisher, Charles

2010-11-01

At least six morphospecies of vestimentiferan tubeworms are associated with cold seeps in the Gulf of Mexico (GOM). The physiology and ecology of the two best-studied species from depths above 1000 m in the upper Louisiana slope (Lamellibrachia luymesi and Seepiophila jonesi) are relatively well understood. The biology of one rare species from the upper slope (escarpiid sp. nov.) and three morphospecies found at greater depths in the GOM (Lamellibrachia sp. 1, L. sp. 2, and Escarpia laminata) are not as well understood. Here we address species distributions and boundaries of cold-seep tubeworms using phylogenetic hypotheses based on two mitochondrial genes. Fragments of the mitochondrial large ribosomal subunit rDNA (16S) and cytochrome oxidase subunit I (COI) genes were sequenced for 167 vestimentiferans collected from the GOM and analyzed in the context of other seep vestimentiferans for which sequence data were available. The analysis supported five monophyletic clades of vestimentiferans in the GOM. Intra-clade variation in both genes was very low, and there was no apparent correlation between the within-clade diversity and collection depth or location. Two of the morphospecies of Lamellibrachia from different depths in the GOM could not be distinguished by either mitochondrial gene. Similarly, E. laminata could not be distinguished from other described species of Escarpia from either the west coast of Africa or the eastern Pacific using COI. We suggest that the mitochondrial COI and 16S genes have little utility as barcoding markers for seep vestimentiferan tubeworms.

Two ATPases

PubMed Central

Senior, Alan E.

2012-01-01

In this article, I reflect on research on two ATPases. The first is F1F0-ATPase, also known as ATP synthase. It is the terminal enzyme in oxidative phosphorylation and famous as a nanomotor. Early work on mitochondrial enzyme involved purification in large amount, followed by deduction of subunit composition and stoichiometry and determination of molecular sizes of holoenzyme and individual subunits. Later work on Escherichia coli enzyme utilized mutagenesis and optical probes to reveal the molecular mechanism of ATP hydrolysis and detailed facets of catalysis. The second ATPase is P-glycoprotein, which confers multidrug resistance, notably to anticancer drugs, in mammalian cells. Purification of the protein in large quantity allowed detailed characterization of catalysis, formulation of an alternating sites mechanism, and recently, advances in structural characterization. PMID:22822068

Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.

PubMed

Zhang, Yuxun; Bharathi, Sivakama S; Rardin, Matthew J; Lu, Jie; Maringer, Katherine V; Sims-Lucas, Sunder; Prochownik, Edward V; Gibson, Bradford W; Goetzman, Eric S

2017-06-16

SIRT5 is a lysine desuccinylase known to regulate mitochondrial fatty acid oxidation and the urea cycle. Here, SIRT5 was observed to bind to cardiolipin via an amphipathic helix on its N terminus. In vitro , succinyl-CoA was used to succinylate liver mitochondrial membrane proteins. SIRT5 largely reversed the succinyl-CoA-driven lysine succinylation. Quantitative mass spectrometry of SIRT5-treated membrane proteins pointed to the electron transport chain, particularly Complex I, as being highly targeted for desuccinylation by SIRT5. Correspondingly, SIRT5 -/- HEK293 cells showed defects in both Complex I- and Complex II-driven respiration. In mouse liver, SIRT5 expression was observed to localize strictly to the periportal hepatocytes. However, homogenates prepared from whole SIRT5 -/- liver did show reduced Complex II-driven respiration. The enzymatic activities of Complex II and ATP synthase were also significantly reduced. Three-dimensional modeling of Complex II suggested that several SIRT5-targeted lysine residues lie at the protein-lipid interface of succinate dehydrogenase subunit B. We postulate that succinylation at these sites may disrupt Complex II subunit-subunit interactions and electron transfer. Lastly, SIRT5 -/- mice, like humans with Complex II deficiency, were found to have mild lactic acidosis. Our findings suggest that SIRT5 is targeted to protein complexes on the inner mitochondrial membrane via affinity for cardiolipin to promote respiratory chain function. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.

PubMed

Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2014-03-01

Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.

A promoter recognition mechanism common to yeast mitochondrial and phage t7 RNA polymerases.

PubMed

Nayak, Dhananjaya; Guo, Qing; Sousa, Rui

2009-05-15

Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family.

Protective effect of hydroxytyrosol in arsenic-induced mitochondrial dysfunction in rat brain.

PubMed

Soni, Manisha; Prakash, Chandra; Sehwag, Sfurti; Kumar, Vijay

2017-07-01

The present study was planned to investigate the protective effect of hydroxytyrosol (HT) against arsenic (As)-induced mitochondrial dysfunction in rat brain. Rats exposed to sodium arsenite (25 ppm for 8 weeks) showed decreased mitochondrial complexes (I, II, IV) activities, mitochondrial superoxide dismutase (MnSOD), and catalase activities in brain mitochondria. As-treated rats showed reduced mRNA expression of complex I (ND-1, ND-2), IV (COX-1, COX-4) subunits, and uncoupling protein-2 (UCP-2). In addition to this, As exposure downregulated the protein expression of MnSOD. Administration of HT with As restored the enzymatic activities of mitochondrial complexes, MnSOD and catalase, increased the mRNA levels of complexes subunits and UCP-2 as well as proteins level of MnSOD. These results suggest that HT efficiently restores mitochondrial dysfunction in As neurotoxicity and might be used as potential mitoprotective agent in future. © 2017 Wiley Periodicals, Inc.

Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

PubMed Central

Hiesel, Rudolf; Brennicke, Axel

1983-01-01

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene. ImagesFig. 5. PMID:16453484

Feeding difficulties, a key feature of the Drosophila NDUFS4 mitochondrial disease model

PubMed Central

Foriel, Sarah; Eidhof, Ilse

2018-01-01

ABSTRACT Mitochondrial diseases are associated with a wide variety of clinical symptoms and variable degrees of severity. Patients with such diseases generally have a poor prognosis and often an early fatal disease outcome. With an incidence of 1 in 5000 live births and no curative treatments available, relevant animal models to evaluate new therapeutic regimes for mitochondrial diseases are urgently needed. By knocking down ND-18, the unique Drosophila ortholog of NDUFS4, an accessory subunit of the NADH:ubiquinone oxidoreductase (Complex I), we developed and characterized several dNDUFS4 models that recapitulate key features of mitochondrial disease. Like in humans, the dNDUFS4 KD flies display severe feeding difficulties, an aspect of mitochondrial disorders that has so far been largely ignored in animal models. The impact of this finding, and an approach to overcome it, will be discussed in the context of interpreting disease model characterization and intervention studies. This article has an associated First Person interview with the first author of the paper. PMID:29590638

Nandrolone decanoate negatively reverses the beneficial effects of exercise on cardiac muscle via sarcolemmal, but not mitochondrial K(ATP) channel.

PubMed

Bayat, Gholamreza; Javan, Mohammad; Safari, Fatemeh; Khalili, Azadeh; Shokri, Saeed; Goudarzvand, Mahdi; Salimi, Mehdi; Hajizadeh, Sohrab

2016-03-01

ATP-sensitive potassium channels are supposed to have a substantial role in improvement of cardiac performance. This study was performed to evaluate whether nandrolone decanoate (ND) and (or) exercise training could affect the expression of cardiac K(ATP) channel subunits. Thirty-five male albino Wistar rats were randomly divided into 5 groups, including sedentary control (SC), sedentary vehicle (SV), sedentary ND (SND), exercise control (EC), and exercise and ND (E+ND). Exercise training was performed on a treadmill 5 times per week. ND was injected (10 mg/kg/week, i.m.) to the rats in the SND and E+ND groups. Following cardiac isolation, the expression of both sarcolemmal and mitochondrial subunits of K(ATP) channel was measured using Western blot method. The expression of sarcolemmal, but not mitochondrial, subunits of K(ATP) channel (Kir6.2 and SUR2) of EC group was significantly higher compared with SC group while ND administration (SND group) did not show any change in their expression. In the E+ND group, ND administration led to decrease of the over-expression of sarcolemmal Kir6.2 and SUR2 which was previously induced by exercise. There was no significant association between the mitochondrial expression of either Kir6.2 or SUR2 proteins and administration of ND or exercise. Supra-physiological dosage of ND negatively reverses the effects of exercise on the cardiac muscle expression of sarcolemmal, but not mitochondrial, K(ATP) channel subunits.

The Mitochondrial Genome of the Prasinophyte Prasinoderma coloniale Reveals Two Trans-Spliced Group I Introns in the Large Subunit rRNA Gene

PubMed Central

Pombert, Jean-François; Otis, Christian; Turmel, Monique; Lemieux, Claude

2013-01-01

Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in cis or trans configuration, and although trans-arrayed group II introns have been known for decades, trans-spliced group I introns have been reported only recently. In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 (Prasinococcales, clade VI), we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes (clades II, III and V). This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications of this interesting observation for trans-splicing of group I introns. PMID:24386369

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

PubMed

Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

2013-11-01

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

Expression of the nuclear gene TaF(A)d is under mitochondrial retrograde regulation in anthers of male sterile wheat plants with timopheevii cytoplasm.

PubMed

Xu, Pei; Yang, Yuwen; Zhang, Zhengzhi; Chen, Weihua; Zhang, Caiqin; Zhang, Lixia; Zou, Sixiang; Ma, Zhengqiang

2008-01-01

Alterations of mitochondrial-encoded subunits of the F(o)F(1)-ATP synthase are frequently associated with cytoplasmic male sterility (CMS) in plants; however, little is known about the relationship of the nuclear encoded subunits of this enzyme with CMS. In the present study, the full cDNA of the gene TaF(A)d that encodes the putative F(A)d subunit of the F(o)F(1)-ATP synthase was isolated from the wheat (Triticum aestivum) fertility restorer '2114' for timopheevii cytoplasm-based CMS. The deduced 238 amino acid polypeptide is highly similar to its counterparts in dicots and other monocots but has low homology to its mammalian equivalents. TaF(A)d is a single copy gene in wheat and maps to the short arm of the group 6 chromosomes. Transient expression of the TaF(A)d-GFP fusion in onion epidermal cells demonstrated TaF(A)d's mitochondrial location. TaF(A)d was expressed abundantly in stem, leaf, anther, and ovary tissues of 2114. Nevertheless, its expression was repressed in anthers of CMS plants with timopheevii cytoplasm. Genic male sterility did not affect its expression in anthers. The expression of the nuclear gene encoding the 20 kDa subunit of F(o) was down-regulated in a manner similar to TaF(A)d in the T-CMS anthers while that of genes encoding the 6 kDa subunit of F(o) and the gamma subunit of F(1) was unaffected. These observations implied that TaF(A)d is under mitochondrial retrograde regulation in the anthers of CMS plants with timopheevii cytoplasm.

A ketogenic diet accelerates neurodegeneration in mice with induced mitochondrial DNA toxicity in the forebrain.

PubMed

Lauritzen, Knut H; Hasan-Olive, Md Mahdi; Regnell, Christine E; Kleppa, Liv; Scheibye-Knudsen, Morten; Gjedde, Albert; Klungland, Arne; Bohr, Vilhelm A; Storm-Mathisen, Jon; Bergersen, Linda H

2016-12-01

Mitochondrial genome maintenance plays a central role in preserving brain health. We previously demonstrated accumulation of mitochondrial DNA damage and severe neurodegeneration in transgenic mice inducibly expressing a mutated mitochondrial DNA repair enzyme (mutUNG1) selectively in forebrain neurons. Here, we examine whether severe neurodegeneration in mutUNG1-expressing mice could be rescued by feeding the mice a ketogenic diet, which is known to have beneficial effects in several neurological disorders. The diet increased the levels of superoxide dismutase 2, and mitochondrial mass, enzymes, and regulators such as SIRT1 and FIS1, and appeared to downregulate N-methyl-D-aspartic acid (NMDA) receptor subunits NR2A/B and upregulate γ-aminobutyric acid A (GABA A ) receptor subunits α 1 . However, unexpectedly, the ketogenic diet aggravated neurodegeneration and mitochondrial deterioration. Electron microscopy showed structurally impaired mitochondria accumulating in neuronal perikarya. We propose that aggravation is caused by increased mitochondrial biogenesis of generally dysfunctional mitochondria. This study thereby questions the dogma that a ketogenic diet is unambiguously beneficial in mitochondrial disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

A ketogenic diet accelerates neurodegeneration in mice with induced mitochondrial DNA toxicity in the forebrain

PubMed Central

Lauritzen, Knut H.; Hasan-Olive, Md Mahdi; Regnell, Christine E.; Kleppa, Liv; Scheibye-Knudsen, Morten; Gjedde, Albert; Klungland, Arne; Bohr, Vilhelm A.; Storm-Mathisen, Jon; Bergersen, Linda H.

2017-01-01

Mitochondrial genome maintenance plays a central role in preserving brain health. We previously demonstrated accumulation of mitochondrial DNA damage and severe neurodegeneration in transgenic mice inducibly expressing a mutated mitochondrial DNA repair enzyme (mutUNG1) selectively in forebrain neurons. Here, we examine whether severe neurodegeneration in mutUNG1-expressing mice could be rescued by feeding the mice a ketogenic diet, which is known to have beneficial effects in several neurological disorders. The diet increased the levels of superoxide dismutase 2, and mitochondrial mass, enzymes, and regulators such as SIRT1 and FIS1, and appeared to downregulate N-methyl-D-aspartic acid (NMDA) receptor subunits NR2A/B and upregulate γ-aminobutyric acid A (GABAA) receptor subunits α1. However, unexpectedly, the ketogenic diet aggravated neurodegeneration and mitochondrial deterioration. Electron microscopy showed structurally impaired mitochondria accumulating in neuronal perikarya. We propose that aggravation is caused by increased mitochondrial biogenesis of generally dysfunctional mitochondria. This study thereby questions the dogma that a ketogenic diet is unambiguously beneficial in mitochondrial disorders. PMID:27639119

Mitochondrial genes are altered in blood early in Alzheimer's disease.

PubMed

Lunnon, Katie; Keohane, Aoife; Pidsley, Ruth; Newhouse, Stephen; Riddoch-Contreras, Joanna; Thubron, Elisabeth B; Devall, Matthew; Soininen, Hikka; Kłoszewska, Iwona; Mecocci, Patrizia; Tsolaki, Magda; Vellas, Bruno; Schalkwyk, Leonard; Dobson, Richard; Malik, Afshan N; Powell, John; Lovestone, Simon; Hodges, Angela

2017-05-01

Although mitochondrial dysfunction is a consistent feature of Alzheimer's disease in the brain and blood, the molecular mechanisms behind these phenomena are unknown. Here we have replicated our previous findings demonstrating reduced expression of nuclear-encoded oxidative phosphorylation (OXPHOS) subunits and subunits required for the translation of mitochondrial-encoded OXPHOS genes in blood from people with Alzheimer's disease and mild cognitive impairment. Interestingly this was accompanied by increased expression of some mitochondrial-encoded OXPHOS genes, namely those residing closest to the transcription start site of the polycistronic heavy chain mitochondrial transcript (MT-ND1, MT-ND2, MT-ATP6, MT-CO1, MT-CO2, MT-C03) and MT-ND6 transcribed from the light chain. Further we show that mitochondrial DNA copy number was unchanged suggesting no change in steady-state numbers of mitochondria. We suggest that an imbalance in nuclear and mitochondrial genome-encoded OXPHOS transcripts may drive a negative feedback loop reducing mitochondrial translation and compromising OXPHOS efficiency, which is likely to generate damaging reactive oxygen species. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration

PubMed Central

Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

2018-01-01

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders. PMID:29451229

m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration.

PubMed

Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

2018-03-01

The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway.

PubMed

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf; Sharma, Reeta Kumari; Verma, Deepika; Priyanka, Kumari; Bal, Amanjit; Gill, Kiran Dip

2015-12-01

The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits - ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host.

PubMed

Iwata, Fumiko; Shinjyo, Noriko; Amino, Hisako; Sakamoto, Kimitoshi; Islam, M Khyrul; Tsuji, Naotoshi; Kita, Kiyoshi

2008-03-01

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.

Mitochondrial hepato-encephalopathy due to deficiency of QIL1/MIC13 (C19orf70), a MICOS complex subunit.

PubMed

Zeharia, Avraham; Friedman, Jonathan R; Tobar, Ana; Saada, Ann; Konen, Osnat; Fellig, Yacov; Shaag, Avraham; Nunnari, Jodi; Elpeleg, Orly

2016-12-01

The mitochondrial inner membrane possesses distinct subdomains including cristae, which are lamellar structures invaginated into the mitochondrial matrix and contain the respiratory complexes. Generation of inner membrane domains requires the complex interplay between the respiratory complexes, mitochondrial lipids and the recently identified mitochondrial contact site and cristae organizing system (MICOS) complex. Proper organization of the mitochondrial inner membrane has recently been shown to be important for respiratory function in yeast. Here we aimed at a molecular diagnosis in a brother and sister from a consanguineous family who presented with a neurodegenerative disorder accompanied by hyperlactatemia, 3-methylglutaconic aciduria, disturbed hepatocellular function with abnormal cristae morphology in liver and cerebellar and vermis atrophy, which suggest mitochondrial dysfunction. Using homozygosity mapping and exome sequencing the patients were found to be homozygous for the p.(Gly15Glufs*75) variant in the QIL1/MIC13 (C19orf70) gene. QIL1/MIC13 is a constituent of MICOS, a six subunit complex that helps to form and/or stabilize cristae junctions and determine the placement, distribution and number of cristae within mitochondria. In patient fibroblasts both MICOS subunits QIL1/MIC13 and MIC10 were absent whereas MIC60 was present in a comparable abundance to that of the control. We conclude that QIL1/MIC13 deficiency in human, is associated with disassembly of the MICOS complex, with the associated aberration of cristae morphology and mitochondrial respiratory dysfunction. 3-Methylglutaconic aciduria is associated with variants in genes encoding mitochondrial inner membrane organizing determinants, including TAZ, DNAJC19, SERAC1 and QIL1/MIC13.

The cytochrome oxidase subunit I and subunit III genes in Oenothera mitochondria are transcribed from identical promoter sequences

PubMed Central

Hiesel, Rudolf; Schobel, Werner; Schuster, Wolfgang; Brennicke, Axel

1987-01-01

Two loci encoding subunit III of the cytochrome oxidase (COX) in Oenothera mitochondria have been identified from a cDNA library of mitochondrial transcripts. A 657-bp sequence block upstream from the open reading frame is also present in the two copies of the COX subunit I gene and is presumably involved in homologous sequence rearrangement. The proximal points of sequence rearrangements are located 3 bp upstream from the COX I and 1139 bp upstream from the COX III initiation codons. The 5'-termini of both COX I and COX III mRNAs have been mapped in this common sequence confining the promoter region for the Oenothera mitochondrial COX I and COX III genes to the homologous sequence block. ImagesFig. 5. PMID:15981332

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

PubMed Central

Babot, Marion; Labarbuta, Paola; Birch, Amanda; Kee, Sara; Fuszard, Matthew; Botting, Catherine H.; Wittig, Ilka; Heide, Heinrich; Galkin, Alexander

2014-01-01

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I + III2 + IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39 kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover. PMID:24560811

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits.

PubMed

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-07-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain‑dead donors. In the present study, two‑dimensional gel electrophoresis and MALDI‑TOF MS‑based comparative proteomic analysis were conducted to profile the differentially‑expressed proteins between brain death and the control group renal tissues. A total of 40 age‑ and sex‑matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two‑dimensional gel electrophoresis, >2‑fold alterations were identified by MALDI‑TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3‑N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b‑c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre‑mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V‑type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin‑3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time‑dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain‑dead donors.

Proteomic analysis of differentially expressed proteins in kidneys of brain dead rabbits

PubMed Central

Li, Ling; Li, Ning; He, Chongxiang; Huang, Wei; Fan, Xiaoli; Zhong, Zibiao; Wang, Yanfeng; Ye, Qifa

2017-01-01

A large number of previous clinical studies have reported a delayed graft function for brain dead donors, when compared with living relatives or cadaveric organ transplantations. However, there is no accurate method for the quality evaluation of kidneys from brain-dead donors. In the present study, two-dimensional gel electrophoresis and MALDI-TOF MS-based comparative proteomic analysis were conducted to profile the differentially-expressed proteins between brain death and the control group renal tissues. A total of 40 age- and sex-matched rabbits were randomly divided into donation following brain death (DBD) and control groups. Following the induction of brain death via intracranial progressive pressure, the renal function and the morphological alterations were measured 2, 6 and 8 h afterwards. The differentially expressed proteins were detected from renal histological evidence at 6 h following brain death. Although 904±19 protein spots in control groups and 916±25 in DBD groups were identified in the two-dimensional gel electrophoresis, >2-fold alterations were identified by MALDI-TOF MS and searched by NCBI database. The authors successfully acquired five downregulated proteins, these were: Prohibitin (isoform CRA_b), beta-1,3-N-acetylgalactosaminyltransferase 1, Annexin A5, superoxide dismutase (mitochondrial) and cytochrome b-c1 complex subunit 1 (mitochondrial precursor). Conversely, the other five upregulated proteins were: PRP38 pre-mRNA processing factor 38 (yeast) domain containing A, calcineurin subunit B type 1, V-type proton ATPase subunit G 1, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and peroxiredoxin-3 (mitochondrial). Immunohistochemical results revealed that the expressions of prohibitin (PHB) were gradually increased in a time-dependent manner. The results indicated that there were alterations in levels of several proteins in the kidneys of those with brain death, even if the primary function and the morphological changes were not obvious. PHB may therefore be a novel biomarker for primary quality evaluation of kidneys from brain-dead donors. PMID:28534953

Studies of the Outer Membrane Proteins of Campylobacter Jejuni for Vaccine Development

DTIC Science & Technology

1991-11-26

Mycobacterium tuberculosis, and M.leprae (66%) and mitochondrial protein p1 precursor of human and Chinese hamster cells (64%), and rubisco subunit binding...175) SAWG--DIgNIISDAP’KXVGRXgVITVK (202) 64% Rubisco subunit binding-protein alpha subunit of wheat (151) SAGN--OELIZGANADAIDOGPOVVLStE (178) 57

DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates.

PubMed

Folmer, O; Black, M; Hoeh, W; Lutz, R; Vrijenhoek, R

1994-10-01

We describe "universal" DNA primers for polymerase chain reaction (PCR) amplification of a 710-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) from 11 invertebrate phyla: Echinodermata, Mollusca, Annelida, Pogonophora, Arthropoda, Nemertinea, Echiura, Sipuncula, Platyhelminthes, Tardigrada, and Coelenterata, as well as the putative phylum Vestimentifera. Preliminary comparisons revealed that these COI primers generate informative sequences for phylogenetic analyses at the species and higher taxonomic levels.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

PubMed

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

2017-01-22

F 1 F o -ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H + flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H + within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F 1 and F O domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia

F{sub 1}F{sub o}-ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H{sup +} flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H{sup +} within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F{sub 1} andmore » F{sub O} domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo.« less

Single channel recording of a mitochondrial calcium uniporter.

PubMed

Wu, Guangyan; Li, Shunjin; Zong, Guangning; Liu, Xiaofen; Fei, Shuang; Shen, Linda; Guan, Xiangchen; Yang, Xue; Shen, Yuequan

2018-01-29

Mitochondrial calcium uniporter (MCU) is the pore-forming subunit of the entire uniporter complex and plays an important role in mitochondrial calcium uptake. However, the single channel recording of MCU remains controversial. Here, we expressed and purified different MCU proteins and then reconstituted them into planar lipid bilayers for single channel recording. We showed that MCU alone from Pyronema omphalodes (pMCU) is active with prominent single channel Ca 2+ currents. In sharp contrast, MCU alone from Homo sapiens (hMCU) is inactive. The essential MCU regulator (EMRE) activates hMCU, and therefore, the complex (hMCU-hEMRE) shows prominent single channel Ca 2+ currents. These single channel currents are sensitive to the specific MCU inhibitor Ruthenium Red. Our results clearly demonstrate that active MCU can conduct large amounts of calcium into the mitochondria. Copyright © 2018 Elsevier Inc. All rights reserved.

Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

PubMed Central

Eya, Jonathan C.; Ukwuaba, Vitalis O.; Yossa, Rodrigue; Gannam, Ann L.

2015-01-01

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish. PMID:25853266

Atrial natriuretic peptide regulates lipid mobilization and oxygen consumption in human adipocytes by activating AMPK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Souza, Sandra C.; Chau, Mary D.L.; Yang, Qing

2011-07-08

Highlights: {yields} Treatment of differentiated human adipocytes with atrial natriuretic peptide (ANP) increased lipolysis and oxygen consumption by activating AMP-activated protein kinase (AMPK). {yields} ANP stimulated lipid mobilization by selective activation of the alpha2 subunit of AMPK and increased energy utilization through activation of both the alpha1 and alpha2 subunits of AMPK. {yields} ANP enhanced adipocyte mitochondrial oxidative capacity as evidenced by induction of oxidative mitochondrial genes and increase in oxygen consumption. {yields} Exposure of human adipocytes to fatty acids and (TNF{alpha}) induced insulin resistance and decreased expression of mitochondrial genes which was restored to normal by ANP. -- Abstract:more » Atrial natriuretic peptide (ANP) has been shown to regulate lipid and carbohydrate metabolism providing a possible link between cardiovascular function and metabolism by mediating the switch from carbohydrate to lipid mobilization and oxidation. ANP exerts a potent lipolytic effect via cGMP-dependent protein kinase (cGK)-I mediated-stimulation of AMP-activated protein kinase (AMPK). Activation of the ANP/cGK signaling cascade also promotes muscle mitochondrial biogenesis and fat oxidation. Here we demonstrate that ANP regulates lipid metabolism and oxygen utilization in differentiated human adipocytes by activating the alpha2 subunit of AMPK. ANP treatment increased lipolysis by seven fold and oxygen consumption by two fold, both of which were attenuated by inhibition of AMPK activity. ANP-induced lipolysis was shown to be mediated by the alpha2 subunit of AMPK as introduction of dominant-negative alpha2 subunit of AMPK attenuated ANP effects on lipolysis. ANP-induced activation of AMPK enhanced mitochondrial oxidative capacity as evidenced by a two fold increase in oxygen consumption and induction of mitochondrial genes, including carnitine palmitoyltransferase 1A (CPT1a) by 1.4-fold, cytochrome C (CytC) by 1.3-fold, and peroxisome proliferator-activated receptor-{gamma} coactivator-1{alpha} (PGC-1{alpha}) by 1.4-fold. Treatment of human adipocytes with fatty acids and tumor necrosis factor {alpha} (TNF{alpha}) induced insulin resistance and down-regulation of mitochondrial genes, which was restored by ANP treatment. These results show that ANP regulates lipid catabolism and enhances energy dissipation through AMPK activation in human adipocytes.« less

Biochemical analysis of human POLG2 variants associated with mitochondrial disease

PubMed Central

Young, Matthew J.; Longley, Matthew J.; Li, Fang-Yuan; Kasiviswanathan, Rajesh; Wong, Lee-Jun; Copeland, William C.

2011-01-01

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic. PMID:21555342

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

PubMed

Lake, Nicole J; Webb, Bryn D; Stroud, David A; Richman, Tara R; Ruzzenente, Benedetta; Compton, Alison G; Mountford, Hayley S; Pulman, Juliette; Zangarelli, Coralie; Rio, Marlene; Boddaert, Nathalie; Assouline, Zahra; Sherpa, Mingma D; Schadt, Eric E; Houten, Sander M; Byrnes, James; McCormick, Elizabeth M; Zolkipli-Cunningham, Zarazuela; Haude, Katrina; Zhang, Zhancheng; Retterer, Kyle; Bai, Renkui; Calvo, Sarah E; Mootha, Vamsi K; Christodoulou, John; Rötig, Agnes; Filipovska, Aleksandra; Cristian, Ingrid; Falk, Marni J; Metodiev, Metodi D; Thorburn, David R

2017-08-03

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32 ∗ ]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Low-intensity laser irradiation at 660 nm stimulates transcription of genes involved in the electron transport chain.

PubMed

Masha, Roland T; Houreld, Nicolette N; Abrahamse, Heidi

2013-02-01

Low-intensity laser irradiation (LILI) has been shown to stimulate cellular functions leading to increased adenosine triphosphate (ATP) synthesis. This study was undertaken to evaluate the effect of LILI on genes involved in the mitochondrial electron transport chain (ETC, complexes I-IV) and oxidative phosphorylation (ATP synthase). Four human skin fibroblast cell models were used in this study: normal non-irradiated cells were used as controls while wounded, diabetic wounded, and ischemic cells were irradiated. Cells were irradiated with a 660 nm diode laser with a fluence of 5 J/cm(2) and gene expression determined by quantitative real-time reverse transcription (RT) polymerase chain reaction (PCR). LILI upregulated cytochrome c oxidase subunit VIb polypeptide 2 (COX6B2), cytochrome c oxidase subunit VIc (COX6C), and pyrophosphatase (inorganic) 1 (PPA1) in diabetic wounded cells; COX6C, ATP synthase, H+transporting, mitochondrial Fo complex, subunit B1 (ATP5F1), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) 1 alpha subcomplex, 11 (NDUFA11), and NADH dehydrogenase (ubiquinone) Fe-S protein 7 (NDUFS7) in wounded cells; and ATPase, H+/K+ exchanging, beta polypeptide (ATP4B), and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C2 (subunit 9) (ATP5G2) in ischemic cells. LILI at 660 nm stimulates the upregulation of genes coding for subunits of enzymes involved in complexes I and IV and ATP synthase.

Chemicals or mutations that target mitochondrial translation can rescue the respiratory deficiency of yeast bcs1 mutants.

PubMed

Panozzo, C; Laleve, A; Tribouillard-Tanvier, D; Ostojić, J; Sellem, C H; Friocourt, G; Bourand-Plantefol, A; Burg, A; Delahodde, A; Blondel, M; Dujardin, G

2017-12-01

Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III 2 /IV in yeast or I/III 2 /IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes. Copyright © 2017. Published by Elsevier B.V.

Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism.

PubMed Central

Fernandes, A P; Nelson, K; Beverley, S M

1993-01-01

Molecular evolutionary relationships within the protozoan order Kinetoplastida were deduced from comparisons of the nuclear small and large subunit ribosomal RNA (rRNA) gene sequences. These studies show that relationships among the trypanosomatid protozoans differ from those previously proposed from studies of organismal characteristics or mitochondrial rRNAs. The genera Leishmania, Endotrypanum, Leptomonas, and Crithidia form a closely related group, which shows progressively more distant relationships to Phytomonas and Blastocrithidia, Trypanosoma cruzi, and lastly Trypanosoma brucei. The rooting of the trypanosomatid tree was accomplished by using Bodo caudatus (family Bodonidae) as an outgroup, a status confirmed by molecular comparisons with other eukaryotes. The nuclear rRNA tree agrees well with data obtained from comparisons of other nuclear genes. Differences with the proposed mitochondrial rRNA tree probably reflect the lack of a suitable outgroup for this tree, as the topologies are otherwise similar. Small subunit rRNA divergences within the trypanosomatids are large, approaching those among plants and animals, which underscores the evolutionary antiquity of the group. Analysis of the distribution of different parasitic life-styles of these species in conjunction with a probable timing of evolutionary divergences suggests that vertebrate parasitism arose multiple times in the trypanosomatids. PMID:8265597

Novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtDNA recombination in the chigger mite, Leptotrombidium pallidum.

PubMed

Shao, Renfu; Mitani, Harumi; Barker, Stephen C; Takahashi, Mamoru; Fukunaga, Masahito

2005-06-01

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

Characterization of the complete mitochondrial genome of Ortleppascaris sinensis (Nematoda: Heterocheilidae) and comparative mitogenomic analysis of eighteen Ascaridida nematodes.

PubMed

Zhao, J H; Tu, G J; Wu, X B; Li, C P

2018-05-01

Ortleppascaris sinensis (Nematoda: Ascaridida) is a dominant intestinal nematode of the captive Chinese alligator. However, the epidemiology, molecular ecology and population genetics of this parasite remain largely unexplored. In this study, the complete mitochondrial (mt) genome sequence of O. sinensis was first determined using a polymerase chain reaction (PCR)-based primer-walking strategy, and this is also the first sequencing of the complete mitochondrial genome of a member of the genus Ortleppascaris. The circular mitochondrial genome (13,828 bp) of O. sinensis contained 12 protein-coding, 22 transfer RNA and 2 ribosomal RNA genes, but lacked the ATP synthetase subunit 8 gene. Finally, phylogenetic analysis of mtDNAs indicated that the genus Ortleppascaris should be attributed to the family Heterocheilidae. It is necessary to sequence more mtNDAs of Ortleppascaris nematodes in the future to test and confirm our conclusion. The complete mitochondrial genome sequence of O. sinensis reported here should contribute to molecular diagnosis, epidemiological investigations and ecological studies of O. sinensis and other related Ascaridida nematodes.

A New Primer Set to Amplify the Mitochondrial Cytochrome C Oxidase Subunit I (COI) Gene in the DHA-Rich Microalgae, the Genus Aurantiochytrium.

PubMed

Nishitani, Goh; Yoshida, Masaki

2018-06-01

This study was performed in order to develop a primer set for mitochondrial cytochrome c oxidase subunit I (COI) in the DHA-rich microalgae of the genus Aurantiochytrium. The performance of the primer set was tested using 12 Aurantiochytrium strains and other thraustochytrid species. There were no genetic polymorphisms in the mitochondrial sequences from the Aurantiochytrium strains, in contrast to the nuclear 18S rRNA gene sequence. This newly developed primer set amplified sequences from Aurantiochytrium and closely related genera, and may be useful for species identification and clarifying the genetic diversity of Aurantiochytrium in the field.

The complete mitochondrial genome sequence of Eimeria magna (Apicomplexa: Coccidia).

PubMed

Tian, Si-Qin; Cui, Ping; Fang, Su-Fang; Liu, Guo-Hua; Wang, Chun-Ren; Zhu, Xing-Quan

2015-01-01

In the present study, we determined the complete mitochondrial DNA (mtDNA) sequence of Eimeria magna from rabbits for the first time, and compared its gene contents and genome organizations with that of seven Eimeria spp. from domestic chickens. The size of the complete mt genome sequence of E. magna is 6249 bp, which consists of 3 protein-coding genes (cytb, cox1 and cox3), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, without transfer RNA genes, in accordance with that of Eimeria spp. from chickens. The putative direction of translation for three genes (cytb, cox1 and cox3) was the same as those of Eimeria species from domestic chickens. The content of A + T is 65.16% for E. magna mt genome (29.73% A, 35.43% T, 17.09 G and 17.75% C). The E. magna mt genome sequence provides novel mtDNA markers for studying the molecular epidemiology and population genetics of Eimeria spp. and has implications for the molecular diagnosis and control of rabbit coccidiosis.

Vps39 Interacts with Tom40 to Establish One of Two Functionally Distinct Vacuole-Mitochondria Contact Sites.

PubMed

González Montoro, Ayelén; Auffarth, Kathrin; Hönscher, Carina; Bohnert, Maria; Becker, Thomas; Warscheid, Bettina; Reggiori, Fulvio; van der Laan, Martin; Fröhlich, Florian; Ungermann, Christian

2018-06-04

The extensive subcellular network of membrane contact sites plays central roles in organelle biogenesis and communication, yet the precise contributions of the involved machineries remain largely enigmatic. The yeast vacuole forms a membrane contact site with mitochondria, called vacuolar and mitochondrial patch (vCLAMP). Formation of vCLAMPs involves the vacuolar Rab GTPase Ypt7 and the Ypt7-interacting Vps39 subunit of the HOPS tethering complex. Here, we uncover the general preprotein translocase of the outer membrane (TOM) subunit Tom40 as the direct binding partner of Vps39 on mitochondria. We identify Vps39 mutants defective in TOM binding, but functional for HOPS. Cells that cannot form vCLAMPs show reduced growth under stress conditions and impaired survival upon starvation. Unexpectedly, our mutant analysis revealed the existence of two functionally independent vacuole-mitochondria MCSs: one formed by the Ypt7-Vps39-Tom40 tether and a second one by Vps13-Mcp1, which is redundant with ER-mitochondrial contacts formed by ERMES. Copyright © 2018 Elsevier Inc. All rights reserved.

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir

2010-10-08

Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-,more » and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.« less

Two nuclear life cycle-regulated genes encode interchangeable subunits c of mitochondrial ATP synthase in Podospora anserina.

PubMed

Déquard-Chablat, Michelle; Sellem, Carole H; Golik, Pawel; Bidard, Frédérique; Martos, Alexandre; Bietenhader, Maïlis; di Rago, Jean-Paul; Sainsard-Chanet, Annie; Hermann-Le Denmat, Sylvie; Contamine, Véronique

2011-07-01

An F(1)F(O) ATP synthase in the inner mitochondrial membrane catalyzes the late steps of ATP production via the process of oxidative phosphorylation. A small protein subunit (subunit c or ATP9) of this enzyme shows a substantial genetic diversity, and its gene can be found in both the mitochondrion and/or nucleus. In a representative set of 26 species of fungi for which the genomes have been entirely sequenced, we found five Atp9 gene repartitions. The phylogenetic distribution of nuclear and mitochondrial Atp9 genes suggests that their evolution has included two independent transfers to the nucleus followed by several independent episodes of the loss of the mitochondrial and/or nuclear gene. Interestingly, we found that in Podospora anserina, subunit c is exclusively produced from two nuclear genes (PaAtp9-5 and PaAtp9-7), which display different expression profiles through the life cycle of the fungus. The PaAtp9-5 gene is specifically and strongly expressed in germinating ascospores, whereas PaAtp9-7 is mostly transcribed during sexual reproduction. Consistent with these observations, deletion of PaAtp9-5 is lethal, whereas PaAtp9-7 deletion strongly impairs ascospore production. The P. anserina PaAtp9-5 and PaAtp9-7 genes are therefore nonredundant. By swapping the 5' and 3' flanking regions between genes we demonstrated, however, that the PaAtp9 coding sequences are functionally interchangeable. These findings show that after transfer to the nucleus, the subunit c gene in Podospora became a key target for the modulation of cellular energy metabolism according to the requirements of the life cycle.

Fungal-specific subunits of the Candida albicans mitochondrial complex I drive diverse cell functions including cell wall synthesis.

PubMed

She, Xiaodong; Khamooshi, Kasra; Gao, Yin; Shen, Yongnian; Lv, Yuxia; Calderone, Richard; Fonzi, William; Liu, Weida; Li, Dongmei

2015-09-01

Our published research has focused on the role of Goa1p, an apparent regulator of the Candida albicans mitochondrial complex I (CI). Lack of Goa1p affects optimum cell growth, CI activity and virulence. Eukaryotic CI is composed of a core of 14 alpha-proteobacterial subunit proteins and a variable number of supernumerary subunit proteins. Of the latter group of proteins, one (NUZM) is fungal specific and the other (NUXM) is found in fungi, algae and plants, but is not a mammalian CI subunit protein. We have established that NUXM is orf19.6607 and NUZM is orf19.287 in C. albicans. Herein, we validate both subunit proteins as NADH:ubiquinone oxidoreductases (NUO) and annotate their gene functions. To accomplish these objectives, we compared null mutants of each with wild type (WT) and gene-reconstituted strains. Genetic mutants of genes NUO1 (orf19.6607) and NUO2 (orf19.287), not surprisingly, each had reduced oxygen consumption, decreased mitochondrial redox potential, decreased CI activity, increased reactive oxidant species (ROS) and decreased chronological ageing in vitro. Loss of either gene results in disassembly of CI. Transcriptional profiling of both mutants indicated significant down-regulation of genes of carbon metabolism, as well as up-regulation of mitochondrial-associated gene families that may occur to compensate for the loss of CI activity. Profiling of both mutants also demonstrated a loss of cell wall β-mannosylation but not in a conserved CI subunit (ndh51Δ). The profiling data may indicate specific functions driven by the enzymatic activity of Nuo1p and Nuo2p. Of importance, each mutant is also avirulent in a murine blood-borne, invasive model of candidiasis associated with their reduced colonization of tissues. Based on their fungal specificity and roles in virulence, we suggest both as drug targets for antifungal drug discovery. © 2015 John Wiley & Sons Ltd.

Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases.

PubMed

Sultana, Shemaila; Solotchi, Mihai; Ramachandran, Aparna; Patel, Smita S

2017-11-03

Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rates of T7 RNAP (2 × 10 -6 ), yeast mitochondrial Rpo41 (6 × 10 -6 ), and human mitochondrial POLRMT (RNA polymerase mitochondrial) (2 × 10 -5 ) indicate high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A→G errors in RNA with rates of ∼10 -4 The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine-purine mismatches were relatively frequent (10 -5 -10 -6 ), whereas C→G, U→A, G→C, and C→A errors from purine-purine and pyrimidine-pyrimidine mismatches were rare (10 -7 -10 -10 ). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (∼10 -4 ). Strikingly, POLRMT shows a high mutagenic bypass rate, which is exacerbated by TEFM (transcription elongation factor mitochondrial). The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM, which allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in the case of T7 RNAP, to assess the quality of in vitro transcribed RNAs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Respiromics - An integrative analysis linking mitochondrial bioenergetics to molecular signatures.

PubMed

Walheim, Ellen; Wiśniewski, Jacek R; Jastroch, Martin

2018-03-01

Energy metabolism is challenged upon nutrient stress, eventually leading to a variety of metabolic diseases that represent a major global health burden. Here, we combine quantitative mitochondrial respirometry (Seahorse technology) and proteomics (LC-MS/MS-based total protein approach) to understand how molecular changes translate to changes in mitochondrial energy transduction during diet-induced obesity (DIO) in the liver. The integrative analysis reveals that significantly increased palmitoyl-carnitine respiration is supported by an array of proteins enriching lipid metabolism pathways. Upstream of the respiratory chain, the increased capacity for ATP synthesis during DIO associates strongest to mitochondrial uptake of pyruvate, which is routed towards carboxylation. At the respiratory chain, robust increases of complex I are uncovered by cumulative analysis of single subunit concentrations. Specifically, nuclear-encoded accessory subunits, but not mitochondrial-encoded or core units, appear to be permissive for enhanced lipid oxidation. Our integrative analysis, that we dubbed "respiromics", represents an effective tool to link molecular changes to functional mechanisms in liver energy metabolism, and, more generally, can be applied for mitochondrial analysis in a variety of metabolic and mitochondrial disease models. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Molecular phylogeny of the Drusinae (Trichoptera: Limnephilidae): preliminary results

NASA Astrophysics Data System (ADS)

Pauls, S.; Lumbsch, T.; Haase, P.

2005-05-01

We examine the phylogenetic relationships within the subfamily of the Drusinae using molecular markers. Sequence data from two mitochondrial loci (mitochondrial cytochrome oxidase I, mitochondrial ribosomal large subunit) are used to infer the relationships within and among the genera of the Drusinae. Sequence data were generated for 21 taxa from five genera from the subfamily. The molecular data were analyzed using a Bayesian Markov Chain Monte Carlo and a Maximum Parsimony approach for both single gene and combined data sets. Several hypotheses of relationships previously inferred based on morphological characters were tested. The study revealed a very close relationship between Drusus discolor and D. romanicus suggesting that divergence between these two species occurred recently. The relationships inferred by molecular data suggest that larval morphology may be an important taxonomic character, which has often been neglected. The data also indicate that the genera Ecclisopteryx and Drusus are polyphyletic with respect to one another.

The complete mitochondrial genome of Chinese green hydra, Hydra sinensis (Hydroida: Hydridae).

PubMed

Pan, Hong-Chun; Qian, Xiao-Cheng; Li, Ping; Li, Xiao-Fei; Wang, An-Tai

2014-02-01

The complete mitochondrial genome of Chinese green hydra, Hydra sinensis (Hydroida: Hydridae) is a linear molecule of 16,189 bp in length, containing 13 protein-coding genes, small and large subunit ribosomal RNAs, methionine and tryptophan transfer RNAs, a pseudogene consisting of a partial copy of COI and terminal sequences at two ends of the linear mitochondrial DNA. The A + T content of the overall base composition of H-strand is 77.2% (T: 41.7%; C: 10.9%; A: 35.5%; and G: 11.9%). COI and ND1 genes begin with GTG as start codon, while other 11 protein-coding genes start with a typical ATG initiation codon. COII, ATP8, ATP6, COIII, ND5, ND6, ND3, ND1, ND4 and COI genes are terminated with TAA as stop codon, ND4L ends with TAG, ND2 ends with TA and Cyt b ends with T.

Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

PubMed

Afolayan, Adeleye J; Eis, Annie; Alexander, Maxwell; Michalkiewicz, Teresa; Teng, Ru-Jeng; Lakshminrusimha, Satyan; Konduri, Girija G

2016-01-01

Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen. Copyright © 2016 the American Physiological Society.

Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

PubMed Central

Burri, Lena; Strahm, Yvan; Hawkins, Christine J.; Gentle, Ian E.; Puryer, Michelle A.; Verhagen, Anne; Callus, Bernard; Vaux, David; Lithgow, Trevor

2005-01-01

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX5P in Imp1 and NX5S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space. PMID:15814844

Acetyl-L-carnitine activates the peroxisome proliferator-activated receptor-γ coactivators PGC-1α/PGC-1β-dependent signaling cascade of mitochondrial biogenesis and decreases the oxidized peroxiredoxins content in old rat liver.

PubMed

Pesce, Vito; Nicassio, Luigi; Fracasso, Flavio; Musicco, Clara; Cantatore, Palmiro; Gadaleta, Maria Nicola

2012-04-01

The behavior of the peroxisome proliferator-activated receptor-γ coactivators PGC-1α/PGC-β-dependent mitochondrial biogenesis signaling pathway, as well as the level of some antioxidant enzymes and proteins involved in mitochondrial dynamics in the liver of old rats before and after 2 months of acetyl-L-carnitine (ALCAR) supplementation, was tested. The results reveal that ALCAR treatment is able to reverse the age-associated decline of PGC-1α, PGC-1β, nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 (ND1), and cytochrome c oxidase subunit IV (COX IV) protein levels, of mitochondrial DNA (mtDNA) content, and of citrate synthase activity. Moreover, it partially reverses the mitochondrial superoxide dismutase 2 (SOD2) decline and reduces the cellular content of oxidized peroxiredoxins. These data demonstrate that ALCAR treatment is able to promote in the old rat liver a new mitochondrial population that can contribute to the cellular oxidative stress reduction. Furthermore, a remarkable decline of Drp1 and of Mfn2 proteins is reported here for the first time, suggesting a reduced mitochondrial dynamics in aging liver with no effect of ALCAR treatment.

Changes in Whole-Body Oxygen Consumption and Skeletal Muscle Mitochondria During Linezolid-Induced Lactic Acidosis.

PubMed

Protti, Alessandro; Ronchi, Dario; Bassi, Gabriele; Fortunato, Francesco; Bordoni, Andreina; Rizzuti, Tommaso; Fumagalli, Roberto

2016-07-01

To better clarify the pathogenesis of linezolid-induced lactic acidosis. Case report. ICU. A 64-year-old man who died with linezolid-induced lactic acidosis. Skeletal muscle was sampled at autopsy to study mitochondrial function. Lactic acidosis developed during continuous infusion of linezolid while oxygen consumption and oxygen extraction were diminishing from 172 to 52 mL/min/m and from 0.27 to 0.10, respectively. Activities of skeletal muscle respiratory chain complexes I, III, and IV, encoded by nuclear and mitochondrial DNA, were abnormally low, whereas activity of complex II, entirely encoded by nuclear DNA, was not. Protein studies confirmed stoichiometric imbalance between mitochondrial (cytochrome c oxidase subunits 1 and 2) and nuclear (succinate dehydrogenase A) DNA-encoded respiratory chain subunits. These findings were not explained by defects in mitochondrial DNA or transcription. There were no compensatory mitochondrial biogenesis (no induction of nuclear respiratory factor 1 and mitochondrial transcript factor A) or adaptive unfolded protein response (reduced concentration of heat shock proteins 60 and 70). Linezolid-induced lactic acidosis is associated with diminished global oxygen consumption and extraction. These changes reflect selective inhibition of mitochondrial protein synthesis (probably translation) with secondary mitonuclear imbalance. One novel aspect of linezolid toxicity that needs to be confirmed is blunting of reactive mitochondrial biogenesis and unfolded protein response.

Mitochondrial NDUFS3 regulates the ROS-mediated onset of metabolic switch in transformed cells

PubMed Central

Suhane, Sonal; Kanzaki, Hirotaka; Arumugaswami, Vaithilingaraja; Murali, Ramachandran; Ramanujan, V. Krishnan

2013-01-01

Summary Aerobic glycolysis in transformed cells is an unique metabolic phenotype characterized by a hyperactivated glycolytic pathway even in the presence of oxygen. It is not clear if the onset of aerobic glycolysis is regulated by mitochondrial dysfunction and, if so, what the metabolic windows of opportunity available to control this metabolic switch (mitochondrial to glycolytic) landscape are in transformed cells. Here we report a genetically-defined model system based on the gene-silencing of a mitochondrial complex I subunit, NDUFS3, where we demonstrate the onset of metabolic switch in isogenic human embryonic kidney cells by differential expression of NDUFS3. By means of extensive metabolic characterization, we demonstrate that NDUFS3 gene silencing systematically introduces mitochondrial dysfunction thereby leading to the onset of aerobic glycolysis in a manner dependent on NDUFS3 protein levels. Furthermore, we show that the sustained imbalance in free radical dynamics is a necessary condition to sustain the observed metabolic switch in cell lines with the most severe NDUFS3 suppression. Together, our data reveal a novel role for mitochondrial complex I subunit NDUFS3 in regulating the degree of mitochondrial dysfunction in living cells, thereby setting a “metabolic threshold” for the observation of aerobic glycolysis phenotype within the confines of mitochondrial dysfunction. PMID:23519235

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.

PubMed

Gong, Guohua; Liu, Xiaoyun; Zhang, Huiliang; Sheu, Shey-Shing; Wang, Wang

2015-10-01

Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions. Copyright © 2015 the American Physiological Society.

Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells*

PubMed Central

Sacoman, Juliana L.; Dagda, Raul Y.; Burnham-Marusich, Amanda R.; Dagda, Ruben K.; Berninsone, Patricia M.

2017-01-01

O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics. PMID:28100784

Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

PubMed

Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

2015-05-04

Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Phenyl-alpha-tert-butyl nitrone reverses mitochondrial decay in acute Chagas' disease.

PubMed

Wen, Jian-Jun; Bhatia, Vandanajay; Popov, Vsevolod L; Garg, Nisha Jain

2006-12-01

In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas' disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respiratory complex subunits, and >60% inhibition of mtDNA-encoded transcripts for respiratory complex subunits in infected myocardium. The antioxidant phenyl-alpha-tert-butyl nitrone (PBN) arrested the oxidative damage-mediated loss in mitochondrial membrane integrity, preserved redox potential-coupled mitochondrial gene expression, and improved respiratory complex activities (47 to 95% increase) and cardiac ATP level (>or=40% increase) in infected myocardium. Importantly, PBN resulted twofold decline in mitochondrial reactive oxygen species production rate in infected myocardium. Taken together, our data demonstrate the pathological significance of oxidative stress in metabolic decay and energy homeostasis in acute chagasic myocarditis and further suggest that oxidative injuries affecting mitochondrial integrity-dependent expression and activity of the respiratory complexes initiate a feedback cycle of electron transport chain inefficiency, increased reactive oxygen species production, and energy homeostasis in acute chagasic hearts. PBN and other mitochondria-targeted antioxidants may be useful in altering mitochondrial decay and oxidative pathology in Chagas' disease.

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

PubMed Central

Schoch, Conrad L.; Seifert, Keith A.; Huhndorf, Sabine; Robert, Vincent; Spouge, John L.; Levesque, C. André; Chen, Wen; Bolchacova, Elena; Voigt, Kerstin; Crous, Pedro W.; Miller, Andrew N.; Wingfield, Michael J.; Aime, M. Catherine; An, Kwang-Deuk; Bai, Feng-Yan; Barreto, Robert W.; Begerow, Dominik; Bergeron, Marie-Josée; Blackwell, Meredith; Boekhout, Teun; Bogale, Mesfin; Boonyuen, Nattawut; Burgaz, Ana R.; Buyck, Bart; Cai, Lei; Cai, Qing; Cardinali, G.; Chaverri, Priscila; Coppins, Brian J.; Crespo, Ana; Cubas, Paloma; Cummings, Craig; Damm, Ulrike; de Beer, Z. Wilhelm; de Hoog, G. Sybren; Del-Prado, Ruth; Dentinger, Bryn; Diéguez-Uribeondo, Javier; Divakar, Pradeep K.; Douglas, Brian; Dueñas, Margarita; Duong, Tuan A.; Eberhardt, Ursula; Edwards, Joan E.; Elshahed, Mostafa S.; Fliegerova, Katerina; Furtado, Manohar; García, Miguel A.; Ge, Zai-Wei; Griffith, Gareth W.; Griffiths, K.; Groenewald, Johannes Z.; Groenewald, Marizeth; Grube, Martin; Gryzenhout, Marieka; Guo, Liang-Dong; Hagen, Ferry; Hambleton, Sarah; Hamelin, Richard C.; Hansen, Karen; Harrold, Paul; Heller, Gregory; Herrera, Cesar; Hirayama, Kazuyuki; Hirooka, Yuuri; Ho, Hsiao-Man; Hoffmann, Kerstin; Hofstetter, Valérie; Högnabba, Filip; Hollingsworth, Peter M.; Hong, Seung-Beom; Hosaka, Kentaro; Houbraken, Jos; Hughes, Karen; Huhtinen, Seppo; Hyde, Kevin D.; James, Timothy; Johnson, Eric M.; Johnson, Joan E.; Johnston, Peter R.; Jones, E.B. Gareth; Kelly, Laura J.; Kirk, Paul M.; Knapp, Dániel G.; Kõljalg, Urmas; Kovács, Gábor M.; Kurtzman, Cletus P.; Landvik, Sara; Leavitt, Steven D.; Liggenstoffer, Audra S.; Liimatainen, Kare; Lombard, Lorenzo; Luangsa-ard, J. Jennifer; Lumbsch, H. Thorsten; Maganti, Harinad; Maharachchikumbura, Sajeewa S. N.; Martin, María P.; May, Tom W.; McTaggart, Alistair R.; Methven, Andrew S.; Meyer, Wieland; Moncalvo, Jean-Marc; Mongkolsamrit, Suchada; Nagy, László G.; Nilsson, R. Henrik; Niskanen, Tuula; Nyilasi, Ildikó; Okada, Gen; Okane, Izumi; Olariaga, Ibai; Otte, Jürgen; Papp, Tamás; Park, Duckchul; Petkovits, Tamás; Pino-Bodas, Raquel; Quaedvlieg, William; Raja, Huzefa A.; Redecker, Dirk; Rintoul, Tara L.; Ruibal, Constantino; Sarmiento-Ramírez, Jullie M.; Schmitt, Imke; Schüßler, Arthur; Shearer, Carol; Sotome, Kozue; Stefani, Franck O.P.; Stenroos, Soili; Stielow, Benjamin; Stockinger, Herbert; Suetrong, Satinee; Suh, Sung-Oui; Sung, Gi-Ho; Suzuki, Motofumi; Tanaka, Kazuaki; Tedersoo, Leho; Telleria, M. Teresa; Tretter, Eric; Untereiner, Wendy A.; Urbina, Hector; Vágvölgyi, Csaba; Vialle, Agathe; Vu, Thuy Duong; Walther, Grit; Wang, Qi-Ming; Wang, Yan; Weir, Bevan S.; Weiß, Michael; White, Merlin M.; Xu, Jianping; Yahr, Rebecca; Yang, Zhu L.; Yurkov, Andrey; Zamora, Juan-Carlos; Zhang, Ning; Zhuang, Wen-Ying; Schindel, David

2012-01-01

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups. PMID:22454494

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.

PubMed

Schoch, Conrad L; Seifert, Keith A; Huhndorf, Sabine; Robert, Vincent; Spouge, John L; Levesque, C André; Chen, Wen

2012-04-17

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.

Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9.

PubMed

Willson, T A; Nagley, P

1987-09-01

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.

Phylogeny, divergence time and historical biogeography of Laetiporus (Basidiomycota, Polyporales).

PubMed

Song, Jie; Cui, Bao-Kai

2017-04-20

The aim of this study was to characterize the molecular relationship, origin and historical biogeography of the species in important brown rot fungal genus Laetiporus from East Asia, Europe, Pan-America, Hawaii and South Africa. We used six genetic markers to estimate a genus-level phylogeny including (1) the internal transcribed spacer (ITS), (2) nuclear large subunit rDNA (nrLSU), (3) nuclear small subunit rDNA (nrSSU), (4) translation elongation factor 1-α (EF-1α), (5) DNA-directed RNA polymerase II subunit 2 (RPB2), and (6) mitochondrial small subunit rDNA (mtSSU). Results of multi-locus phylogenetic analyses show clade support for at least seventeen species-level lineages including two new Laetiporus in China. Molecular dating using BEAST estimated the present crown group diverged approximately 20.16 million years ago (Mya) in the early Miocene. Biogeographic analyses using RASP indicated that Laetiporus most likely originated in temperate zones with East Asia and North America having the highest probability (48%) of being the ancestral area. Four intercontinental dispersal routes and a possible concealed dispersal route were established for the first time.

Long-term tracing of Rhizophagus irregularis isolate BEG140 inoculated on Phalaris arundinacea in a coal mine spoil bank, using mitochondrial large subunit rDNA markers.

PubMed

Sýkorová, Zuzana; Börstler, Boris; Zvolenská, Soňa; Fehrer, Judith; Gryndler, Milan; Vosátka, Miroslav; Redecker, Dirk

2012-01-01

During the last decade, the application of arbuscular mycorrhizal fungi (AMF) as bioenhancers has increased significantly. However, until now, it has been difficult to verify the inoculation success in terms of fungal symbiont establishment in roots of inoculated plants because specific fungal strains could not be detected within colonized roots. Using mitochondrial large subunit ribosomal DNA, we show that Rhizophagus irregularis (formerly known as Glomus intraradices) isolate BEG140 consists of two different haplotypes. We developed nested PCR assays to specifically trace each of the two haplotypes in the roots of Phalaris arundinacea from a field experiment in a spoil bank of a former coal mine, where BEG140 was used as inoculant. We revealed that despite the relatively high diversity of native R. irregularis strains, R. irregularis BEG140 survived and proliferated successfully in the field experiment and was found significantly more often in the inoculated than control plots. This work is the first one to show tracing of an inoculated AMF isolate in the roots of target plants and to verify its survival and propagation in the field. These results will have implications for basic research on the ecology of AMF at the intraspecific level as well as for commercial users of mycorrhizal inoculation.

Fulminant neurological deterioration in a neonate with Leigh syndrome due to a maternally transmitted missense mutation in the mitochondrial ND3 gene.

PubMed

Leshinsky-Silver, E; Lev, D; Tzofi-Berman, Z; Cohen, S; Saada, A; Yanoov-Sharav, M; Gilad, E; Lerman-Sagie, T

2005-08-26

Leigh syndrome can result from both nuclear and mitochondrial DNA defects. Mutations in complex V genes of the respiratory chain were considered until recently as the most frequent cause for mitochondrial inherited Leigh syndrome, while gene defects in complex I were related to recessive Leigh syndrome. Recently few reports of mutations in the mitochondrial-encoded complex I subunit genes causing Leigh syndrome have been reported. We describe a 1-month-old baby who acutely deteriorated, with abrupt onset of brainstem dysfunction, due to basal ganglia lesions extending to the brainstem. A muscle biopsy demonstrated complex I deficiency. Subsequent analysis of the mitochondrial genome revealed a homoplastic T10191C mutation in the ND3 gene (in blood and muscle), resulting in a substitution of serine to proline. Hair root analysis revealed a 50% mutant load, reflecting heteroplasmy in early embryonic stages. The mutation was also detected in his mother (5%). Western blot analysis revealed a decrease of the 20 kDa subunit (likely ND6) and of the 30 kDa subunit (NDUFA9), which is probably due to instability attributed to the inability to form subcomplexes with ND3. This is the first description of infantile Leigh syndrome due to a maternally transmitted T10191C substitution in ND3 and not due to a de novo mutation. This mutation is age and tissue dependent and therefore may not be amenable to prenatal testing.

The translational landscape of Arabidopsis mitochondria.

PubMed

Planchard, Noelya; Bertin, Pierre; Quadrado, Martine; Dargel-Graffin, Céline; Hatin, Isabelle; Namy, Olivier; Mireau, Hakim

2018-06-05

Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosome footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated at higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V, where higher ribosome coverage corroborated with higher requirements for specific subunits. In complex I respiratory mutants, a reorganization of mitochondrial mRNAs ribosome association was detected involving increased ribosome densities for certain ribosomal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding translation in higher plant mitochondria.

Haploinsufficiency of the 22q11.2 microdeletion gene Mrpl40 disrupts short-term synaptic plasticity and working memory through dysregulation of mitochondrial calcium.

PubMed

Devaraju, P; Yu, J; Eddins, D; Mellado-Lagarde, M M; Earls, L R; Westmoreland, J J; Quarato, G; Green, D R; Zakharenko, S S

2017-09-01

Hemizygous deletion of a 1.5- to 3-megabase region on chromosome 22 causes 22q11.2 deletion syndrome (22q11DS), which constitutes one of the strongest genetic risks for schizophrenia. Mouse models of 22q11DS have abnormal short-term synaptic plasticity that contributes to working-memory deficiencies similar to those in schizophrenia. We screened mutant mice carrying hemizygous deletions of 22q11DS genes and identified haploinsufficiency of Mrpl40 (mitochondrial large ribosomal subunit protein 40) as a contributor to abnormal short-term potentiation (STP), a major form of short-term synaptic plasticity. Two-photon imaging of the genetically encoded fluorescent calcium indicator GCaMP6, expressed in presynaptic cytosol or mitochondria, showed that Mrpl40 haploinsufficiency deregulates STP via impaired calcium extrusion from the mitochondrial matrix through the mitochondrial permeability transition pore. This led to abnormally high cytosolic calcium transients in presynaptic terminals and deficient working memory but did not affect long-term spatial memory. Thus, we propose that mitochondrial calcium deregulation is a novel pathogenic mechanism of cognitive deficiencies in schizophrenia.

Maternal High Fat Diet Alters Skeletal Muscle Mitochondrial Catalytic Activity in Adult Male Rat Offspring

PubMed Central

Pileggi, Chantal A.; Hedges, Christopher P.; Segovia, Stephanie A.; Markworth, James F.; Durainayagam, Brenan R.; Gray, Clint; Zhang, Xiaoyuan D.; Barnett, Matthew P. G.; Vickers, Mark H.; Hickey, Anthony J. R.; Reynolds, Clare M.; Cameron-Smith, David

2016-01-01

A maternal high-fat (HF) diet during pregnancy can lead to metabolic compromise, such as insulin resistance in adult offspring. Skeletal muscle mitochondrial dysfunction is one mechanism contributing to metabolic impairments in insulin resistant states. Therefore, the present study aimed to investigate whether mitochondrial dysfunction is evident in metabolically compromised offspring born to HF-fed dams. Sprague-Dawley dams were randomly assigned to receive a purified control diet (CD; 10% kcal from fat) or a high fat diet (HFD; 45% kcal from fat) for 10 days prior to mating, throughout pregnancy and during lactation. From weaning, all male offspring received a standard chow diet and soleus muscle was collected at day 150. Expression of the mitochondrial transcription factors nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor A (mtTFA) were downregulated in HF offspring. Furthermore, genes encoding the mitochondrial electron transport system (ETS) respiratory complex subunits were suppressed in HF offspring. Moreover, protein expression of the complex I subunit, NDUFB8, was downregulated in HF offspring (36%), which was paralleled by decreased maximal catalytic linked activity of complex I and III (40%). Together, these results indicate that exposure to a maternal HF diet during development may elicit lifelong mitochondrial alterations in offspring skeletal muscle. PMID:27917127

The genome and transcriptome of perennial ryegrass mitochondria

PubMed Central

2013-01-01

Background Perennial ryegrass (Lolium perenne L.) is one of the most important forage and turf grass species of temperate regions worldwide. Its mitochondrial genome is inherited maternally and contains genes that can influence traits of agricultural importance. Moreover, the DNA sequence of mitochondrial genomes has been established and compared for a large number of species in order to characterize evolutionary relationships. Therefore, it is crucial to understand the organization of the mitochondrial genome and how it varies between and within species. Here, we report the first de novo assembly and annotation of the complete mitochondrial genome from perennial ryegrass. Results Intact mitochondria from perennial ryegrass leaves were isolated and used for mtDNA extraction. The mitochondrial genome was sequenced to a 167-fold coverage using the Roche 454 GS-FLX Titanium platform, and assembled into a circular master molecule of 678,580 bp. A total of 34 proteins, 14 tRNAs and 3 rRNAs are encoded by the mitochondrial genome, giving a total gene space of 48,723 bp (7.2%). Moreover, we identified 149 open reading frames larger than 300 bp and covering 67,410 bp (9.93%), 250 SSRs, 29 tandem repeats, 5 pairs of large repeats, and 96 pairs of short inverted repeats. The genes encoding subunits of the respiratory complexes – nad1 to nad9, cob, cox1 to cox3 and atp1 to atp9 – all showed high expression levels both in absolute numbers and after normalization. Conclusions The circular master molecule of the mitochondrial genome from perennial ryegrass presented here constitutes an important tool for future attempts to compare mitochondrial genomes within and between grass species. Our results also demonstrate that mitochondria of perennial ryegrass contain genes crucial for energy production that are well conserved in the mitochondrial genome of monocotyledonous species. The expression analysis gave us first insights into the transcriptome of these mitochondrial genes in perennial ryegrass. PMID:23521852

DOE Office of Scientific and Technical Information (OSTI.GOV)

Desai, S.; Ruff, V.; DuBrul, E.F.

The pyruvate dehydrogenase complex (PDC) plays a pivotal role in the anaerobic metabolism of Ascaris suum mitochondria. They have initiated a series of studies on the in vitro synthesis and mitochondrial import of PDC. PDC has been purified from adult Ascaris body wall muscle, fully phosphorylated in vitro, and separated into its component subunits on SDS/PAGE. The individual components were electroeluted from the gels and used to immunize rabbits. IgG's to the individual subunits were prepared from antisera and their specificities were verified by immuno-blotting. Each IgG identified a single specific band at the appropriate location in extracts of adultmore » Ascaris body wall muscle mitochondria. Poly A/sup +/-RNA was prepared from body wall muscle and translated in a reticylocyte lysate system using /sup 35/S-methionine. Translation products were immunoprecipitated with specific IgG's, electrophoresed, and fluorographed. Each immunoprecipitation gave rise to a single radioactive polypeptide that was slightly larger than the specific PDC subunit isolated from the adult mitochondria. This system has demonstrated its feasibility for the study of mitochondrial import of a multienzyme complex that is critical for the anaerobic mitochondrial metabolism of Ascaris suum.« less

Binding of the immunomodulatory drug Bz-423 to mitochondrial FoF1-ATP synthase in living cells by FRET acceptor photobleaching

NASA Astrophysics Data System (ADS)

Starke, Ilka; Johnson, Kathryn M.; Petersen, Jan; Gräber, Peter; Opipari, Anthony W.; Glick, Gary D.; Börsch, Michael

2016-03-01

Bz-423 is a promising new drug for treatment of autoimmune diseases. This small molecule binds to subunit OSCP of the mitochondrial enzyme FoF1-ATP synthase and modulates its catalytic activities. We investigate the binding of Bz-423 to mitochondria in living cells and how subunit rotation in FoF1-ATP synthase, i.e. the mechanochemical mechanism of this enzyme, is affected by Bz-423. Therefore, the enzyme was marked selectively by genetic fusion with the fluorescent protein EGFP to the C terminus of subunit γ. Imaging the threedimensional arrangement of mitochondria in living yeast cells was possible at superresolution using structured illumination microscopy, SIM. We measured uptake and binding of a Cy5-labeled Bz-423 derivative to mitochondrial FoF1-ATP synthase in living yeast cells using FRET acceptor photobleaching microscopy. Our data confirmed the binding of Cy5-labeled Bz-423 to the top of the F1 domain of the enzyme in mitochondria of living Saccharomyces cerevisiae cells.

Mutant NDUFS3 subunit of mitochondrial complex I causes Leigh syndrome.

PubMed

Bénit, P; Slama, A; Cartault, F; Giurgea, I; Chretien, D; Lebon, S; Marsac, C; Munnich, A; Rötig, A; Rustin, P

2004-01-01

Respiratory chain complex I deficiency represents a genetically heterogeneous group of diseases resulting from mutations in mitochondrial or nuclear genes. Mutations have been reported in 13 of the 14 subunits encoding the core of complex I (seven mitochondrial and six nuclear genes) and these result in Leigh or Leigh-like syndromes or cardiomyopathy. In this study, a combination of denaturing high performance liquid chromatography and sequence analysis was used to study the NDUFS3 gene in a series of complex I deficient patients. Mutations found in this gene (NADH dehydrogenase iron-sulphur protein 3), coding for the seventh and last subunit of complex I core, were shown to cause late onset Leigh syndrome, optic atrophy, and complex I deficiency. A biochemical diagnosis of complex I deficiency on cultured amniocytes from a later pregnancy was confirmed through the identification of disease causing NDUFS3 mutations in these cells. While mutations in the NDUFS3 gene thus result in Leigh syndrome, a dissimilar clinical phenotype is observed in mutations in the NDUFV2 and NDUFS2 genes, resulting in encephalomyopathy and cardiomyopathy. The reasons for these differences are uncertain.

A MELAS syndrome family harboring two mutations in mitochondrial genome.

PubMed

Choi, Byung-Ok; Hwang, Jung Hee; Kim, Joonki; Cho, Eun Min; Cho, Sun Young; Hwang, Su Jin; Lee, Hyang Woon; Kim, Song Ja; Chung, Ki Wha

2008-06-30

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T>C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A>C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T>C mutation is pathogenic, however, the 13849A>C mutation is of unclear significance. It is likely that the 13849A>C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders.

A MELAS syndrome family harboring two mutations in mitochondrial genome

PubMed Central

Choi, Byung-Ok; Hwang, Jung Hee; Kim, Joonki; Cho, Eun Min; Cho, Sun Young; Hwang, Su Jin; Lee, Hyang Woon; Kim, Song Ja

2008-01-01

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T > C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A > C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T > C mutation is pathogenic, however, the 13849A > C mutation is of unclear significance. It is likely that the 13849A > C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders. PMID:18587274

The amino terminal extension of mammalian mitochondrial RNA polymerase ensures promoter specific transcription initiation

PubMed Central

Posse, Viktor; Hoberg, Emily; Dierckx, Anke; Shahzad, Saba; Koolmeister, Camilla; Larsson, Nils-Göran; Wilhelmsson, L. Marcus; Hällberg, B. Martin; Gustafsson, Claes M.

2014-01-01

Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated. PMID:24445803

Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase*║

PubMed Central

Wang, Yamin; Singh, Usha; Mueller, David M.

2013-01-01

The mitochondrial ATP synthase is a molecular motor, which couples the flow of rotons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. PMID:17244612

Aim-less translation: loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis.

PubMed

Kuzmenko, Anton; Derbikova, Ksenia; Salvatori, Roger; Tankov, Stoyan; Atkinson, Gemma C; Tenson, Tanel; Ott, Martin; Kamenski, Piotr; Hauryliuk, Vasili

2016-01-05

The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system's components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability. mIF3 was thought to be absent in Saccharomyces cerevisiae until we recently identified mitochondrial protein Aim23 as the missing orthologue. Here we show that, surprisingly, loss of mIF3/Aim23 in S. cerevisiae does not indiscriminately abrogate mitochondrial translation but rather causes an imbalance in protein production: the rate of synthesis of the Atp9 subunit of F1F0 ATP synthase (complex V) is increased, while expression of Cox1, Cox2 and Cox3 subunits of cytochrome c oxidase (complex IV) is repressed. Our results provide one more example of deviation of mitochondrial translation from its bacterial origins.

Fine-scale mergers of chloroplast and mitochondrial genes create functional, transcompartmentally chimeric mitochondrial genes.

PubMed

Hao, Weilong; Palmer, Jeffrey D

2009-09-29

The mitochondrial genomes of flowering plants possess a promiscuous proclivity for taking up sequences from the chloroplast genome. All characterized chloroplast integrants exist apart from native mitochondrial genes, and only a few, involving chloroplast tRNA genes that have functionally supplanted their mitochondrial counterparts, appear to be of functional consequence. We developed a novel computational approach to search for homologous recombination (gene conversion) in a large number of sequences and applied it to 22 mitochondrial and chloroplast gene pairs, which last shared common ancestry some 2 billion years ago. We found evidence of recurrent conversion of short patches of mitochondrial genes by chloroplast homologs during angiosperm evolution, but no evidence of gene conversion in the opposite direction. All 9 putative conversion events involve the atp1/atpA gene encoding the alpha subunit of ATP synthase, which is unusually well conserved between the 2 organelles and the only shared gene that is widely sequenced across plant mitochondria. Moreover, all conversions were limited to the 2 regions of greatest nucleotide and amino acid conservation of atp1/atpA. These observations probably reflect constraints operating on both the occurrence and fixation of recombination between ancient homologs. These findings indicate that recombination between anciently related sequences is more frequent than previously appreciated and creates functional mitochondrial genes of chimeric origin. These results also have implications for the widespread use of mitochondrial atp1 in phylogeny reconstruction.

Proteome Imbalance of Mitochondrial Electron Transport Chain in Brown Adipocytes Leads to Metabolic Benefits.

PubMed

Masand, Ruchi; Paulo, Esther; Wu, Dongmei; Wang, Yangmeng; Swaney, Danielle L; Jimenez-Morales, David; Krogan, Nevan J; Wang, Biao

2018-03-06

Brown adipose tissue (BAT) thermogenesis is critical for thermoregulation and contributes to total energy expenditure. However, whether BAT has non-thermogenic functions is largely unknown. Here, we describe that BAT-specific liver kinase b1 knockout (Lkb1 BKO ) mice exhibited impaired BAT mitochondrial respiration and thermogenesis but reduced adiposity and liver triglyceride accumulation under high-fat-diet feeding at room temperature. Importantly, these metabolic benefits were also present in Lkb1 BKO mice at thermoneutrality, where BAT thermogenesis was not required. Mechanistically, decreased mRNA levels of mtDNA-encoded electron transport chain (ETC) subunits and ETC proteome imbalance led to defective BAT mitochondrial respiration in Lkb1 BKO mice. Furthermore, reducing mtDNA gene expression directly in BAT by removing mitochondrial transcription factor A (Tfam) in BAT also showed ETC proteome imbalance and the trade-off between BAT thermogenesis and systemic metabolism at room temperature and thermoneutrality. Collectively, our data demonstrate that ETC proteome imbalance in BAT regulates systemic metabolism independently of thermogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Aging impact on biochemical activities and gene expression of Drosophila melanogaster mitochondria.

PubMed

Dubessay, Pascal; Garreau-Balandier, Isabelle; Jarrousse, Anne-Sophie; Fleuriet, Annie; Sion, Benoit; Debise, Roger; Alziari, Serge

2007-08-01

The consequences of aging are characterized by a decline in the main cellular functions, including those of the mitochondria. Although these consequences have been much studied, efforts have often focused solely on a few parameters used to assess the "state" of mitochondrial function during aging. We performed comparative measurements of several parameters in young (a few days) and old (8 and 12 weeks) adult male Drosophila melanogaster: respiratory complex activities, mitochondrial respiration, ATP synthesis, lipid composition of the inner membrane, concentrations of respiratory complex subunits, expression of genes (nuclear and mitochondrial) coding for mitochondrial proteins. Our results show that, in the mitochondria of "old" flies, the activities of three respiratory complexes (I, III, IV) are greatly diminished, ATP synthesis is decreased, and the lipid composition of the inner membrane (fatty acids, cardiolipin) is modified. However, the respiration rate and subunit concentrations measured by Western blot are unaffected. Although cellular mitochondrial DNA (mtDNA) content remains constant, there is a decrease in concentrations of nuclear and mitochondrial transcripts apparently coordinated. The expression of nuclear genes encoding the transcription factors TFAM, TFB1, TFB2, and DmTTF, which are essential for the maintenance and expression of mtDNA are also decreased. The decrease in nuclear and mitochondrial transcript concentrations may be one of the principal effects of aging on mitochondria, and could explain observed decreases in mitochondrial efficiency.

Maintenance of mitochondrial DNA copy number and expression are essential for preservation of mitochondrial function and cell growth.

PubMed

Jeng, Jaan-Yeh; Yeh, Tien-Shun; Lee, Jing-Wen; Lin, Shyh-Hsiang; Fong, Tsorng-Han; Hsieh, Rong-Hong

2008-02-01

To examine whether a reduction in the mtDNA level will compromise mitochondrial biogenesis and mitochondrial function, we created a cell model with depleted mtDNA. Stable transfection of small interfering (si)RNA of mitochondrial transcription factor A (Tfam) was used to interfere with Tfam gene expression. Selected stable clones showed 60-95% reduction in Tfam gene expression and 50-90% reduction in cytochrome b (Cyt b) gene expression. Tfam gene knockdown clones also showed decreased mtDNA-encoded cytochrome c oxidase subunit I (COX I) protein expression. However, no significant differences in protein expression were observed in nuclear DNA (nDNA)-encoded mitochondrial respiratory enzyme subunits. The cell morphology changed from a rhombus-like to a spindle-like form as determined in clones with decreased expressions of Tfam, mtRNA, and mitochondrial proteins. The mitochondrial respiratory enzyme activities and ATP production in such clones were significantly lower. The proportions of mtDNA mutations including 8-hydroxy-2'-deoxyguanosine (8-OHdG), a 4,977-bp deletion, and a 3,243-point mutation were also examined in these clones. No obvious increase in mtDNA mutations was observed in mitochondrial dysfunctional cell clones. The mitochondrial respiratory activity and ATP production ability recovered in cells with increased mtDNA levels after removal of the specific siRNA treatment. These experimental results provide direct evidence to substantiate that downregulation of mtDNA copy number and expression may compromise mitochondrial function and subsequent cell growth and morphology. (c) 2007 Wiley-Liss, Inc.

Mitochondrial Copper Metabolism and Delivery to Cytochrome c Oxidase

PubMed Central

Horn, Darryl; Barrientos, Antoni

2010-01-01

Summary Metals are essential elements of all living organisms. Among them, copper is required for a multiplicity of functions including mitochondrial oxidative phosphorylation and protection against oxidative stress. Here we will focus on describing the pathways involved in the delivery of copper to cytochrome c oxidase (COX), a mitochondrial metalloenzyme acting as the terminal enzyme of the mitochondrial respiratory chain. The catalytic core of COX is formed by three mitochondrially-encoded subunits and contains three copper atoms. Two copper atoms bound to subunit 2 constitute the CuA site, the primary acceptor of electrons from ferrocytochrome c. The third copper, CuB, is associated with the high-spin heme a3 group of subunit 1. Recent studies, mostly performed in the yeast Saccharomyces cerevisiae, have provided new clues about 1- the source of the copper used for COX metallation; 2- the roles of Sco1p and Cox11p, the proteins involved in the direct delivery of copper to the CuA and CuB sites, respectively; 3- the action mechanism of Cox17p, a copper chaperone that provides copper to Sco1p and Cox11p; 4- the existence of at least four Cox17p homologues carrying a similar twin CX9C domain suggestive of metal binding, Cox19p, Cox23p, Pet191p and Cmc1p, that could be part of the same pathway; and 5- the presence of a disulfide relay system in the intermembrane space of mitochondria that mediates import of proteins with conserved cysteines motifs such as the CX9C characteristic of Cox17p and its homologues. The different pathways are reviewed and discussed in the context of both mitochondrial COX assembly and copper homeostasis. PMID:18459161

Genetic variability of Baylisascaris schroederi from the Qinling subspecies of the giant panda in China revealed by sequences of three mitochondrial genes.

PubMed

Zhao, Zhong-Hui; Bian, Qing-Qing; Ren, Wan-Xin; Cheng, Wen-Yu; Jia, Yan-Qing; Fang, Yan-Qin; Zhao, Guang-Hui

2014-06-01

The present study examined the variations in three mitochondrial (mt) DNA sequences, namely cytochrome b (cytb), cytochrome c oxidase subunit 3 (cox3) and NADH dehydrogenase subunit 5 (nad5), among Baylisascaris schroederi isolates from the Qinling subspecies of the giant panda in Shaanxi province, northwestern China. No differences in length were detected in the three mt fragments from different isolates. The intra-specific sequence variations within all B. schroederi samples were 0-2.6% for pcytb, 0-1.8% for pcox3 and 0-2.1% for pnad5, while the inter-specific sequence differences among members of the genus Baylisascaris were 8.2-15.2%, 6.2-15.9% and 8.4-16.0% for pcytb, pcox3, pnad5, respectively. A phylogenetic analysis of the combined sequences of pcytb, pcox3 and pnad 5 showed that all B. schroederi samples in the present study were located in two large clusters, with one cluster containing samples from giant pandas in Sichuan province. These findings provide basic information for further study of molecular epidemiology and control of B. schroederi infection in the Qinling subspecies of the giant panda and throughout China.

Functional and composition differences between mitochondrial complex II in Arabidopsis and rice are correlated with the complex genetic history of the enzyme.

PubMed

Huang, Shaobai; Taylor, Nicolas L; Narsai, Reena; Eubel, Holger; Whelan, James; Millar, A Harvey

2010-02-01

Complex II plays a central role in mitochondrial metabolism as a component of both the electron transport chain and the tricarboxylic acid cycle. However, the composition and function of the plant enzyme has been elusive and differs from the well-characterised enzymes in mammals and bacteria. Herewith, we demonstrate that mitochondrial Complex II from Arabidopsis and rice differ significantly in several aspects: (1) Stability-Rice complex II in contrast to Arabidopsis is not stable when resolved by native electrophoresis and activity staining. (2) Composition-Arabidopsis complex II contains 8 subunits, only 7 of which have homologs in the rice genome. SDH 1 and 2 subunits display high levels of amino acid identity between two species, while the remainder of the subunits are not well conserved at a sequence level, indicating significant divergence. (3) Gene expression-the pairs of orthologous SDH1 and SDH2 subunits were universally expressed in both Arabidopsis and rice. The very divergent genes for SDH3 and SDH4 were co-expressed in both species, consistent with their functional co-ordination to form the membrane anchor. The plant-specific SDH5, 6 and 7 subunits with unknown functions appeared to be differentially expressed in both species. (4) Biochemical regulation -succinate-dependent O(2) consumption and SDH activity of isolated Arabidopsis mitochondria were substantially stimulated by ATP, but a much more minor effect of ATP was observed for the rice enzyme. The ATP activation of succinate-dependent reduction of DCPIP in frozen-thawed and digitonin-solubilised mitochondrial samples, and with or without the uncoupler CCCP, indicate that the differential ATP effect on SDH is not via the protonmotive force but likely due to an allosteric effect on the plant SDH enzyme itself, in contrast to the enzyme in other organisms.

MPC1-like Is a Placental Mammal-specific Mitochondrial Pyruvate Carrier Subunit Expressed in Postmeiotic Male Germ Cells.

PubMed

Vanderperre, Benoît; Cermakova, Kristina; Escoffier, Jessica; Kaba, Mayis; Bender, Tom; Nef, Serge; Martinou, Jean-Claude

2016-08-05

Selective transport of pyruvate across the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) is a fundamental step that couples cytosolic and mitochondrial metabolism. The recent molecular identification of the MPC complex has revealed two interacting subunits, MPC1 and MPC2. Although in yeast, an additional subunit, MPC3, can functionally replace MPC2, no alternative MPC subunits have been described in higher eukaryotes. Here, we report for the first time the existence of a novel MPC subunit termed MPC1-like (MPC1L), which is present uniquely in placental mammals. MPC1L shares high sequence, structural, and topological homology with MPC1. In addition, we provide several lines of evidence to show that MPC1L is functionally equivalent to MPC1: 1) when co-expressed with MPC2, it rescues pyruvate import in a MPC-deleted yeast strain; 2) in mammalian cells, it can associate with MPC2 to form a functional carrier as assessed by bioluminescence resonance energy transfer; 3) in MPC1 depleted mouse embryonic fibroblasts, MPC1L rescues the loss of pyruvate-driven respiration and stabilizes MPC2 expression; and 4) MPC1- and MPC1L-mediated pyruvate imports show similar efficiency. However, we show that MPC1L has a highly specific expression pattern and is localized almost exclusively in testis and more specifically in postmeiotic spermatids and sperm cells. This is in marked contrast to MPC1/MPC2, which are ubiquitously expressed throughout the organism. To date, the biological importance of this alternative MPC complex during spermatogenesis in placental mammals remains unknown. Nevertheless, these findings open up new avenues for investigating the structure-function relationship within the MPC complex. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial Biogenesis in Diverse Cauliflower Cultivars under Mild and Severe Drought. Impaired Coordination of Selected Transcript and Proteomic Responses, and Regulation of Various Multifunctional Proteins

PubMed Central

Rurek, Michał; Czołpińska, Magdalena; Staszak, Aleksandra Maria; Nowak, Witold; Krzesiński, Włodzimierz; Spiżewski, Tomasz

2018-01-01

Mitochondrial responses under drought within Brassica genus are poorly understood. The main goal of this study was to investigate mitochondrial biogenesis of three cauliflower (Brassica oleracea var. botrytis) cultivars with varying drought tolerance. Diverse quantitative changes (decreases in abundance mostly) in the mitochondrial proteome were assessed by two-dimensional gel electrophoresis (2D PAGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Respiratory (e.g., complex II, IV (CII, CIV) and ATP synthase subunits), transporter (including diverse porin isoforms) and matrix multifunctional proteins (e.g., components of RNA editing machinery) were diversely affected in their abundance under two drought levels. Western immunoassays showed additional cultivar-specific responses of selected mitochondrial proteins. Dehydrin-related tryptic peptides (found in several 2D spots) immunopositive with dehydrin-specific antisera highlighted the relevance of mitochondrial dehydrin-like proteins for the drought response. The abundance of selected mRNAs participating in drought response was also determined. We conclude that mitochondrial biogenesis was strongly, but diversely affected in various cauliflower cultivars, and associated with drought tolerance at the proteomic and functional levels. However, discussed alternative oxidase (AOX) regulation at the RNA and protein level were largely uncoordinated due to the altered availability of transcripts for translation, mRNA/ribosome interactions, and/or miRNA impact on transcript abundance and translation. PMID:29642585

Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria.

PubMed

Santamaria, Monica; Vicario, Saverio; Pappadà, Graziano; Scioscia, Gaetano; Scazzocchio, Claudio; Saccone, Cecilia

2009-06-16

A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers. The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries. After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals. The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode Initiative. The large scale investigation of Ascomycota mitochondrial introns performed through this tool, allowing to exclude the introns-rich sequences from the barcode candidates exploration, could be the first step towards a mitochondrial barcoding strategy for these organisms, similar to the standard approach employed in metazoans.

A cannabinoid link between mitochondria and memory.

PubMed

Hebert-Chatelain, Etienne; Desprez, Tifany; Serrat, Román; Bellocchio, Luigi; Soria-Gomez, Edgar; Busquets-Garcia, Arnau; Pagano Zottola, Antonio Christian; Delamarre, Anna; Cannich, Astrid; Vincent, Peggy; Varilh, Marjorie; Robin, Laurie M; Terral, Geoffrey; García-Fernández, M Dolores; Colavita, Michelangelo; Mazier, Wilfrid; Drago, Filippo; Puente, Nagore; Reguero, Leire; Elezgarai, Izaskun; Dupuy, Jean-William; Cota, Daniela; Lopez-Rodriguez, Maria-Luz; Barreda-Gómez, Gabriel; Massa, Federico; Grandes, Pedro; Bénard, Giovanni; Marsicano, Giovanni

2016-11-24

Cellular activity in the brain depends on the high energetic support provided by mitochondria, the cell organelles which use energy sources to generate ATP. Acute cannabinoid intoxication induces amnesia in humans and animals, and the activation of type-1 cannabinoid receptors present at brain mitochondria membranes (mtCB 1 ) can directly alter mitochondrial energetic activity. Although the pathological impact of chronic mitochondrial dysfunctions in the brain is well established, the involvement of acute modulation of mitochondrial activity in high brain functions, including learning and memory, is unknown. Here, we show that acute cannabinoid-induced memory impairment in mice requires activation of hippocampal mtCB 1 receptors. Genetic exclusion of CB 1 receptors from hippocampal mitochondria prevents cannabinoid-induced reduction of mitochondrial mobility, synaptic transmission and memory formation. mtCB 1 receptors signal through intra-mitochondrial Gα i protein activation and consequent inhibition of soluble-adenylyl cyclase (sAC). The resulting inhibition of protein kinase A (PKA)-dependent phosphorylation of specific subunits of the mitochondrial electron transport system eventually leads to decreased cellular respiration. Hippocampal inhibition of sAC activity or manipulation of intra-mitochondrial PKA signalling or phosphorylation of the Complex I subunit NDUFS2 inhibit bioenergetic and amnesic effects of cannabinoids. Thus, the G protein-coupled mtCB 1 receptors regulate memory processes via modulation of mitochondrial energy metabolism. By directly linking mitochondrial activity to memory formation, these data reveal that bioenergetic processes are primary acute regulators of cognitive functions.

Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.

PubMed

Hirawake, H; Taniwaki, M; Tamura, A; Amino, H; Tomitsuka, E; Kita, K

1999-08-04

We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.

Description of Groenewaldozyma gen. nov. for placement of Candida auringiensis, Candida salmanticensis and Candida tartarivorans.

PubMed

Kurtzman, Cletus P

2016-07-01

DNA sequence analyses have demonstrated that species of the polyphyletic anamorphic ascomycete genus Candida may be members of described teleomorphic genera, members of the Candida tropicalis clade upon which the genus Candida is circumscribed, or members of isolated clades that represent undescribed genera. From phylogenetic analysis of gene sequences from nuclear large subunit rRNA, mitochondrial small subunit rRNA and cytochrome oxidase II, Candida auringiensis (NRRL Y-17674(T), CBS 6913(T)), Candida salmanticensis (NRRL Y-17090(T), CBS 5121(T)), and Candida tartarivorans (NRRL Y-27291(T), CBS 7955(T)) were shown to be members of an isolated clade and are proposed for reclassification in the genus Groenewaldozyma gen. nov. (MycoBank MB 815817). Neighbouring taxa include species of the Wickerhamiella clade and Candida blankii.

Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

PubMed

Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

2015-06-01

Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic derangement. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Partial kinetoplast-mitochondrial gene organization and expression in the respiratory deficient plant trypanosomatid Phytomonas serpens.

PubMed

Maslov, D A; Nawathean, P; Scheel, J

1999-04-30

In plant-dwelling trypanosomatids from the genus Phytomonas, mitochondrial functions, such as cytochrome mediated respiration, ATP production and Krebs cycle, are missing, and cell energetics is based on the glycolysis. Using Blue Native/Tricine-SDS two-dimensional gel electrophoretic analysis, we observed that mitochondrial respiratory Complexes III (cytochrome bc1) and IV (cytochrome c oxidase) were absent in Phytomonas serpens; however, Complex V (ATPase) was present. A deletion of the genes for cytochrome c oxidase subunit III (COIII) and apocytochrome b (Cyb) was identified within the 6234 bp sequenced region of the 31 kb maxicircle kinetoplast DNA. Genes, found in this region, include 12S and 9S ribosomal RNAs, subunits 7, 8 and 9 of NADH dehydrogenase (ND7, ND8 and ND9) and subunit 6 of ATPase (A6 or MURF4), as well as the genes (MURF1, MURF5 and G3) with unknown function. Most genes are actively transcribed and some mRNAs are edited. Fully edited mRNAs for A6 and G3 were abundant, while edited ND7 transcripts were rare, and only partially edited and pre-edited transcripts for ND8 were detected. The data show that the mitochondrial genome of P. serpens is functional, although its functions may be limited to expressing the ATPase and, possibly, NADH dehydrogenase complexes.

The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying.

PubMed

Wang, Shuaiyu; Jacquemyn, Julie; Murru, Sara; Martinelli, Paola; Barth, Esther; Langer, Thomas; Niessen, Carien M; Rugarli, Elena I

2016-12-01

The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both Afg3l2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leshinsky-Silver, E.; Mitochondrial Disease Center, Wolfson Medical Center, Holon; E-mail: leshinsky@wolfson.health.gov.il

Leigh syndrome can result from both nuclear and mitochondrial DNA defects. Mutations in complex V genes of the respiratory chain were considered until recently as the most frequent cause for mitochondrial inherited Leigh syndrome, while gene defects in complex I were related to recessive Leigh syndrome. Recently few reports of mutations in the mitochondrial-encoded complex I subunit genes causing Leigh syndrome have been reported. We describe a 1-month-old baby who acutely deteriorated, with abrupt onset of brainstem dysfunction, due to basal ganglia lesions extending to the brainstem. A muscle biopsy demonstrated complex I deficiency. Subsequent analysis of the mitochondrial genomemore » revealed a homoplastic T10191C mutation in the ND3 gene (in blood and muscle), resulting in a substitution of serine to proline. Hair root analysis revealed a 50% mutant load, reflecting heteroplasmy in early embryonic stages. The mutation was also detected in his mother (5%). Western blot analysis revealed a decrease of the 20 kDa subunit (likely ND6) and of the 30 kDa subunit (NDUFA9), which is probably due to instability attributed to the inability to form subcomplexes with ND3. This is the first description of infantile Leigh syndrome due to a maternally transmitted T10191C substitution in ND3 and not due to a de novo mutation. This mutation is age and tissue dependent and therefore may not be amenable to prenatal testing.« less

Cardiac metabolic pathways affected in the mouse model of barth syndrome.

PubMed

Huang, Yan; Powers, Corey; Madala, Satish K; Greis, Kenneth D; Haffey, Wendy D; Towbin, Jeffrey A; Purevjav, Enkhsaikhan; Javadov, Sabzali; Strauss, Arnold W; Khuchua, Zaza

2015-01-01

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F0F1-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber’s hereditary optic neuropathy

PubMed Central

Iommarini, Luisa; Giordano, Luca; Maresca, Alessandra; Pisano, Annalinda; Valentino, Maria Lucia; Caporali, Leonardo; Liguori, Rocco; Deceglie, Stefania; Roberti, Marina; Fanelli, Francesca; Fracasso, Flavio; Ross-Cisneros, Fred N.; D’Adamo, Pio; Hudson, Gavin; Pyle, Angela; Yu-Wai-Man, Patrick; Chinnery, Patrick F.; Zeviani, Massimo; Salomao, Solange R.; Berezovsky, Adriana; Belfort, Rubens; Ventura, Dora Fix; Moraes, Milton; Moraes Filho, Milton; Barboni, Piero; Sadun, Federico; De Negri, Annamaria; Sadun, Alfredo A.; Tancredi, Andrea; Mancini, Massimiliano; d’Amati, Giulia; Loguercio Polosa, Paola; Cantatore, Palmiro

2014-01-01

Leber’s hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber’s hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber’s hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies. PMID:24369379

Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress.

PubMed

Hiller, Sylvia; DeKroon, Robert; Hamlett, Eric D; Xu, Longquan; Osorio, Cristina; Robinette, Jennifer; Winnik, Witold; Simington, Stephen; Maeda, Nobuyo; Alzate, Oscar; Yi, Xianwen

2016-01-01

S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

Acute high-altitude hypoxic brain injury: Identification of ten differential proteins

PubMed Central

Li, Jianyu; Qi, Yuting; Liu, Hui; Cui, Ying; Zhang, Li; Gong, Haiying; Li, Yaxiao; Li, Lingzhi; Zhang, Yongliang

2013-01-01

Hypobaric hypoxia can cause severe brain damage and mitochondrial dysfunction, and is involved in hypoxic brain injury. However, little is currently known about the mechanisms responsible for mitochondrial dysfunction in hypobaric hypoxic brain damage. In this study, a rat model of hypobaric hypoxic brain injury was established to investigate the molecular mechanisms associated with mitochondrial dysfunction. As revealed by two-dimensional electrophoresis analysis, 16, 21, and 36 differential protein spots in cerebral mitochondria were observed at 6, 12, and 24 hours post-hypobaric hypoxia, respectively. Furthermore, ten protein spots selected from each hypobaric hypoxia subgroup were similarly regulated and were identified by mass spectrometry. These detected proteins included dihydropyrimidinase-related protein 2, creatine kinase B-type, isovaleryl-CoA dehydrogenase, elongation factor Ts, ATP synthase beta-subunit, 3-mercaptopyruvate sulfurtransferase, electron transfer flavoprotein alpha-subunit, Chain A of 2-enoyl-CoA hydratase, NADH dehydrogenase iron-sulfur protein 8 and tropomyosin beta chain. These ten proteins are all involved in the electron transport chain and the function of ATP synthase. Our findings indicate that hypobaric hypoxia can induce the differential expression of several cerebral mitochondrial proteins, which are involved in the regulation of mitochondrial energy production. PMID:25206614

Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus

PubMed Central

Hao, Xiao-Dan; Chen, Zhao-Li; Qu, Ming-Li; Zhao, Xiao-Wen; Li, Su-Xia; Chen, Peng

2016-01-01

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC. PMID:27783701

Each Monomer of the Dimeric Accessory Protein for Human Mitochondrial DNA Polymerase Has a Distinct Role in Conferring Processivity*

PubMed Central

Lee, Young-Sam; Lee, Sujin; Demeler, Borries; Molineux, Ian J.; Johnson, Kenneth A.; Yin, Y. Whitney

2010-01-01

The accessory protein polymerase (pol) γB of the human mitochondrial DNA polymerase stimulates the synthetic activity of the catalytic subunit. pol γB functions by both accelerating the polymerization rate and enhancing polymerase-DNA interaction, thereby distinguishing itself from the accessory subunits of other DNA polymerases. The molecular basis for the unique functions of human pol γB lies in its dimeric structure, where the pol γB monomer proximal to pol γA in the holoenzyme strengthens the interaction with DNA, and the distal pol γB monomer accelerates the reaction rate. We further show that human pol γB exhibits a catalytic subunit- and substrate DNA-dependent dimerization. By duplicating the monomeric pol γB of lower eukaryotes, the dimeric mammalian proteins confer additional processivity to the holoenzyme polymerase. PMID:19858216

New features of mitochondrial DNA replication system in yeast and man.

PubMed

Lecrenier, N; Foury, F

2000-04-04

In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens. Brief incursions into systems of other organisms have also been achieved when they provide new information.S. cerevisiae and H. sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms. However, recent studies suggest that mitochondrial genome of S. cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular. Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident. As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast. Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously. Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products. Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections. These links are presented on a map. Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication.

Nutrient-gene interactions determine mitochondrial function: effect of dietary fat.

PubMed

Kim, M J; Berdanier, C D

1998-02-01

The effect on mitochondrial respiration of feeding hydrogenated coconut oil, corn oil, or menhaden oil (MO) to diabetes-prone BHE/cdb rats and normal Sprague Dawley (SD) rats was studied. Both fat source and strain affected the temperature dependence of succinate-supported respiration. The transition temperature was greater in BHE/cdb rats than in the SD rats. The efficiency of ATP synthesis as reflected by the ADP:O ratio was decreased in the BHE/cdb rats compared to SD rats, with the exception of the comparison made at 37 degrees C with the MO-fed rats; at this temperature, the ADP:O ratios were similar. The diet and strain differences suggest a dietary lipid-gene interaction with respect to the mobility of subunit 6 of the F1F0ATPase. This subunit has two errors in its gene: one that affects the proton channel and another that likely affects its mobility within the inner mitochondrial membrane.

Resolution of the African hominoid trichotomy by use of a mitochondrial gene sequence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruvolo, M.; Disotell, T.R.; Allard, M.W.

1991-02-15

Mitochondrial DNA sequences encoding the cytochrome oxidase subunit II gene have been determined for five primate species, siamang (Hylobates syndactylus), lowland gorilla (Gorilla gorilla), pygmy chimpanzee (Pan paniscus), crab-eating macaque (Macaca fascicularis), and green monkey (Cercopithecus aethiops), and compared with published sequences of other primate and nonprimate species. Comparisons of cytochrome oxidase subunit II gene sequences provide clear-cut evidence from the mitochondrial genome for the separation of the African ape trichotomy into two evolutionary lineages, one leading to gorillas and the other to humans and chimpanzees. Several different tree-building methods support this same phylogenetic tree topology. The comparisons also yieldmore » trees in which a substantial length separates the divergence point of gorillas from that of humans and chimpanzees, suggesting that the lineage most immediately ancestral to humans and chimpanzees may have been in existence for a relatively long time.« less

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM.

PubMed

Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

2015-10-06

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

MELAS syndrome: Clinical manifestations, pathogenesis, and treatment options.

PubMed

El-Hattab, Ayman W; Adesina, Adekunle M; Jones, Jeremy; Scaglia, Fernando

2015-01-01

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. MELAS syndrome is a multi-organ disease with broad manifestations including stroke-like episodes, dementia, epilepsy, lactic acidemia, myopathy, recurrent headaches, hearing impairment, diabetes, and short stature. The most common mutation associated with MELAS syndrome is the m.3243A>G mutation in the MT-TL1 gene encoding the mitochondrial tRNA(Leu(UUR)). The m.3243A>G mutation results in impaired mitochondrial translation and protein synthesis including the mitochondrial electron transport chain complex subunits leading to impaired mitochondrial energy production. The inability of dysfunctional mitochondria to generate sufficient energy to meet the needs of various organs results in the multi-organ dysfunction observed in MELAS syndrome. Energy deficiency can also stimulate mitochondrial proliferation in the smooth muscle and endothelial cells of small blood vessels leading to angiopathy and impaired blood perfusion in the microvasculature of several organs. These events will contribute to the complications observed in MELAS syndrome particularly the stroke-like episodes. In addition, nitric oxide deficiency occurs in MELAS syndrome and can contribute to its complications. There is no specific consensus approach for treating MELAS syndrome. Management is largely symptomatic and should involve a multidisciplinary team. Unblinded studies showed that l-arginine therapy improves stroke-like episode symptoms and decreases the frequency and severity of these episodes. Additionally, carnitine and coenzyme Q10 are commonly used in MELAS syndrome without proven efficacy. Copyright © 2015 Elsevier Inc. All rights reserved.

Dolabra nepheliae on rambutan and lychee represents a novel lineage of phytopathogenic Eurotiomycetes

PubMed Central

Schoch, Conrad L.; Farr, David F.; Nishijima, Kate; Keith, Lisa; Goenaga, Ricardo

2010-01-01

Rambutan (Nephelium lappaceum) and lychee (Litchi chinensis) are tropical trees in the Sapindaceae that produce delicious edible fruits and are increasingly cultivated in tropical regions. These trees are afflicted with a stem canker disease associated with the ascomycete Dolabra nepheliae. Previously known from Asia and Australia, this fungus was recently reported from Hawaii and Puerto Rico. The sexual and asexual states of Dolabra nepheliae are redescribed and illustrated. In addition, the ITS and large subunit of the nuclear ribosomal DNA plus fragments from the genes RPB2, TEF1, and the mitochondrial small ribosomal subunit were sequenced for three isolates of D. nepheliae and compared with other sequences of ascomycetes. It was determined that D. nepheliae represents a new lineage within the Eurotiomycetes allied with Phaeomoniella chlamydospora, the causal agent of Petri grapevine decline. PMID:20802819

Dolabra nepheliae on rambutan and lychee represents a novel lineage of phytopathogenic Eurotiomycetes.

PubMed

Rossman, Amy Y; Schoch, Conrad L; Farr, David F; Nishijima, Kate; Keith, Lisa; Goenaga, Ricardo

2010-07-01

Rambutan (Nephelium lappaceum) and lychee (Litchi chinensis) are tropical trees in the Sapindaceae that produce delicious edible fruits and are increasingly cultivated in tropical regions. These trees are afflicted with a stem canker disease associated with the ascomycete Dolabra nepheliae. Previously known from Asia and Australia, this fungus was recently reported from Hawaii and Puerto Rico. The sexual and asexual states of Dolabra nepheliae are redescribed and illustrated. In addition, the ITS and large subunit of the nuclear ribosomal DNA plus fragments from the genes RPB2, TEF1, and the mitochondrial small ribosomal subunit were sequenced for three isolates of D. nepheliae and compared with other sequences of ascomycetes. It was determined that D. nepheliae represents a new lineage within the Eurotiomycetes allied with Phaeomoniella chlamydospora, the causal agent of Petri grapevine decline.

Age-related decline of the cytochrome c oxidase subunit expression in the auditory cortex of the mimetic aging rat model associated with the common deletion.

PubMed

Zhong, Yi; Hu, Yujuan; Peng, Wei; Sun, Yu; Yang, Yang; Zhao, Xueyan; Huang, Xiang; Zhang, Honglian; Kong, Weijia

2012-12-01

The age-related deterioration in the central auditory system is well known to impair the abilities of sound localization and speech perception. However, the mechanisms involved in the age-related central auditory deficiency remain unclear. Previous studies have demonstrated that mitochondrial DNA (mtDNA) deletions accumulated with age in the auditory system. Also, a cytochrome c oxidase (CcO) deficiency has been proposed to be a causal factor in the age-related decline in mitochondrial respiratory activity. This study was designed to explore the changes of CcO activity and to investigate the possible relationship between the mtDNA common deletion (CD) and CcO activity as well as the mRNA expression of CcO subunits in the auditory cortex of D-galactose (D-gal)-induced mimetic aging rats at different ages. Moreover, we explored whether peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) were involved in the changes of nuclear- and mitochondrial-encoded CcO subunits in the auditory cortex during aging. Our data demonstrated that d-gal-induced mimetic aging rats exhibited an accelerated accumulation of the CD and a gradual decline in the CcO activity in the auditory cortex during the aging process. The reduction in the CcO activity was correlated with the level of CD load in the auditory cortex. The mRNA expression of CcO subunit III was reduced significantly with age in the d-gal-induced mimetic aging rats. In contrast, the decline in the mRNA expression of subunits I and IV was relatively minor. Additionally, significant increases in the mRNA and protein levels of PGC-1α, NRF-1 and TFAM were observed in the auditory cortex of D-gal-induced mimetic aging rats with aging. These findings suggested that the accelerated accumulation of the CD in the auditory cortex may induce a substantial decline in CcO subunit III and lead to a significant decline in the CcO activity progressively with age despite compensatory increases of PGC-1α, NRF-1 and TFAM. Therefore, CcO may be a specific intramitochondrial site of age-related deterioration in the auditory cortex, and CcO subunit III might be a target in the development of presbycusis. Copyright © 2012 Elsevier B.V. All rights reserved.

Mechanisms of Mitochondrial Defects in Gulf War Syndrome

DTIC Science & Technology

2014-10-01

parameters: uncoupling ratio, net routine flux control ratio, respiratory control ratio, leak flux control ratio, phosphorylation respiratory... oxidative phosphorylation subunit) Quantitative analysis of individual mitochondrial proteins. The technique has been established and validated for muscle...Blue Native and Clear Native Analyses (non-denatured analysis of supercomplex formation and monomeric oxidative phosphorylation enzyme assembly

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

USDA-ARS?s Scientific Manuscript database

The mitochondrial pyruvate dehydrogenase complex is regulated by reversible seryl-phosphorylation of the E1alpha subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated...

Biogenesis of mitochondria in cauliflower (Brassica oleracea var. botrytis) curds subjected to temperature stress and recovery involves regulation of the complexome, respiratory chain activity, organellar translation and ultrastructure.

PubMed

Rurek, Michal; Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2015-01-01

The biogenesis of the cauliflower curd mitochondrial proteome was investigated under cold, heat and the recovery. For the first time, two dimensional fluorescence difference gel electrophoresis was used to study the plant mitochondrial complexome in heat and heat recovery. Particularly, changes in the complex I and complex III subunits and import proteins, and the partial disintegration of matrix complexes were observed. The presence of unassembled subunits of ATP synthase was accompanied by impairment in mitochondrial translation of its subunit. In cold and heat, the transcription profiles of mitochondrial genes were uncorrelated. The in-gel activities of respiratory complexes were particularly affected after stress recovery. Despite a general stability of respiratory chain complexes in heat, functional studies showed that their activity and the ATP synthesis yield were affected. Contrary to cold stress, heat stress resulted in a reduced efficiency of oxidative phosphorylation likely due to changes in alternative oxidase (AOX) activity. Stress and stress recovery differently modulated the protein level and activity of AOX. Heat stress induced an increase in AOX activity and protein level, and AOX1a and AOX1d transcript level, while heat recovery reversed the AOX protein and activity changes. Conversely, cold stress led to a decrease in AOX activity (and protein level), which was reversed after cold recovery. Thus, cauliflower AOX is only induced by heat stress. In heat, contrary to the AOX activity, the activity of rotenone-insensitive internal NADH dehydrogenase was diminished. The relevance of various steps of plant mitochondrial biogenesis to temperature stress response and recovery is discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Biallelic Mutations in ATP5F1D , which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle

ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1F O ATP synthase andmore » subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.« less

Biallelic Mutations in ATP5F1D, which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder.

PubMed

Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle; Jangam, Sharayu; Fernandez, Liliana; Davidson, Jean M; Kyle, Jennifer E; Grove, Megan E; Fisk, Dianna G; Kohler, Jennefer N; Holmes, Matthew; Dries, Annika M; Huang, Yong; Zhao, Chunli; Contrepois, Kévin; Zappala, Zachary; Frésard, Laure; Waggott, Daryl; Zink, Erika M; Kim, Young-Mo; Heyman, Heino M; Stratton, Kelly G; Webb-Robertson, Bobbie-Jo M; Snyder, Michael; Merker, Jason D; Montgomery, Stephen B; Fisher, Paul G; Feichtinger, René G; Mayr, Johannes A; Hall, Julie; Barbosa, Ines A; Simpson, Michael A; Deshpande, Charu; Waters, Katrina M; Koeller, David M; Metz, Thomas O; Morris, Andrew A; Schelley, Susan; Cowan, Tina; Friederich, Marisa W; McFarland, Robert; Van Hove, Johan L K; Enns, Gregory M; Yamamoto, Shinya; Ashley, Euan A; Wangler, Michael F; Taylor, Robert W; Bellen, Hugo J; Bernstein, Jonathan A; Wheeler, Matthew T

2018-03-01

ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1 F O ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Biallelic Mutations in ATP5F1D , which Encodes a Subunit of ATP Synthase, Cause a Metabolic Disorder

DOE PAGES

Oláhová, Monika; Yoon, Wan Hee; Thompson, Kyle; ...

2018-02-22

ATP synthase, H + transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F 1F O ATP synthase andmore » subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.« less

Disruptive mitochondrial DNA mutations in complex I subunits are markers of oncocytic phenotype in thyroid tumors.

PubMed

Gasparre, Giuseppe; Porcelli, Anna Maria; Bonora, Elena; Pennisi, Lucia Fiammetta; Toller, Matteo; Iommarini, Luisa; Ghelli, Anna; Moretti, Massimo; Betts, Christine M; Martinelli, Giuseppe Nicola; Ceroni, Alberto Rinaldi; Curcio, Francesco; Carelli, Valerio; Rugolo, Michela; Tallini, Giovanni; Romeo, Giovanni

2007-05-22

Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P=0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.

Protective role of Parkin in skeletal muscle contractile and mitochondrial function.

PubMed

Gouspillou, Gilles; Godin, Richard; Piquereau, Jérome; Picard, Martin; Mofarrahi, Mahroo; Mathew, Jasmin; Purves-Smith, Fennigje M; Sgarioto, Nicolas; Hepple, Russell T; Burelle, Yan; Hussain, Sabah N A

2018-04-22

Parkin, an E3 ubiquitin ligase encoded by the Park2 gene, has been implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are degraded. The exact physiological significance of Parkin in regulating mitochondrial function and contractility in skeletal muscle remains largely unexplored. Using Park2 -/- mice, we show that Parkin ablation causes a decrease in muscle specific force, a severe decrease in mitochondrial respiration, mitochondrial uncoupling and an increased susceptibility to opening of the permeability transition pore. These results demonstrate that Parkin plays a protective role in the maintenance of normal mitochondrial and contractile functions in skeletal muscles. Parkin is an E3 ubiquitin ligase encoded by the Park2 gene. Parkin has been implicated in the regulation of mitophagy, a quality control process in which defective mitochondria are sequestered in autophagosomes and delivered to lysosomes for degradation. Although Parkin has been mainly studied for its implication in neuronal degeneration in Parkinson disease, its role in other tissues remains largely unknown. In the present study, we investigated the skeletal muscles of Park2 knockout (Park2 -/- ) mice to test the hypothesis that Parkin plays a physiological role in mitochondrial quality control in normal skeletal muscle, a tissue highly reliant on mitochondrial content and function. We first show that the tibialis anterior (TA) of Park2 -/- mice display a slight but significant decrease in its specific force. Park2 -/ - muscles also show a trend for type IIB fibre hypertrophy without alteration in muscle fibre type proportion. Compared to Park2 +/+ muscles, the mitochondrial function of Park2 -/- skeletal muscles was significantly impaired, as indicated by the significant decrease in ADP-stimulated mitochondrial respiratory rates, uncoupling, reduced activities of respiratory chain complexes containing mitochondrial DNA (mtDNA)-encoded subunits and increased susceptibility to opening of the permeability transition pore. Muscles of Park2 -/- mice also displayed a decrease in the content of the mitochondrial pro-fusion protein Mfn2 and an increase in the pro-fission protein Drp1 suggesting an increase in mitochondrial fragmentation. Finally, Park2 ablation resulted in an increase in basal autophagic flux in skeletal muscles. Overall, the results of the present study demonstrate that Parkin plays a protective role in the maintenance of normal mitochondrial and contractile functions in normal skeletal muscles. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons.

PubMed

Aschrafi, Armaz; Kar, Amar N; Gale, Jenna R; Elkahloun, Abdel G; Vargas, Jose Noberto S; Sales, Naomi; Wilson, Gabriel; Tompkins, Miranda; Gioio, Anthony E; Kaplan, Barry B

2016-09-01

Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function. Published by Elsevier B.V.

Intraspecific differentiation of Paramecium novaurelia strains (Ciliophora, Protozoa) inferred from phylogenetic analysis of ribosomal and mitochondrial DNA variation.

PubMed

Tarcz, Sebastian

2013-01-01

Paramecium novaurelia Beale and Schneller, 1954, was first found in Scotland and is known to occur mainly in Europe, where it is the most common species of the P. aurelia complex. In recent years, two non-European localities have been described: Turkey and the United States of America. This article presents the analysis of intraspecific variability among 25 strains of P. novaurelia with the application of ribosomal and mitochondrial loci (ITS1-5.8S-ITS2, 5' large subunit rDNA (5'LSU rDNA) and cytochrome c oxidase subunit 1 (COI) mtDNA). The mean distance observed for all of the studied P. novaurelia sequence pairs was p=0.008/0.016/0.092 (ITS1-5.8S-ITS2/5'LSU rDNA/COI). Phylogenetic trees (NJ/MP/BI) based on a comparison of all of the analysed sequences show that the studied strains of P. novaurelia form a distinct clade, separate from the P. caudatum outgroup, and are divided into two clusters (A and B) and two branches (C and D). The occurrence of substantial genetic differentiation within P. novaurelia, confirmed by the analysed DNA fragments, indicates a rapid evolution of particular species within the Paramecium genus. Copyright © 2012 Elsevier GmbH. All rights reserved.

The complete mitochondrial genomes of five Eimeria species infecting domestic rabbits.

PubMed

Liu, Guo-Hua; Tian, Si-Qin; Cui, Ping; Fang, Su-Fang; Wang, Chun-Ren; Zhu, Xing-Quan

2015-12-01

Rabbit coccidiosis caused by members of the genus Eimeria can cause enormous economic impact worldwide, but the genetics, epidemiology and biology of these parasites remain poorly understood. In the present study, we sequenced and annotated the complete mitochondrial (mt) genomes of five Eimeria species that commonly infect the domestic rabbits. The complete mt genomes of Eimeria intestinalis, Eimeria flavescens, Eimeria media, Eimeria vejdovskyi and Eimeria irresidua were 6261bp, 6258bp, 6168bp, 6254bp, 6259bp in length, respectively. All of the mt genomes consist of 3 genes for proteins (cytb, cox1, and cox3), 14 gene fragments for the large subunit (LSU) rRNA and 11 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA (tRNA) genes. The gene order of the mt genomes is similar to that of Plasmodium, but distinct from Haemosporida and Theileria. Phylogenetic analyses based on full nucleotide sequences using Bayesian analysis revealed that the monophyly of the Eimeria of rabbits was strongly statistically supported with a Bayesian posterior probabilities. These data provide novel mtDNA markers for studying the population genetics and molecular epidemiology of the Eimeria species, and should have implications for the molecular diagnosis, prevention and control of coccidiosis in rabbits. Copyright © 2015 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Heung M.; Reed, Jason; Greeley, George H.

Survivors of massive inhalation of combustion smoke endure critical injuries, including lasting neurological complications. We have previously reported that acute inhalation of combustion smoke disrupts the nitric oxide homeostasis in the rat brain. In this study, we extend our findings and report that a 30-minute exposure of awake rats to ambient wood combustion smoke induces protein nitration in the rat hippocampus and that mitochondrial proteins are a sensitive nitration target in this setting. Mitochondria are central to energy metabolism and cellular signaling and are critical to proper cell function. Here, analyses of the mitochondrial proteome showed elevated protein nitration inmore » the course of a 24-hour recovery following exposure to smoke. Mass spectrometry identification of several significantly nitrated mitochondrial proteins revealed diverse functions and involvement in central aspects of mitochondrial physiology. The nitrated proteins include the ubiquitous mitochondrial creatine kinase, F1-ATP synthase {alpha} subunit, dihydrolipoamide dehydrogenase (E3), succinate dehydrogenase Fp subunit, and voltage-dependent anion channel (VDAC1) protein. Furthermore, acute exposure to combustion smoke significantly compromised the respiratory capacity of hippocampal mitochondria. Importantly, elevated protein nitration and reduced mitochondrial respiration in the hippocampus persisted beyond the time required for restoration of normal oxygen and carboxyhemoglobin blood levels after the cessation of exposure to smoke. Thus, the time frame for intensification of the various smoke-induced effects differs between blood and brain tissues. Taken together, our findings suggest that nitration of essential mitochondrial proteins may contribute to the reduction in mitochondrial respiratory capacity and underlie, in part, the brain pathophysiology after acute inhalation of combustion smoke.« less

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

PubMed Central

MJ, Falk; Z, Zhang; Rosenjack; Nissim; E, Daikhin; Nissim; MM, Sedensky; M, Yudkoff; PG, Morgan

2008-01-01

C. elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear gene that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD+ dependent oxidation of ketoacids occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease. PMID:18178500

Genome-wide analysis of signal transducers and regulators of mitochondrial dysfunction in Saccharomyces cerevisiae.

PubMed

Singh, Keshav K; Rasmussen, Anne Karin; Rasmussen, Lene Juel

2004-04-01

Mitochondrial dysfunction is a hallmark of cancer cells. However, genetic response to mitochondrial dysfunction during carcinogenesis is unknown. To elucidate genetic response to mitochondrial dysfunction we used Saccharomyces cerevisiae as a model system. We analyzed genome-wide expression of nuclear genes involved in signal transduction and transcriptional regulation in a wild-type yeast and a yeast strain lacking the mitochondrial genome (rho(0)). Our analysis revealed that the gene encoding cAMP-dependent protein kinase subunit 3 (PKA3) was upregulated. However, the gene encoding cAMP-dependent protein kinase subunit 2 (PKA2) and the VTC1, PTK2, TFS1, CMK1, and CMK2 genes, involved in signal transduction, were downregulated. Among the known transcriptional factors, OPI1, MIG2, INO2, and ROX1 belonged to the upregulated genes, whereas MSN4, MBR1, ZMS1, ZAP1, TFC3, GAT1, ADR1, CAT8, and YAP4 including RFA1 were downregulated. RFA1 regulates DNA repair genes at the transcriptional level. RFA is also involved directly in DNA recombination, DNA replication, and DNA base excision repair. Downregulation of RFA1 in rho(0) cells is consistent with our finding that mitochondrial dysfunction leads to instability of the nuclear genome. Together, our data suggest that gene(s) involved in mitochondria-to-nucleus communication play a role in mutagenesis and may be implicated in carcinogenesis.

THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

PubMed Central

Lizardi, Paul M.; Luck, David J. L.

1972-01-01

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes. PMID:4261038

Mutant POLG2 Disrupts DNA Polymerase γ Subunits and Causes Progressive External Ophthalmoplegia

PubMed Central

Longley, Matthew J.; Clark, Susanna; Yu Wai Man, Cynthia; Hudson, Gavin; Durham, Steve E.; Taylor, Robert W.; Nightingale, Simon; Turnbull, Douglass M.; Copeland, William C.; Chinnery, Patrick F.

2006-01-01

DNA polymerase γ (pol γ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol γ (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G→A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol γ, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)–deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID:16685652

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM.

PubMed

Mager, Frauke; Sokolova, Lucie; Lintzel, Julia; Brutschy, Bernhard; Nussberger, Stephan

2010-11-17

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOM

NASA Astrophysics Data System (ADS)

Mager, Frauke; Sokolova, Lucie; Lintzel, Julia; Brutschy, Bernhard; Nussberger, Stephan

2010-11-01

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

Impaired Muscle Mitochondrial Biogenesis and Myogenesis in Spinal Muscular Atrophy

PubMed Central

Ripolone, Michela; Ronchi, Dario; Violano, Raffaella; Vallejo, Dionis; Fagiolari, Gigliola; Barca, Emanuele; Lucchini, Valeria; Colombo, Irene; Villa, Luisa; Berardinelli, Angela; Balottin, Umberto; Morandi, Lucia; Mora, Marina; Bordoni, Andreina; Fortunato, Francesco; Corti, Stefania; Parisi, Daniela; Toscano, Antonio; Sciacco, Monica; DiMauro, Salvatore; Comi, Giacomo P.; Moggio, Maurizio

2016-01-01

IMPORTANCE The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively). OBJECTIVE To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA. DESIGN, SETTING, AND PARTICIPANTS We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca’ Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015. EXPOSURES We used histochemical, biochemical, and molecular techniques to examine the muscle samples. MAIN OUTCOMES AND MEASURES Respiratory chain activity and mitochondrial content. RESULTS Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P < .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P < .05). We linked these alterations to downregulation of peroxisome proliferator–activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P < .001), COX4 (26.6%; P < .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P < .001). Conversely, the levels of expression of 3 myogenic regulatory factors—muscle-specificmyogenic factor 5, myoblast determination 1, and myogenin—were higher in muscles from patients with SMA compared with muscles from age-matched controls (P < .05). CONCLUSIONS AND RELEVANCE Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes. PMID:25844556

Broad genomic and transcriptional analysis reveals a highly derived genome in dinoflagellate mitochondria

PubMed Central

Jackson, Christopher J; Norman, John E; Schnare, Murray N; Gray, Michael W; Keeling, Patrick J; Waller, Ross F

2007-01-01

Background Dinoflagellates comprise an ecologically significant and diverse eukaryotic phylum that is sister to the phylum containing apicomplexan endoparasites. The mitochondrial genome of apicomplexans is uniquely reduced in gene content and size, encoding only three proteins and two ribosomal RNAs (rRNAs) within a highly compacted 6 kb DNA. Dinoflagellate mitochondrial genomes have been comparatively poorly studied: limited available data suggest some similarities with apicomplexan mitochondrial genomes but an even more radical type of genomic organization. Here, we investigate structure, content and expression of dinoflagellate mitochondrial genomes. Results From two dinoflagellates, Crypthecodinium cohnii and Karlodinium micrum, we generated over 42 kb of mitochondrial genomic data that indicate a reduced gene content paralleling that of mitochondrial genomes in apicomplexans, i.e., only three protein-encoding genes and at least eight conserved components of the highly fragmented large and small subunit rRNAs. Unlike in apicomplexans, dinoflagellate mitochondrial genes occur in multiple copies, often as gene fragments, and in numerous genomic contexts. Analysis of cDNAs suggests several novel aspects of dinoflagellate mitochondrial gene expression. Polycistronic transcripts were found, standard start codons are absent, and oligoadenylation occurs upstream of stop codons, resulting in the absence of termination codons. Transcripts of at least one gene, cox3, are apparently trans-spliced to generate full-length mRNAs. RNA substitutional editing, a process previously identified for mRNAs in dinoflagellate mitochondria, is also implicated in rRNA expression. Conclusion The dinoflagellate mitochondrial genome shares the same gene complement and fragmentation of rRNA genes with its apicomplexan counterpart. However, it also exhibits several unique characteristics. Most notable are the expansion of gene copy numbers and their arrangements within the genome, RNA editing, loss of stop codons, and use of trans-splicing. PMID:17897476

Acetyl-L-carnitine supplementation to old rats partially reverts the age-related mitochondrial decay of soleus muscle by activating peroxisome proliferator-activated receptor gamma coactivator-1alpha-dependent mitochondrial biogenesis.

PubMed

Pesce, Vito; Fracasso, Flavio; Cassano, Pierluigi; Lezza, Angela Maria Serena; Cantatore, Palmiro; Gadaleta, Maria Nicola

2010-01-01

The age-related decay of mitochondrial function is a major contributor to the aging process. We tested the effects of 2-month-daily acetyl-L-carnitine (ALCAR) supplementation on mitochondrial biogenesis in the soleus muscle of aged rats. This muscle is heavily dependent on oxidative metabolism. Mitochondrial (mt) DNA content, citrate synthase activity, transcript levels of some nuclear- and mitochondrial-coded genes (cytochrome c oxidase subunit IV [COX-IV], 16S rRNA, COX-I) and of some factors involved in the mitochondrial biogenesis signaling pathway (peroxisome proliferator-activated receptor gamma [PPARgamma] coactivator-1alpha [PGC-1alpha], mitochondrial transcription factor A mitochondrial [TFAM], mitochondrial transcription factor 2B [TFB2]), as well as the protein content of PGC-1alpha were determined. The results suggest that the ALCAR treatment in old rats activates PGC-1alpha-dependent mitochondrial biogenesis, thus partially reverting the age-related mitochondrial decay.

Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

NASA Astrophysics Data System (ADS)

Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

1988-12-01

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

Lipopolysaccharide-induced mitochondrial dysfunction in boar sperm is mediated by activation of oxidative phosphorylation.

PubMed

He, Bin; Guo, Huiduo; Gong, Yabin; Zhao, Ruqian

2017-01-01

Lipopolysaccharide (LPS) has been reported to exert detrimental effects on boar sperm viability. In the present study, LPS was detected in boar semen samples at an average level of 0.62 ± 0.14 μg/mL. We treated boar sperm with 1 μg/mL LPS for 6 hours and examined alterations in sperm motility and apoptosis, together with mitochondrial functionality and mitochondrial reactive oxygen species generation. The expression and the location of toll-like receptor 4 (TLR4) and mitochondrial transcription factor A (TFAM) were determined to reveal possible mechanisms. LPS-treated sperm showed significant reduction in motility (P < 0.05) and viability (P < 0.05). LPS induced sperm mitochondrial damage via oxidative stress which is indicated by marked ultrastructural changes in the mitochondria including swelling, disorientation and vacuole, a decrease of mitochondrial membrane potential (ΔΨm; P < 0.05), as well as an increase of malondialdehyde levels (P < 0.01). Moreover, the production of mitochondrial reactive oxygen species through oxidative phosphorylation (OXPHOS) was significantly (P < 0.05) increased, which leads to oxidative stress. The copy number of mitochondrial DNA was significantly (P < 0.05) higher in LPS-treated sperm. Moreover, cytochrome c oxidase subunit IV (COXIV), an important subunit in mitochondrial electron transport chain and OXPHOS, was significantly (P < 0.05) upregulated after LPS treatment. TFAM, the key transcription factor that activates mitochondrial DNA replication and transcription, was translocated from the head to the midpiece of sperm where mitochondria are distributed in LPS-treated sperm. Taken together, these results indicate that LPS-induced decrease of motility and viability in boar sperm is mediated by abnormal activation of OXPHOS and mitochondrial membrane lipid peroxidation. These findings may provide new insights in understanding the mechanisms underlying the bacterial infection-induced sperm damage. Copyright © 2016 Elsevier Inc. All rights reserved.

Mitochondrial disease associated with complex I (NADH-CoQ oxidoreductase) deficiency.

PubMed

Scheffler, Immo E

2015-05-01

Mitochondrial diseases due to a reduced capacity for oxidative phosphorylation were first identified more than 20 years ago, and their incidence is now recognized to be quite significant. In a large proportion of cases the problem can be traced to a complex I (NADH-CoQ oxidoreductase) deficiency (Phenotype MIM #252010). Because the complex consists of 44 subunits, there are many potential targets for pathogenic mutations, both on the nuclear and mitochondrial genomes. Surprisingly, however, almost half of the complex I deficiencies are due to defects in as yet unidentified genes that encode proteins other than the structural proteins of the complex. This review attempts to summarize what we know about the molecular basis of complex I deficiencies: mutations in the known structural genes, and mutations in an increasing number of genes encoding "assembly factors", that is, proteins required for the biogenesis of a functional complex I that are not found in the final complex I. More such genes must be identified before definitive genetic counselling can be applied in all cases of affected families.

Cardiac Med1 deletion promotes early lethality, cardiac remodeling, and transcriptional reprogramming

PubMed Central

Spitler, Kathryn M.; Ponce, Jessica M.; Oudit, Gavin Y.; Hall, Duane D.

2017-01-01

The mediator complex, a multisubunit nuclear complex, plays an integral role in regulating gene expression by acting as a bridge between transcription factors and RNA polymerase II. Genetic deletion of mediator subunit 1 (Med1) results in embryonic lethality, due in large part to impaired cardiac development. We first established that Med1 is dynamically expressed in cardiac development and disease, with marked upregulation of Med1 in both human and murine failing hearts. To determine if Med1 deficiency protects against cardiac stress, we generated two cardiac-specific Med1 knockout mouse models in which Med1 is conditionally deleted (Med1cKO mice) or inducibly deleted in adult mice (Med1cKO-MCM mice). In both models, cardiac deletion of Med1 resulted in early lethality accompanied by pronounced changes in cardiac function, including left ventricular dilation, decreased ejection fraction, and pathological structural remodeling. We next defined how Med1 deficiency alters the cardiac transcriptional profile using RNA-sequencing analysis. Med1cKO mice demonstrated significant dysregulation of genes related to cardiac metabolism, in particular genes that are coordinated by the transcription factors Pgc1α, Pparα, and Errα. Consistent with the roles of these transcription factors in regulation of mitochondrial genes, we observed significant alterations in mitochondrial size, mitochondrial gene expression, complex activity, and electron transport chain expression under Med1 deficiency. Taken together, these data identify Med1 as an important regulator of vital cardiac gene expression and maintenance of normal heart function. NEW & NOTEWORTHY Disruption of transcriptional gene expression is a hallmark of dilated cardiomyopathy; however, its etiology is not well understood. Cardiac-specific deletion of the transcriptional coactivator mediator subunit 1 (Med1) results in dilated cardiomyopathy, decreased cardiac function, and lethality. Med1 deletion disrupted cardiac mitochondrial and metabolic gene expression patterns. PMID:28159809

Diversity of mitochondrial large subunit rDNA haplotypes of Glomus intraradices in two agricultural field experiments and two semi-natural grasslands.

PubMed

Börstler, Boris; Thiéry, Odile; Sýkorová, Zuzana; Berner, Alfred; Redecker, Dirk

2010-04-01

Glomus intraradices, an arbuscular mycorrhizal fungus (AMF), is frequently found in a surprisingly wide range of ecosystems all over the world. It is used as model organism for AMF and its genome is being sequenced. Despite the ecological importance of AMF, little has been known about their population structure, because no adequate molecular markers have been available. In the present study we analyse for the first time the intraspecific genetic structure of an AMF directly from colonized roots in the field. A recently developed PCR-RFLP approach for the mitochondrial rRNA large subunit gene (mtLSU) of these obligate symbionts was used and complemented by sequencing and primers specific for a particularly frequent mtLSU haplotype. We analysed root samples from two agricultural field experiments in Switzerland and two semi-natural grasslands in France and Switzerland. RFLP type composition of G. intraradices (phylogroup GLOM A-1) differed strongly between agricultural and semi-natural sites and the G. intraradices populations of the two agricultural sites were significantly differentiated. RFLP type richness was higher in the agricultural sites compared with the grasslands. Detailed sequence analyses which resolved multiple sequence haplotypes within some RFLP types even revealed that there was no overlap of haplotypes among any of the study sites except between the two grasslands. Our results demonstrate a surprisingly high differentiation among semi-natural and agricultural field sites for G. intraradices. These findings will have major implications on our views of processes of adaptation and specialization in these plant/fungus associations.

The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon▿

PubMed Central

Graef, Katy M.; Vreede, Frank T.; Lau, Yuk-Fai; McCall, Amber W.; Carr, Simon M.; Subbarao, Kanta; Fodor, Ervin

2010-01-01

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-β) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-β and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence. PMID:20538852

Correlation of mitochondrial protein expression in complexes I to V with natural and induced forms of canine idiopathic dilated cardiomyopathy.

PubMed

Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

2006-06-01

To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated. Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

A novel mutation m.8561C>G in MT-ATP6/8 causing a mitochondrial syndrome with ataxia, peripheral neuropathy, diabetes mellitus, and hypergonadotropic hypogonadism.

PubMed

Kytövuori, Laura; Lipponen, Joonas; Rusanen, Harri; Komulainen, Tuomas; Martikainen, Mika H; Majamaa, Kari

2016-11-01

Defects in the respiratory chain or mitochondrial ATP synthase (complex V) result in mitochondrial dysfunction that is an important cause of inherited neurological disease. Two of the subunits of complex V are encoded by MT-ATP6 and MT-ATP8 in the mitochondrial genome. Pathogenic mutations in MT-ATP6 are associated with the Leigh syndrome, the syndrome of neuropathy, ataxia, and retinitis pigmentosa (NARP), as well as with non-classical phenotypes, while MT-ATP8 is less frequently mutated in patients with mitochondrial disease. We investigated two adult siblings presenting with features of cerebellar ataxia, peripheral neuropathy, diabetes mellitus, sensorineural hearing impairment, and hypergonadotropic hypogonadism. As the phenotype was suggestive of mitochondrial disease, mitochondrial DNA was sequenced and a novel heteroplasmic mutation m.8561C>G in the overlapping region of the MT-ATP6 and MT-ATP8 was found. The mutation changed amino acids in both subunits. Mutation heteroplasmy correlated with the disease phenotype in five family members. An additional assembly intermediate of complex V and increased amount of subcomplex F 1 were observed in myoblasts of the two patients, but the total amount of complex V was unaffected. Furthermore, intracellular ATP concentration was lower in patient myoblasts indicating defective energy production. We suggest that the m.8561C>G mutation in MT-ATP6/8 is pathogenic, leads biochemically to impaired assembly and decreased ATP production of complex V, and results clinically in a phenotype with the core features of cerebellar ataxia, peripheral neuropathy, diabetes mellitus, and hypergonadotropic hypogonadism.

Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

PubMed Central

Zhou, Anna; Rohou, Alexis; Schep, Daniel G; Bason, John V; Montgomery, Martin G; Walker, John E; Grigorieff, Nikolaus; Rubinstein, John L

2015-01-01

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases. DOI: http://dx.doi.org/10.7554/eLife.10180.001 PMID:26439008

Yeast Mitoribosome Large Subunit Assembly Proceeds by Hierarchical Incorporation of Protein Clusters and Modules on the Inner Membrane.

PubMed

Zeng, Rui; Smith, Erin; Barrientos, Antoni

2018-03-06

Mitoribosomes are specialized for the synthesis of hydrophobic membrane proteins encoded by mtDNA, all essential for oxidative phosphorylation. Despite their linkage to human mitochondrial diseases and the recent cryoelectron microscopy reconstruction of yeast and mammalian mitoribosomes, how they are assembled remains obscure. Here, we dissected the yeast mitoribosome large subunit (mtLSU) assembly process by systematic genomic deletion of 44 mtLSU proteins (MRPs). Analysis of the strain collection unveiled 37 proteins essential for functional mtLSU assembly, three of which are critical for mtLSU 21S rRNA stability. Hierarchical cluster analysis of mtLSU subassemblies accumulated in mutant strains revealed co-operative assembly of protein sets forming structural clusters and preassembled modules. It also indicated crucial roles for mitochondrion-specific membrane-binding MRPs in anchoring newly transcribed 21S rRNA to the inner membrane, where assembly proceeds. Our results define the yeast mtLSU assembly landscape in vivo and provide a foundation for studies of mitoribosome assembly across evolution. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetic differentiation of the stingless bee Tetragonula pagdeni in Thailand using SSCP analysis of a large subunit of mitochondrial ribosomal DNA.

PubMed

Thummajitsakul, Sirikul; Klinbunga, Sirawut; Sittipraneed, Siriporn

2011-08-01

Genetic diversity and population differentiation of the stingless bee Tetragonula pagdeni (Schwarz) was assessed using single-strand conformational polymorphism (SSCP) analysis of a large subunit of the ribosomal RNA gene (16S rRNA). High levels of genetic variation among individuals within each population (North, Northeast, Central, Prachuap Khiri Khan, Chumphon, and Peninsular Thailand) of T. pagdeni were observed. Analysis of molecular variance indicated significant genetic differentiation among the six geographic populations (Φ (PT) = 0.28, P < 0.001) and between samples collected from north and south of the Isthmus of Kra (Φ (PT) = 0.18, P < 0.001). In addition, Φ (PT) values between all pairwise comparisons were statistically significant (P < 0.01), indicating strong degrees of intraspecific population differentiation. Therefore, PCR-SSCP is a simple and cost-effective technique applicable for routine population genetic analyses in T. pagdeni and other stingless bees. The results also provide an important baseline for the conservation and management of this ecologically important species.

[Molecular identification of human Diphyllobothrium nihonkaiense using mitochondrial cytochrome c oxidase subunit 1 (cox1) gene sequence].

PubMed

Ono, Sayaka; Morimoto, Norihito; Korenaga, Masataka; Kumazawa, Hideo; Komatsu, Yutaka; Kuge, Itsu; Higashidani, Yoshihumi; Ogura, Katsumi; Sugiura, Tetsuro

2010-11-01

Identification of Diphyllobothrium species has been carried out based on their morphology, especially sexual organs. In addition to these criteria, PCR-based identification methods have been developed recently. A 20 year-old Japanese living in Kochi Prefecture passed tapeworm. He was successfully treated with single dose of gastrografin. We examined the morphologic features of the proglottids and eggs using histology and scanning electron microscope. We also analyzed mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the proglottids. The causative tapeworm species was identified as D. nihonkaiense based on the results of morphologic features and genetic analysis. We discussed the advantage of PCR-based identification methods of Diphyllobothrium species using cox1 sequence in the clinical laboratory.

Atomistic simulations indicate the c-subunit ring of the F1Fo ATP synthase is not the mitochondrial permeability transition pore

PubMed Central

Zhou, Wenchang; Marinelli, Fabrizio; Nief, Corrine; Faraldo-Gómez, José D

2017-01-01

Pathological metabolic conditions such as ischemia induce the rupture of the mitochondrial envelope and the release of pro-apoptotic proteins, leading to cell death. At the onset of this process, the inner mitochondrial membrane becomes depolarized and permeable to osmolytes, proposedly due to the opening of a non-selective protein channel of unknown molecular identity. A recent study purports that this channel, referred to as Mitochondrial Permeability Transition Pore (MPTP), is formed within the c-subunit ring of the ATP synthase, upon its dissociation from the catalytic domain of the enzyme. Here, we examine this claim for two c-rings of different lumen width, through calculations of their ion conductance and selectivity based on all-atom molecular dynamics simulations. We also quantify the likelihood that the lumen of these c-rings is in a hydrated, potentially conducting state rather than empty or blocked by lipid molecules. These calculations demonstrate that the structure and biophysical properties of a correctly assembled c-ring are inconsistent with those attributed to the MPTP. DOI: http://dx.doi.org/10.7554/eLife.23781.001 PMID:28186490

Ubiquitin-Dependent Degradation of Mitochondrial Proteins Regulates Energy Metabolism.

PubMed

Lavie, Julie; De Belvalet, Harmony; Sonon, Sessinou; Ion, Ana Madalina; Dumon, Elodie; Melser, Su; Lacombe, Didier; Dupuy, Jean-William; Lalou, Claude; Bénard, Giovanni

2018-06-05

The ubiquitin proteasome system (UPS) regulates many cellular functions by degrading key proteins. Notably, the role of UPS in regulating mitochondrial metabolic functions is unclear. Here, we show that ubiquitination occurs in different mitochondrial compartments, including the inner mitochondrial membrane, and that turnover of several metabolic proteins is UPS dependent. We specifically detailed mitochondrial ubiquitination and subsequent UPS-dependent degradation of succinate dehydrogenase subunit A (SDHA), which occurred when SDHA was minimally involved in mitochondrial energy metabolism. We demonstrate that SDHA ubiquitination occurs inside the organelle. In addition, we show that the specific inhibition of SDHA degradation by UPS promotes SDHA-dependent oxygen consumption and increases ATP, malate, and citrate levels. These findings suggest that the mitochondrial metabolic machinery is also regulated by the UPS. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

PubMed

Veenman, Leo; Alten, Julia; Linnemannstöns, Karen; Shandalov, Yulia; Zeno, Sivan; Lakomek, Max; Gavish, Moshe; Kugler, Wilfried

2010-07-01

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

PubMed Central

Zíková, Alena; Schnaufer, Achim; Dalley, Rachel A.; Panigrahi, Aswini K.; Stuart, Kenneth D.

2009-01-01

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought. PMID:19436713

Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

PubMed

Shao, Zhiyong; Graf, Shannon; Chaga, Oleg Y; Lavrov, Dennis V

2006-10-15

The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease

PubMed Central

Elson, Joanna L.; Smith, Paul M.; Greaves, Laura C.; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M.A.; Taylor, Robert W.; Vila-Sanjurjo, Antón

2015-01-01

Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge. Only two disease causing mutations have been identified at these loci, both mapping to the small subunit (SSU). On the large subunit (LSU), however, the evidence for the presence of pathogenic LSU mt-rRNA changes is particularly sparse. We have previously expanded the list of deleterious SSU mt-rRNA mutations by identifying highly disruptive base changes capable of blocking the activity of the mitoribosomal SSU. To do this, we used a new methodology named heterologous inferential analysis (HIA). The recent arrival of near-atomic-resolution structures of the human mitoribosomal LSU, has enhanced the power of our approach by permitting the analysis of the corresponding sites of mutation within their natural structural context. Here, we have used these tools to determine whether LSU mt-rRNA mutations found in the context of human disease and/or ageing could disrupt the function of the mitoribosomal LSU. Our results clearly show that, much like the for SSU mt-rRNA, LSU mt-rRNAs mutations capable of compromising the function of the mitoribosomal LSU are indeed present in clinical samples. Thus, our work constitutes an important contribution to an emerging view of the mitoribosome as an important element in human health. PMID:26349026

The adaptive evolution of the mammalian mitochondrial genome

PubMed Central

da Fonseca, Rute R; Johnson, Warren E; O'Brien, Stephen J; Ramos, Maria João; Antunes, Agostinho

2008-01-01

Background The mitochondria produce up to 95% of a eukaryotic cell's energy through oxidative phosphorylation. The proteins involved in this vital process are under high functional constraints. However, metabolic requirements vary across species, potentially modifying selective pressures. We evaluate the adaptive evolution of 12 protein-coding mitochondrial genes in 41 placental mammalian species by assessing amino acid sequence variation and exploring the functional implications of observed variation in secondary and tertiary protein structures. Results Wide variation in the properties of amino acids were observed at functionally important regions of cytochrome b in species with more-specialized metabolic requirements (such as adaptation to low energy diet or large body size, such as in elephant, dugong, sloth, and pangolin, and adaptation to unusual oxygen requirements, for example diving in cetaceans, flying in bats, and living at high altitudes in alpacas). Signatures of adaptive variation in the NADH dehydrogenase complex were restricted to the loop regions of the transmembrane units which likely function as protons pumps. Evidence of adaptive variation in the cytochrome c oxidase complex was observed mostly at the interface between the mitochondrial and nuclear-encoded subunits, perhaps evidence of co-evolution. The ATP8 subunit, which has an important role in the assembly of F0, exhibited the highest signal of adaptive variation. ATP6, which has an essential role in rotor performance, showed a high adaptive variation in predicted loop areas. Conclusion Our study provides insight into the adaptive evolution of the mtDNA genome in mammals and its implications for the molecular mechanism of oxidative phosphorylation. We present a framework for future experimental characterization of the impact of specific mutations in the function, physiology, and interactions of the mtDNA encoded proteins involved in oxidative phosphorylation. PMID:18318906

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

PubMed Central

Fearnley, I M; Finel, M; Skehel, J M; Walker, J E

1991-01-01

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit. Images Fig. 1. PMID:1832859

Redox and Reactive Oxygen Species Regulation of Mitochondrial Cytochrome c Oxidase Biogenesis

PubMed Central

Bourens, Myriam; Fontanesi, Flavia; Soto, Iliana C.; Liu, Jingjing

2013-01-01

Abstract Significance: Cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is the major oxygen consumer enzyme in the cell. COX biogenesis involves several redox-regulated steps. The process is highly regulated to prevent the formation of pro-oxidant intermediates. Recent Advances: Regulation of COX assembly involves several reactive oxygen species and redox-regulated steps. These include: (i) Intricate redox-controlled machineries coordinate the expression of COX isoenzymes depending on the environmental oxygen concentration. (ii) COX is a heme A-copper metalloenzyme. COX copper metallation involves the copper chaperone Cox17 and several other recently described cysteine-rich proteins, which are oxidatively folded in the mitochondrial intermembrane space. Copper transfer to COX subunits 1 and 2 requires concomitant transfer of redox power. (iii) To avoid the accumulation of reactive assembly intermediates, COX is regulated at the translational level to minimize synthesis of the heme A-containing Cox1 subunit when assembly is impaired. Critical Issues: An increasing number of regulatory pathways converge to facilitate efficient COX assembly, thus preventing oxidative stress. Future Directions: Here we will review on the redox-regulated COX biogenesis steps and will discuss their physiological relevance. Forthcoming insights into the precise regulation of mitochondrial COX biogenesis in normal and stress conditions will likely open future perspectives for understanding mitochondrial redox regulation and prevention of oxidative stress. Antioxid. Redox Signal. 19, 1940–1952. PMID:22937827

Muscle biopsies from human muscle diseases with myopathic pathology reveal common alterations in mitochondrial function.

PubMed

Sunitha, Balaraju; Gayathri, Narayanappa; Kumar, Manish; Keshava Prasad, Thottethodi Subrahmanya; Nalini, Atchayaram; Padmanabhan, Balasundaram; Srinivas Bharath, Muchukunte Mukunda

2016-07-01

Muscle diseases are clinically and genetically heterogeneous and manifest as dystrophic, inflammatory and myopathic pathologies, among others. Our previous study on the cardiotoxin mouse model of myodegeneration and inflammation linked muscle pathology with mitochondrial damage and oxidative stress. In this study, we investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from muscle disease patients, represented by dysferlinopathy (dysfy) (dystrophic pathology; n = 43), polymyositis (PM) (inflammatory pathology; n = 24), and distal myopathy with rimmed vacuoles (DMRV) (distal myopathy; n = 31) were analyzed. Mitochondrial damage (ragged blue and COX-deficient fibers) was revealed in dysfy, PM, and DMRV cases by enzyme histochemistry (SDH and COX-SDH), electron microscopy (vacuolation and altered cristae) and biochemical assays (significantly increased ADP/ATP ratio). Proteomic analysis of muscle mitochondria from all three muscle diseases by isobaric tag for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis demonstrated down-regulation of electron transport chain (ETC) complex subunits, assembly factors and Krebs cycle enzymes. Interestingly, 80 of the under-expressed proteins were common among the three pathologies. Assay of ETC and Krebs cycle enzyme activities validated the MS data. Mitochondrial proteins from muscle pathologies also displayed higher tryptophan (Trp) oxidation and the same was corroborated in the cardiotoxin model. Molecular modeling predicted Trp oxidation to alter the local structure of mitochondrial proteins. Our data highlight mitochondrial alterations in muscle pathologies, represented by morphological changes, altered mitochondrial proteome and protein oxidation, thereby establishing the role of mitochondrial damage in human muscle diseases. We investigated whether human muscle diseases display mitochondrial changes. Muscle biopsies from dysferlinopathy (Dysfy), polymyositis (PM), and distal myopathy with rimmed vacuoles (DMRV) displayed morphological and biochemical evidences of mitochondrial dysfunction. Proteomic analysis revealed down-regulation of electron transport chain (ETC) subunits, assembly factors, and tricarboxylic acid (TCA) cycle enzymes, with 80 proteins common among the three pathologies. Mitochondrial proteins from muscle pathologies also displayed higher Trp oxidation that could alter the local structure. Cover image for this issue: doi: 10.1111/jnc.13324. © 2016 International Society for Neurochemistry.

The clinical maze of mitochondrial neurology

PubMed Central

DiMauro, Salvatore; Schon, Eric A.; Carelli, Valerio; Hirano, Michio

2014-01-01

Mitochondrial diseases involve the respiratory chain, which is under the dual control of nuclear and mitochondrial DNA (mtDNA). The complexity of mitochondrial genetics provides one explanation for the clinical heterogeneity of mitochondrial diseases, but our understanding of disease pathogenesis remains limited. Classification of Mendelian mitochondrial encephalomyopathies has been laborious, but whole-exome sequencing studies have revealed unexpected molecular aetiologies for both typical and atypical mitochondrial disease phenotypes. Mendelian mitochondrial defects can affect five components of mitochondrial biology: subunits of respiratory chain complexes (direct hits); mitochondrial assembly proteins; mtDNA translation; phospholipid composition of the inner mitochondrial membrane; or mitochondrial dynamics. A sixth category—defects of mtDNA maintenance—combines features of Mendelian and mitochondrial genetics. Genetic defects in mitochondrial dynamics are especially important in neurology as they cause optic atrophy, hereditary spastic paraplegia, and Charcot–Marie–Tooth disease. Therapy is inadequate and mostly palliative, but promising new avenues are being identified. Here, we review current knowledge on the genetics and pathogenesis of the six categories of mitochondrial disorders outlined above, focusing on their salient clinical manifestations and highlighting novel clinical entities. An outline of diagnostic clues for the various forms of mitochondrial disease, as well as potential therapeutic strategies, is also discussed. PMID:23835535

Identification of Novel Mitochondrial Protein Components of Chlamydomonas reinhardtii. A Proteomic Approach1

PubMed Central

van Lis, Robert; Atteia, Ariane; Mendoza-Hernández, Guillermo; González-Halphen, Diego

2003-01-01

Pure mitochondria of the photosynthetic alga Chlamydomonas reinhardtii were analyzed using blue native-polyacrylamide gel electrophoresis (BN-PAGE). The major oxidative phosphorylation complexes were resolved: F1F0-ATP synthase, NADH-ubiquinone oxidoreductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase. The oligomeric states of these complexes were determined. The F1F0-ATP synthase runs exclusively as a dimer, in contrast to the C. reinhardtii chloroplast enzyme, which is present as a monomer and subcomplexes. The sequence of a 60-kD protein, associated with the mitochondrial ATP synthase and with no known counterpart in any other organism, is reported. This protein may be related to the strong dimeric character of the algal F1F0-ATP synthase. The oxidative phosphorylation complexes resolved by BN-PAGE were separated into their subunits by second dimension sodium dodecyl sulfate-PAGE. A number of polypeptides were identified mainly on the basis of their N-terminal sequence. Core I and II subunits of complex III were characterized, and their proteolytic activities were predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits in cytochrome c oxidase was demonstrated. Other mitochondrial proteins like the chaperone HSP60, the alternative oxidase, the aconitase, and the ADP/ATP carrier were identified. BN-PAGE was also used to approach the analysis of the major chloroplast protein complexes of C. reinhardtii. PMID:12746537

MicroRNA-138 and MicroRNA-25 Down-regulate Mitochondrial Calcium Uniporter, Causing the Pulmonary Arterial Hypertension Cancer Phenotype

PubMed Central

Hong, Zhigang; Chen, Kuang-Hueih; DasGupta, Asish; Potus, Francois; Dunham-Snary, Kimberly; Bonnet, Sebastien; Tian, Lian; Fu, Jennifer; Breuils-Bonnet, Sandra; Provencher, Steeve; Wu, Danchen; Mewburn, Jeffrey; Ormiston, Mark L.

2017-01-01

Rationale: Pulmonary arterial hypertension (PAH) is an obstructive vasculopathy characterized by excessive pulmonary artery smooth muscle cell (PASMC) proliferation, migration, and apoptosis resistance. This cancer-like phenotype is promoted by increased cytosolic calcium ([Ca2+]cyto), aerobic glycolysis, and mitochondrial fission. Objectives: To determine how changes in mitochondrial calcium uniporter (MCU) complex (MCUC) function influence mitochondrial dynamics and contribute to PAH’s cancer-like phenotype. Methods: PASMCs were isolated from patients with PAH and healthy control subjects and assessed for expression of MCUC subunits. Manipulation of the pore-forming subunit, MCU, in PASMCs was achieved through small interfering RNA knockdown or MCU plasmid-mediated up-regulation, as well as through modulation of the upstream microRNAs (miRs) miR-138 and miR-25. In vivo, nebulized anti-miRs were administered to rats with monocrotaline-induced PAH. Measurements and Main Results: Impaired MCUC function, resulting from down-regulation of MCU and up-regulation of an inhibitory subunit, mitochondrial calcium uptake protein 1, is central to PAH’s pathogenesis. MCUC dysfunction decreases intramitochondrial calcium ([Ca2+]mito), inhibiting pyruvate dehydrogenase activity and glucose oxidation, while increasing [Ca2+]cyto, promoting proliferation, migration, and fission. In PAH PASMCs, increasing MCU decreases cell migration, proliferation, and apoptosis resistance by lowering [Ca2+]cyto, raising [Ca2+]mito, and inhibiting fission. In normal PASMCs, MCUC inhibition recapitulates the PAH phenotype. In PAH, elevated miRs (notably miR-138) down-regulate MCU directly and also by decreasing MCU’s transcriptional regulator cAMP response element–binding protein 1. Nebulized anti-miRs against miR-25 and miR-138 restore MCU expression, reduce cell proliferation, and regress established PAH in the monocrotaline model. Conclusions: These results highlight miR-mediated MCUC dysfunction as a unifying mechanism in PAH that can be therapeutically targeted. PMID:27648837

Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

PubMed Central

Merkwirth, Carsten; Morbin, Michela; Brönneke, Hella S.; Jordan, Sabine D.; Rugarli, Elena I.; Langer, Thomas

2012-01-01

Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies caused by a dysfunction of mitochondria and raise the possibility that tau pathologies are associated with other neurodegenerative disorders caused by deficiencies in mitochondrial dynamics. PMID:23144624

Plastid–Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae

PubMed Central

Weng, Mao-Lun; Ruhlman, Tracey A.; Jansen, Robert K.

2016-01-01

Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid–nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

ATP-sensitive potassium currents from channels formed by Kir6 and a modified cardiac mitochondrial SUR2 variant

PubMed Central

Aggarwal, Nitin T; Shi, Nian-Qing; Makielski, Jonathan C

2013-01-01

Cardiac ATP-sensitive potassium channels (KATP) are found in both the sarcoplasmic reticulum (sarcKATP) and the inner membrane of mitochondria (mitoKATP). SarcKATP are composed of a pore containing subunit Kir6.2 and a regulatory sulfonylurea receptor subunit (SUR2), but the composition of mitoKATP remains unclear. An unusual intra-exonic splice variant of SUR2 (SUR2A-55) was previously identified in mitochondria of mammalian heart and brain, and by analogy with sarcKATP we proposed SUR2A-55 as a candidate regulatory subunit of mitoKATP. Although SUR2A-55 lacks the first nucleotide binding domain (NBD) and 2 transmembrane domains (TMD), it has a hybrid TMD and retains the second NBD. It resembles a hemi-ABC transporter suggesting it could multimerize to function as a regulatory subunit. A putative mitochondrial targeting signal in the N-terminal domain of SUR2A-55 was removed by truncation and when co-expressed with Kir6.1 and Kir6.2 it targeted to the plasma membrane and yielded KATP currents. Single channel conductance, mean open time, and burst open time of SUR2A-55 based KATP was similar to the full-length SUR2A based KATP. However, the SUR2A-55 KATP were 70-fold less sensitive to block by ATP, and twice as resistant to intracellular Ca2+ inhibition compared with the SUR2A KATP, and were markedly insensitive to KATP drugs, pinacidil, diazoxide, and glybenclamide. These results suggest that the SUR2A-55 based channels would tend to be open under physiological conditions and in ischemia, and could account for cardiac and mitochondrial phenotypes protective for ischemia. PMID:24037327

In vitro reconstitution and characterization of the yeast mitochondrial degradosome complex unravels tight functional interdependence.

PubMed

Malecki, Michal; Jedrzejczak, Robert; Stepien, Piotr P; Golik, Pawel

2007-09-07

The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.

How the nucleus and mitochondria communicate in energy production during stress: nuclear MtATP6, an early-stress responsive gene, regulates the mitochondrial F₁F₀-ATP synthase complex.

PubMed

Moghadam, Ali Asghar; Ebrahimie, Eemaeil; Taghavi, Seyed Mohsen; Niazi, Ali; Babgohari, Mahbobeh Zamani; Deihimi, Tahereh; Djavaheri, Mohammad; Ramezani, Amin

2013-07-01

A small number of stress-responsive genes, such as those of the mitochondrial F1F0-ATP synthase complex, are encoded by both the nucleus and mitochondria. The regulatory mechanism of these joint products is mysterious. The expression of 6-kDa subunit (MtATP6), a relatively uncharacterized nucleus-encoded subunit of F0 part, was measured during salinity stress in salt-tolerant and salt-sensitive cultivated wheat genotypes, as well as in the wild wheat genotypes, Triticum and Aegilops using qRT-PCR. The MtATP6 expression was suddenly induced 3 h after NaCl treatment in all genotypes, indicating an early inducible stress-responsive behavior. Promoter analysis showed that the MtATP6 promoter includes cis-acting elements such as ABRE, MYC, MYB, GTLs, and W-boxes, suggesting a role for this gene in abscisic acid-mediated signaling, energy metabolism, and stress response. It seems that 6-kDa subunit, as an early response gene and nuclear regulatory factor, translocates to mitochondria and completes the F1F0-ATP synthase complex to enhance ATP production and maintain ion homeostasis under stress conditions. These communications between nucleus and mitochondria are required for inducing mitochondrial responses to stress pathways. Dual targeting of 6-kDa subunit may comprise as a mean of inter-organelle communication and save energy for the cell. Interestingly, MtATP6 showed higher and longer expression in the salt-tolerant wheat and the wild genotypes compared to the salt-sensitive genotype. Apparently, salt-sensitive genotypes have lower ATP production efficiency and weaker energy management than wild genotypes; a stress tolerance mechanism that has not been transferred to cultivated genotypes.

Mitochondrial Reactive Oxygen Species Mediate Cardiac Structural, Functional, and Mitochondrial Consequences of Diet-Induced Metabolic Heart Disease.

PubMed

Sverdlov, Aaron L; Elezaby, Aly; Qin, Fuzhong; Behring, Jessica B; Luptak, Ivan; Calamaras, Timothy D; Siwik, Deborah A; Miller, Edward J; Liesa, Marc; Shirihai, Orian S; Pimentel, David R; Cohen, Richard A; Bachschmid, Markus M; Colucci, Wilson S

2016-01-11

Mitochondrial reactive oxygen species (ROS) are associated with metabolic heart disease (MHD). However, the mechanism by which ROS cause MHD is unknown. We tested the hypothesis that mitochondrial ROS are a key mediator of MHD. Mice fed a high-fat high-sucrose (HFHS) diet develop MHD with cardiac diastolic and mitochondrial dysfunction that is associated with oxidative posttranslational modifications of cardiac mitochondrial proteins. Transgenic mice that express catalase in mitochondria and wild-type mice were fed an HFHS or control diet for 4 months. Cardiac mitochondria from HFHS-fed wild-type mice had a 3-fold greater rate of H2O2 production (P=0.001 versus control diet fed), a 30% decrease in complex II substrate-driven oxygen consumption (P=0.006), 21% to 23% decreases in complex I and II substrate-driven ATP synthesis (P=0.01), and a 62% decrease in complex II activity (P=0.002). In transgenic mice that express catalase in mitochondria, all HFHS diet-induced mitochondrial abnormalities were ameliorated, as were left ventricular hypertrophy and diastolic dysfunction. In HFHS-fed wild-type mice complex II substrate-driven ATP synthesis and activity were restored ex vivo by dithiothreitol (5 mmol/L), suggesting a role for reversible cysteine oxidative posttranslational modifications. In vitro site-directed mutation of complex II subunit B Cys100 or Cys103 to redox-insensitive serines prevented complex II dysfunction induced by ROS or high glucose/high palmitate in the medium. Mitochondrial ROS are pathogenic in MHD and contribute to mitochondrial dysfunction, at least in part, by causing oxidative posttranslational modifications of complex I and II proteins including reversible oxidative posttranslational modifications of complex II subunit B Cys100 and Cys103. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

PubMed Central

Chen, Chwen-Lih; Zhang, Liwen; Yeh, Alexander; Chen, Chun-An; Green-Church, Kari B.; Zweier, Jay L.; Chen, Yeong-Renn

2008-01-01

The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2·-) and O2·--derived oxidants change the redox status of the mitochondrial GSH pool. An electron transport protein, Mitochondrial Complex I, is the major host of reactive/regulatory protein thiols. An important response of protein thiols to oxidative stress is to reversibly form protein mixed disulfide via S-glutathiolation. Exposure of Complex I to oxidized GSH, GSSG, resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits. Here, to investigate the molecular mechanism of S-glutathiolation of Complex I, we prepared isolated bovine Complex I under non-reducing conditions and employed the techniques of mass spectrometry and EPR spin trapping for analysis. LC/MS/MS analysis of tryptic digests of the 51 kDa and 75 kDa polypeptides from glutathiolated Complex I (GS-NQR) revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of GS-NQR in catalyzing NADH oxidation by Q1 was significantly enhanced. However, O2·- generation activity (SGA) mediated by GS-NQR suffered a mild loss as measured by EPR spin trapping, suggesting the protective role of S-glutathiolation in the intact Complex I. Exposure of NADH dehydrogenase (NDH), the flavin subcomplex of Complex I, to GSSG resulted in specific S-glutathiolation on the 51 kDa subunit. Both ETA and SGA of S-glutathiolated NDH (GS-NDH) decreased in parallel as the dosage of GSSG increased. LC/MS/MS analysis of a tryptic digest of the 51 kDa subunit from GS-NDH revealed that C206, C187, and C425 were glutathiolated. C425 of the 51 kDa subunit is a ligand residue of the 4Fe-4S N3 center, suggesting that destruction of 4Fe-4S is the major mechanism involved in the inhibiton of NDH. The result also implies that S-glutathiolation of the 75 kDa subunit may play a role in protecting the 4Fe-4S cluster of the 51 kDa subunit from redox modification when Complex I is exposed to redox change in the GSH pool. PMID:17444656

The mitochondrial genome of the plant-pathogenic fungus Stemphylium lycopersici uncovers a dynamic structure due to repetitive and mobile elements.

PubMed

Franco, Mario Emilio Ernesto; López, Silvina Marianela Yanil; Medina, Rocio; Lucentini, César Gustavo; Troncozo, Maria Inés; Pastorino, Graciela Noemí; Saparrat, Mario Carlos Nazareno; Balatti, Pedro Alberto

2017-01-01

Stemphylium lycopersici (Pleosporales) is a plant-pathogenic fungus that has been associated with a broad range of plant-hosts worldwide. It is one of the causative agents of gray leaf spot disease in tomato and pepper. The aim of this work was to characterize the mitochondrial genome of S. lycopersici CIDEFI-216, to use it to trace taxonomic relationships with other fungal taxa and to get insights into the evolutionary history of this phytopathogen. The complete mitochondrial genome was assembled into a circular double-stranded DNA molecule of 75,911 bp that harbors a set of 37 protein-coding genes, 2 rRNA genes (rns and rnl) and 28 tRNA genes, which are transcribed from both sense and antisense strands. Remarkably, its gene repertoire lacks both atp8 and atp9, contains a free-standing gene for the ribosomal protein S3 (rps3) and includes 13 genes with homing endonuclease domains that are mostly located within its 15 group I introns. Strikingly, subunits 1 and 2 of cytochrome oxidase are encoded by a single continuous open reading frame (ORF). A comparative mitogenomic analysis revealed the large extent of structural rearrangements among representatives of Pleosporales, showing the plasticity of their mitochondrial genomes. Finally, an exhaustive phylogenetic analysis of the subphylum Pezizomycotina based on mitochondrial data reconstructed their relationships in concordance with several studies based on nuclear data. This is the first report of a mitochondrial genome belonging to a representative of the family Pleosporaceae.

The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

PubMed Central

Bakkaiova, Jana; Arata, Kosuke; Matsunobu, Miki; Ono, Bungo; Aoki, Tomoyo; Lajdova, Dana; Nebohacova, Martina; Nosek, Jozef; Miyakawa, Isamu

2014-01-01

Mitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a Δmhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the Δmhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. PMID:24972935

Effect of thyroid status on the expression of metabolic enzymes during chronic stimulation.

PubMed

Hood, D A; Simoneau, J A; Kelly, A M; Pette, D

1992-10-01

The effect of thyroid status on the expression of cytochrome c oxidase (CYTOX) and the activities of citrate synthase (CS) and phosphofructokinase (PFK) were examined in chronically stimulated (10 Hz; 35 days) and contralateral, nonstimulated rat tibialis anterior muscle of hypothyroid, hyperthyroid, and euthyroid animals. Stimulation increased CYTOX activity by 2.7-, 3.2-, and 4.9-fold in hyperthyroid, euthyroid, and hypothyroid animals, respectively, to similar absolute values. CS displayed similar increases. Stimulation reduced PFK activity in hypothyroid and euthyroid animals to 45% and 60% of control values. This effect was abolished with hyperthyroidism. Thus stimulation and thyroid hormone act antagonistically on PFK activity. Stimulation increased CYTOX subunit III (mitochondrially encoded) mRNA by 2.5- and 2.9-fold in hyperthyroid and euthyroid animals. Similar increases were observed in the nuclear-encoded mRNAs of CYTOX subunit VIc in euthyroid muscle. In hyperthyroid and euthyroid conditions, the mRNA changes paralleled the increases in enzyme activity. In hypothyroid muscle, the increase in mRNA was less for subunit VIc than III, suggesting that hypothyroidism upsets the coordinate expression of nuclear and mitochondrial genes. Further, the increases in CYTOX activity exceeded that of both subunit mRNAs in hypothyroid muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

The structural analysis of the mitochondrial SSUrRNA implies a close phylogenetic relationship between mitochondria from plants and from the heterotrophic alga Prototheca wickerhamii.

PubMed

Wolff, G; Kück, U

1990-04-01

The gene for the mitochondrial small subunit rRNA (SSUrRNA) from the heterotrophic alga Prototheca wickerhamii has been isolated from a gene library of extranuclear DNA. Sequence and structural analyses allow the determination of a secondary structure model for this rRNA. In addition, several sequence motifs are present which are typically found in SSUrRNAs of various mitochondrial origins. Unexpectedly, the Prototheca RNA sequence has more features in common with mitochondrial SSUrRNAs from plants than with that from the green alga Chlamydomonas reinhardtii. The phylogenetic relationship between mitochondria from plants and algae is discussed.

Genotypic variation of Pneumocystis jirovecii isolates in India based on sequence diversity at mitochondrial large subunit rRNA.

PubMed

Gupta, Rashmi; Mirdha, Bijay Ranjan; Guleria, Randeep; Agarwal, Sanjay Kumar; Samantaray, Jyotish Chandra; Kumar, Lalit; Kabra, Sushil Kumar; Luthra, Kalpana; Sreenivas, Vishnubhatla; Iyer, Venkateswaran K

2011-03-01

Pneumocystis pneumonia (PCP), a common and serious opportunistic infection in immunocompromised patients, is caused by Pneumocystis jirovecii (formerly known as Pneumocystis carinii f. sp. hominis). The aim of the present study was to describe the prevalence and distribution of genotypes of P. jirovecii based on sequence polymorphisms at mitochondrial large subunit ribosomal RNA (mt LSU rRNA) region in both HIV and non-HIV immunocompromised individuals with a positive PCR result for PCP in a tertiary health care centre in northern India. From January 2005 to October 2008, 50 patients [22 HIV-seropositive individuals, 10 post-renal transplant (PRT) recipients, 3 cancer patients, and 15 patients with various other kinds of immunosuppression] were found to be positive for P. jirovecii using PCR at the mt LSU rRNA gene. Genotyping of the positive samples was performed at the mt LSU rRNA locus. Genotype 2 was the most common accounting for 42% of total types. This was followed by the genotypes 3 (24%), 1 (20%), and 4 (8%). Mixed infection was observed in 3 cases (6%). The rates of genotype distribution were similar in HIV-seropositive individuals, cancer patients, and in patients with other kinds of immunosuppression. In the PRT recipients, genotype 1 was the most prevalent type (80%). This is the first study describing the prevalence of genotypes in HIV-infected and HIV-uninfected, immunocompromised patients based on the mt LSU rRNA gene from the Indian subcontinent. The most prevalent genotype observed was type 2 in contrast to many studies from other parts of the world where genotype 1 was the most prevalent type, suggesting geographical variation. Copyright © 2010 Elsevier GmbH. All rights reserved.

Novel Insights into the Role of Neurospora crassa NDUFAF2, an Evolutionarily Conserved Mitochondrial Complex I Assembly Factor

PubMed Central

Pereira, Bruno; Videira, Arnaldo

2013-01-01

Complex I deficiency is commonly associated with mitochondrial oxidative phosphorylation diseases. Mutations in nuclear genes encoding structural subunits or assembly factors of complex I have been increasingly identified as the cause of the diseases. One such factor, NDUFAF2, is a paralog of the NDUFA12 structural subunit of the enzyme, but the mechanism by which it exerts its function remains unknown. Herein, we demonstrate that the Neurospora crassa NDUFAF2 homologue, the 13.4L protein, is a late assembly factor that associates with complex I assembly intermediates containing the membrane arm and the connecting part but lacking the N module of the enzyme. Furthermore, we provide evidence that dissociation of the assembly factor is dependent on the incorporation of the putative regulatory module composed of the subunits of 13.4 (NDUFA12), 18.4 (NDUFS6), and 21 (NDUFS4) kDa. Our results demonstrate that the 13.4L protein is a complex I assembly factor functionally conserved from fungi to mammals. PMID:23648483

Regulation of succinate-ubiquinone reductase and fumarate reductase activities in human complex II by phosphorylation of its flavoprotein subunit.

PubMed

Tomitsuka, Eriko; Kita, Kiyoshi; Esumi, Hiroyasu

2009-01-01

Complex II (succinate-ubiquinone reductase; SQR) is a mitochondrial respiratory chain enzyme that is directly involved in the TCA cycle. Complex II exerts a reverse reaction, fumarate reductase (FRD) activity, in various species such as bacteria, parasitic helminths and shellfish, but the existence of FRD activity in humans has not been previously reported. Here, we describe the detection of FRD activity in human cancer cells. The activity level was low, but distinct, and it increased significantly when the cells were cultured under hypoxic and glucose-deprived conditions. Treatment with phosphatase caused the dephosphorylation of flavoprotein subunit (Fp) with a concomitant increase in SQR activity, whereas FRD activity decreased. On the other hand, treatment with protein kinase caused an increase in FRD activity and a decrease in SQR activity. These data suggest that modification of the Fp subunit regulates both the SQR and FRD activities of complex II and that the phosphorylation of Fp might be important for maintaining mitochondrial energy metabolism within the tumor microenvironment.

COX16 promotes COX2 metallation and assembly during respiratory complex IV biogenesis

PubMed Central

Aich, Abhishek; Wang, Cong; Chowdhury, Arpita; Ronsör, Christin; Pacheu-Grau, David; Richter-Dennerlein, Ricarda; Dennerlein, Sven

2018-01-01

Cytochrome c oxidase of the mitochondrial oxidative phosphorylation system reduces molecular oxygen with redox equivalent-derived electrons. The conserved mitochondrial-encoded COX1- and COX2-subunits are the heme- and copper-center containing core subunits that catalyze water formation. COX1 and COX2 initially follow independent biogenesis pathways creating assembly modules with subunit-specific, chaperone-like assembly factors that assist in redox centers formation. Here, we find that COX16, a protein required for cytochrome c oxidase assembly, interacts specifically with newly synthesized COX2 and its copper center-forming metallochaperones SCO1, SCO2, and COA6. The recruitment of SCO1 to the COX2-module is COX16- dependent and patient-mimicking mutations in SCO1 affect interaction with COX16. These findings implicate COX16 in CuA-site formation. Surprisingly, COX16 is also found in COX1-containing assembly intermediates and COX2 recruitment to COX1. We conclude that COX16 participates in merging the COX1 and COX2 assembly lines. PMID:29381136

Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.

PubMed

Roloff, Gabrielle A; Henry, Michael F

2015-08-15

Three mitochondrial DNA-encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. © 2015 Roloff and Henry. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Mitochondrial Structure and Reactive Oxygen Species in Mammary Oncogenesis

DTIC Science & Technology

2007-04-01

for the subunits of this Complex responsible for the hereditary paraganglioma and pheochromocytoma (2- 4,12,13,30,31,32) suggest that Complex II...familial pheochromocytoma and to familial paraganglioma. Am J Hum Genet. 2001, 69:49-54. 3. Baysal BE, Ferrell RE, Willett-Brozick JE, Lawrence EC...Eng C. Somatic and occult germ-line mutations in SDHD, a mitochondrial complex II gene, in nonfamilial pheochromocytoma . Cancer Res. 2000, 60

Changing phenotypic expression in a patient with a mitochondrial encephalopathy due to 13042G>A de novo mutation--a 5 year follow up.

PubMed

Schinwelski, M; Kierdaszuk, B; Dulski, J; Tońska, K; Kodroń, A; Sitek, E J; Bartnik, E; Kamińska, A; Kwieciński, H; Sławek, J

2015-08-01

Mutations in NADH dehydrogenase (ND) subunits of complex I lead to mitochondrial encephalomyopathies associated with various phenotypes. This report aims to present the patient's clinical symptomatology in the context of a very rare 13042G>A de novo mutation and with an emphasis on changing phenotypic expression and pronounced, long-standing response to levetiracetam.

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain.

PubMed

Mu, Hong-Na; Li, Quan; Pan, Chun-Shui; Liu, Yu-Ying; Yan, Li; Hu, Bai-He; Sun, Kai; Chang, Xin; Zhao, Xin-Rong; Fan, Jing-Yu; Han, Jing-Yan

2015-08-01

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain. Copyright © 2015 Elsevier Inc. All rights reserved.

The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize

PubMed Central

Bonen, Linda; Boer, Poppo H.; Gray, Michael W.

1984-01-01

We have determined the sequence of the wheat mitochondrial gene for cytochrome oxidase subunit II (COII) and find that its derived protein sequence differs from that of maize at only three amino acid positions. Unexpectedly, all three replacements are non-conservative ones. The wheat COII gene has a highly-conserved intron at the same position as in maize, but the wheat intron is 1.5 times longer because of an insert relative to its maize counterpart. Hybridization analysis of mitochondrial DNA from rye, pea, broad bean and cucumber indicates strong sequence conservation of COII coding sequences among all these higher plants. However, only rye and maize mitochondrial DNA show homology with wheat COII intron sequences and rye alone with intron-insert sequences. We find that a sequence identical to the region of the 5' exon corresponding to the transmembrane domain of the COII protein is present at a second genomic location in wheat mitochondria. These variations in COII gene structure and size, as well as the presence of repeated COII sequences, illustrate at the DNA sequence level, factors which contribute to higher plant mitochondrial DNA diversity and complexity. ImagesFig. 3.Fig. 4.Fig. 5. PMID:16453565

Human mitochondrial pyruvate carrier 2 as an autonomous membrane transporter.

PubMed

Nagampalli, Raghavendra Sashi Krishna; Quesñay, José Edwin Neciosup; Adamoski, Douglas; Islam, Zeyaul; Birch, James; Sebinelli, Heitor Gobbi; Girard, Richard Marcel Bruno Moreira; Ascenção, Carolline Fernanda Rodrigues; Fala, Angela Maria; Pauletti, Bianca Alves; Consonni, Sílvio Roberto; de Oliveira, Juliana Ferreira; Silva, Amanda Cristina Teixeira; Franchini, Kleber Gomes; Leme, Adriana Franco Paes; Silber, Ariel Mariano; Ciancaglini, Pietro; Moraes, Isabel; Dias, Sandra Martha Gomes; Ambrosio, Andre Luis Berteli

2018-02-22

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

Cyclin D1 Determines Mitochondrial Function In Vivo†

PubMed Central

Sakamaki, Toshiyuki; Casimiro, Mathew C.; Ju, Xiaoming; Quong, Andrew A.; Katiyar, Sanjay; Liu, Manran; Jiao, Xuanmao; Li, Anping; Zhang, Xueping; Lu, Yinan; Wang, Chenguang; Byers, Stephen; Nicholson, Robert; Link, Todd; Shemluck, Melvin; Yang, Jianguo; Fricke, Stanley T.; Novikoff, Phyllis M.; Papanikolaou, Alexandros; Arnold, Andrew; Albanese, Christopher; Pestell, Richard

2006-01-01

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function. PMID:16809779

CDK1 enhances mitochondrial bioenergetics for radiation-induced DNA repair

DOE PAGES

Qin, Lili; Fan, Ming; Candas, Demet; ...

2015-12-06

Nuclear DNA repair capacity is a critical determinant of cell fate under genotoxic stress conditions. DNA repair is a well-defined energy-consuming process. However, it is unclear how DNA repair is fueled and whether mitochondrial energy production contributes to nuclear DNA repair. Here, we report a dynamic enhancement of oxygen consumption and mitochondrial ATP generation in irradiated normal cells, paralleled with increased mitochondrial relocation of the cell-cycle kinase CDK1 and nuclear DNA repair. The basal and radiation-induced mitochondrial ATP generation is reduced significantly in cells harboring CDK1 phosphorylation-deficient mutant complex I subunits. Similarly, mitochondrial ATP generation and nuclear DNA repair aremore » also compromised severely in cells harboring mitochondrially targeted, kinase-deficient CDK1. These findings demonstrate a mechanism governing the communication between mitochondria and the nucleus by which CDK1 boosts mitochondrial bioenergetics to meet the increased cellular fuel demand for DNA repair and cell survival under genotoxic stress conditions.« less

The Complete Mitochondrial DNA Sequence of Scenedesmus obliquus Reflects an Intermediate Stage in the Evolution of the Green Algal Mitochondrial Genome

PubMed Central

Nedelcu, Aurora M.; Lee, Robert W.; Lemieux, Claude; Gray, Michael W.; Burger, Gertraud

2000-01-01

Two distinct mitochondrial genome types have been described among the green algal lineages investigated to date: a reduced–derived, Chlamydomonas-like type and an ancestral, Prototheca-like type. To determine if this unexpected dichotomy is real or is due to insufficient or biased sampling and to define trends in the evolution of the green algal mitochondrial genome, we sequenced and analyzed the mitochondrial DNA (mtDNA) of Scenedesmus obliquus. This genome is 42,919 bp in size and encodes 42 conserved genes (i.e., large and small subunit rRNA genes, 27 tRNA and 13 respiratory protein-coding genes), four additional free-standing open reading frames with no known homologs, and an intronic reading frame with endonuclease/maturase similarity. No 5S rRNA or ribosomal protein-coding genes have been identified in Scenedesmus mtDNA. The standard protein-coding genes feature a deviant genetic code characterized by the use of UAG (normally a stop codon) to specify leucine, and the unprecedented use of UCA (normally a serine codon) as a signal for termination of translation. The mitochondrial genome of Scenedesmus combines features of both green algal mitochondrial genome types: the presence of a more complex set of protein-coding and tRNA genes is shared with the ancestral type, whereas the lack of 5S rRNA and ribosomal protein-coding genes as well as the presence of fragmented and scrambled rRNA genes are shared with the reduced–derived type of mitochondrial genome organization. Furthermore, the gene content and the fragmentation pattern of the rRNA genes suggest that this genome represents an intermediate stage in the evolutionary process of mitochondrial genome streamlining in green algae. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF204057.] PMID:10854413

First genetic characterization of Fasciola hepatica in Argentina by nuclear and mitochondrial gene markers.

PubMed

Carnevale, Silvana; Malandrini, Jorge Bruno; Pantano, María Laura; Soria, Claudia Cecilia; Rodrigues-Silva, Rosângela; Machado-Silva, José Roberto; Velásquez, Jorge Néstor; Kamenetzky, Laura

2017-10-15

Fasciola hepatica is a trematode showing genetic variation among isolates from different regions of the world. The objective of this work was to characterize for the first time F. hepatica isolates circulating in different regions of Argentina. Twenty-two adult flukes were collected from naturally infected bovine livers in different areas from Argentina and used for DNA extraction. We carried out PCR amplification and sequence analysis of the ribosomal internal transcribed spacer 1 (ITS1), mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunits 4 and 5 (nad4 and nad5) and mitochondrial cytochrome c oxidase subunit I (cox1) genes as genetic markers. Phylogenies were reconstructed using maximum parsimony algorithm. A total of 6 haplotypes were found for cox1, 4 haplotypes for nad4 and 3 haplotypes for nad5. The sequenced ITS1 fragment was identical in all samples. The analyzed cox1 gene fragment is the most variable marker and is recommended for future analyses. No geographic association was found in the Argentinean samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants

PubMed Central

Pujol, Claire; Bailly, Marc; Kern, Daniel; Maréchal-Drouard, Laurence; Becker, Hubert; Duchêne, Anne-Marie

2008-01-01

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNAGln is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNAGln is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNAGln, which is the only tRNAGln present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNAGln by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNAGln formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts. PMID:18441100

A large deletion in the succinate dehydrogenase B gene (SDHB) in a Japanese patient with abdominal paraganglioma and concomitant metastasis.

PubMed

Kodama, Hitomi; Iihara, Masatoshi; Nissato, Sumiko; Isobe, Kazumasa; Kawakami, Yasushi; Okamoto, Takahiro; Takekoshi, Kazuhiro

2010-01-01

Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.

The Mitochondrial Cytochrome Oxidase Subunit I Gene Occurs on a Minichromosome with Extensive Heteroplasmy in Two Species of Chewing Lice, Geomydoecus aurei and Thomomydoecus minor

PubMed Central

Pietan, Lucas L.; Spradling, Theresa A.

2016-01-01

In animals, mitochondrial DNA (mtDNA) typically occurs as a single circular chromosome with 13 protein-coding genes and 22 tRNA genes. The various species of lice examined previously, however, have shown mitochondrial genome rearrangements with a range of chromosome sizes and numbers. Our research demonstrates that the mitochondrial genomes of two species of chewing lice found on pocket gophers, Geomydoecus aurei and Thomomydoecus minor, are fragmented with the 1,536 base-pair (bp) cytochrome-oxidase subunit I (cox1) gene occurring as the only protein-coding gene on a 1,916–1,964 bp minicircular chromosome in the two species, respectively. The cox1 gene of T. minor begins with an atypical start codon, while that of G. aurei does not. Components of the non-protein coding sequence of G. aurei and T. minor include a tRNA (isoleucine) gene, inverted repeat sequences consistent with origins of replication, and an additional non-coding region that is smaller than the non-coding sequence of other lice with such fragmented mitochondrial genomes. Sequences of cox1 minichromosome clones for each species reveal extensive length and sequence heteroplasmy in both coding and noncoding regions. The highly variable non-gene regions of G. aurei and T. minor have little sequence similarity with one another except for a 19-bp region of phylogenetically conserved sequence with unknown function. PMID:27589589

Leigh disease presenting in utero due to a novel missense mutation in the mitochondrial DNA-ND3.

PubMed

Leshinsky-Silver, Esther; Lev, Dorit; Malinger, Gustavo; Shapira, Daniel; Cohen, Sarit; Lerman-Sagie, Tally; Saada, Ann

2010-05-01

Leigh syndrome can be caused by defects in both nuclear and mitochondrial genes involved in energy metabolism. Recently, an increasing number of mutations in mitochondrial DNA encoding regions, especially in NADH dehydrogenase (respiratory chain complex I) subunits, have been reported as causative of early onset Leigh syndrome. We describe a patient whose fetal brain ultrasound demonstrated periventricular pseudocyst suggestive of a possible mitochondrial disorder who presented postnatally with Leigh syndrome. A muscle biopsy demonstrated a partial decrease in complex I and pyruvate dehydrogenase (PDH-E1 alpha) activity. Sequencing of the PDH-E1 alpha gene did not reveal any mutation. Sequencing of the mtDNA revealed a novel heteroplasmic G10254A (D66N) mutation in the ND3 gene. This change results in a substitution of aspartic acid to asparagine in a highly conserved domain of the ND3 subunit. The mutation could not be detected in the mother's blood or urine sediment. Blue native gel electrophoresis of muscle mitochondria revealed a normal size, albeit a decreased level of complex I. The G10254A substitution in the mtDNA-ND3 gene is another cause of maternally inherited Leigh syndrome. This case demonstrates that periventricular pseudocysts may be the initial in utero presentation in patients with mitochondrial disorders. We emphasize the importance of screening the mtDNA in pediatric patients as the first step in molecular diagnosis of Leigh syndrome. (c) 2010 Elsevier Inc. All rights reserved.

Intragenic inversion of mtDNA: a new type of pathogenic mutation in a patient with mitochondrial myopathy.

PubMed Central

Musumeci, O; Andreu, A L; Shanske, S; Bresolin, N; Comi, G P; Rothstein, R; Schon, E A; DiMauro, S

2000-01-01

We report an unusual molecular defect in the mitochondrially encoded ND1 subunit of NADH ubiquinone oxidoreductase (complex I) in a patient with mitochondrial myopathy and isolated complex I deficiency. The mutation is an inversion of seven nucleotides within the ND1 gene, which maintains the reading frame. The inversion, which alters three highly conserved amino acids in the polypeptide, was heteroplasmic in the patient's muscle but was not detectable in blood. This is the first report of a pathogenic inversion mutation in human mtDNA. PMID:10775530

Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high-intensity exercise training.

PubMed

Wyckelsma, Victoria L; Levinger, Itamar; McKenna, Michael J; Formosa, Luke E; Ryan, Michael T; Petersen, Aaron C; Anderson, Mitchell J; Murphy, Robyn M

2017-06-01

Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres. These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE). Following 12 weeks of high-intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres. We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria. Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High-intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin-2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70-100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

The HO-1/CO system regulates mitochondrial-capillary density relationships in human skeletal muscle.

PubMed

Pecorella, Shelly R H; Potter, Jennifer V F; Cherry, Anne D; Peacher, Dionne F; Welty-Wolf, Karen E; Moon, Richard E; Piantadosi, Claude A; Suliman, Hagir B

2015-10-15

The heme oxygenase-1 (HO-1)/carbon monoxide (CO) system induces mitochondrial biogenesis, but its biological impact in human skeletal muscle is uncertain. The enzyme system generates CO, which stimulates mitochondrial proliferation in normal muscle. Here we examined whether CO breathing can be used to produce a coordinated metabolic and vascular response in human skeletal muscle. In 19 healthy subjects, we performed vastus lateralis muscle biopsies and tested one-legged maximal O2 uptake (V̇o2max) before and after breathing air or CO (200 ppm) for 1 h daily for 5 days. In response to CO, there was robust HO-1 induction along with increased mRNA levels for nuclear-encoded mitochondrial transcription factor A (Tfam), cytochrome c, cytochrome oxidase subunit IV (COX IV), and mitochondrial-encoded COX I and NADH dehydrogenase subunit 1 (NDI). CO breathing did not increase V̇o2max (1.96 ± 0.51 pre-CO, 1.87 ± 0.50 post-CO l/min; P = not significant) but did increase muscle citrate synthase, mitochondrial density (139.0 ± 34.9 pre-CO, 219.0 ± 36.2 post-CO; no. of mitochondrial profiles/field), myoglobin content and glucose transporter (GLUT4) protein level and led to GLUT4 localization to the myocyte membrane, all consistent with expansion of the tissue O2 transport system. These responses were attended by increased cluster of differentiation 31 (CD31)-positive muscle capillaries (1.78 ± 0.16 pre-CO, 2.37 ± 0.59 post-CO; capillaries/muscle fiber), implying the enrichment of microvascular O2 reserve. The findings support that induction of the HO-1/CO system by CO not only improves muscle mitochondrial density, but regulates myoglobin content, GLUT4 localization, and capillarity in accordance with current concepts of skeletal muscle plasticity. Copyright © 2015 the American Physiological Society.

Plastid-Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae.

PubMed

Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

2016-06-27

Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid-nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Rescuing the Rescuer: On the Protein Complex between the Human Mitochondrial Acyl Carrier Protein and ISD11.

PubMed

Herrera, María Georgina; Pignataro, María Florencia; Noguera, Martín Ezequiel; Cruz, Karen Magalí; Santos, Javier

2018-05-16

Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier protein (ACP). We carried out the coexpression of human mitochondrial ACP and ISD11 in E. coli. This work shows that ACP and ISD11 form a soluble, structured, and stable complex able to bind to the human NFS1 subunit modulating its activity. Results suggest that ACP plays a key-role in ISD11 folding and stability in vitro. These findings offer the opportunity to study the mechanism of interaction between ISD11 and NFS1.

Systematic relationships among Lutzomyia sand flies (Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA sequences.

PubMed

Beati, Lorenza; Cáceres, Abraham G; Lee, Jamie A; Munstermann, Leonard E

2004-02-01

Lutzomyia spp. are New World phlebotomine sand flies, many of which are involved in the transmission of human diseases, such as leishmaniases and bartonellosis. The systematic classification of the approximately 400 species in the genus has been based on morphological characters, but the relationships within the genus are still very much in question. We have inferred phylogenies of 32 species of phlebotomine sand flies belonging to seven sub-genera and two species groups, by using fragments of the mitochondrial small subunit (12SrRNA) and of the nuclear large subunit (28SrRNA) ribosomal gene sequences. The subgenus Helcocyrtomyia and the Verrucarum species group, prominent representatives of the Peruvian sand fly fauna, were represented by 11 and 7 species, respectively. Although based on a limited number of taxa, the resulting phylogenies, based on 837 characters, provide an initial phylogenetic backbone for the progressive reconstruction of infrageneric relationships within Lutzomyia.

The mitochondrial genome of Hydra oligactis (Cnidaria, Hydrozoa) sheds new light on animal mtDNA evolution and cnidarian phylogeny.

PubMed

Kayal, Ehsan; Lavrov, Dennis V

2008-02-29

The 16,314-nuceotide sequence of the linear mitochondrial DNA (mtDNA) molecule of Hydra oligactis (Cnidaria, Hydrozoa)--the first from the class Hydrozoa--has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs, as is typical for cnidarians. All genes have the same transcriptional orientation and their arrangement in the genome is similar to that of the jellyfish Aurelia aurita. In addition, a partial copy of cox1 is present at one end of the molecule in a transcriptional orientation opposite to the rest of the genes, forming a part of inverted terminal repeat characteristic of linear mtDNA and linear mitochondrial plasmids. The sequence close to at least one end of the molecule contains several homonucleotide runs as well as small inverted repeats that are able to form strong secondary structures and may be involved in mtDNA maintenance and expression. Phylogenetic analysis of mitochondrial genes of H. oligactis and other cnidarians supports the Medusozoa hypothesis but also suggests that Anthozoa may be paraphyletic, with octocorallians more closely related to the Medusozoa than to the Hexacorallia. The latter inference implies that Anthozoa is paraphyletic and that the polyp (rather than a medusa) is the ancestral body type in Cnidaria.

Molecular identification of Echinococcus granulosus on the Tibetan Plateau using mitochondrial DNA markers.

PubMed

Hu, D; Song, X; Wang, N; Zhong, X; Wang, J; Liu, T; Jiang, Z; Dawa, T; Gu, X; Peng, X; Yang, G

2015-10-30

Cystic echinococcosis (CE) is an important worldwide zoonotic disease that causes large economic losses and human suffering. Echinococcus granulosus, the causative agent of CE, exhibits different genotypes in different locations. In order to identify its genotypes and analyze its genetic structure on the Tibetan Plateau, we collected 72 hydatid cysts from different intermediate hosts and amplified and sequenced their mitochondrial cytochrome c oxidase subunit 2 (cox2) genes. Seventy isolates were identified as the E. granulosus G1 genotype, while two isolates belonged to the G6 genotype. There were 18 haplotypes among the 70 E. granulosus isolates, which exhibited a star-like network pattern and shared a common haplotype (H1). There was little difference between geographical sub-populations. Our results suggest that a recent E. granulosus population expansion occurred on the Tibetan Plateau, suggesting that E. granulosus was introduced into China. This study increases the basic molecular data needed for the molecular diagnosis, epidemiology, prevention, and control of Echinococcus diseases.

The complete mitochondrial genome of Glaucidium brodiei (Strigiformes: Strigidae).

PubMed

Sun, Xiaonan; Zhou, Wenliang; Sun, Zhonglou; Qian, Lifu; Zhang, Yanan; Pan, Tao; Zhang, Baowei

2016-07-01

In this paper, the complete mitochondrial genome of Glaucidium brodiei is sequenced and reported for the first time. The mitochondrial genome is a circular molecule of 17,318 bp in length, consisting of 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and a control region. Overall base composition of the complete mitochondrial DNA is A (29.9%), G (14.1%), C (32.1%) and T (23.9%), the percentage of A and T (53.8%) is slightly higher than G and C (46.2%). All the genes in G. brodiei are distributed on the H-strand, except for the ND6 subunit gene and nine tRNA genes, which are encoded on the L-strand.

Reduced Mitochondrial Activity is Early and Steady in the Entorhinal Cortex but it is Mainly Unmodified in the Frontal Cortex in Alzheimer's Disease.

PubMed

Armand-Ugon, Mercedes; Ansoleaga, Belen; Berjaoui, Sara; Ferrer, Isidro

2017-01-01

It is well established that mitochondrial damage plays a role in the pathophysiology of Alzheimer's disease (AD). However, studies carried out in humans barely contemplate regional differences with disease progression. To study the expression of selected nuclear genes encoding subunits of the mitochondrial complexes and the activity of mitochondrial complexes in AD, in two regions: the entorhinal cortex (EC) and frontal cortex area 8 (FC). Frozen samples from 148 cases processed for gene expression by qRT-PCR and determination of individual activities of mitochondrial complexes I, II, IV and V using commercial kits and home-made assays. Decreased expression of NDUFA2, NDUFB3, UQCR11, COX7C, ATPD, ATP5L and ATP50, covering subunits of complex I, II, IV and V, occurs in total homogenates of the EC in AD stages V-VI when compared with stages I-II. However reduced activity of complexes I, II and V of isolated mitochondria occurs as early as stages I-II when compared with middle-aged individuals in the EC. In contrast, no alterations in the expression of the same genes and no alterations in the activity of mitochondrial complexes are found in the FC in the same series. Different mechanisms of impaired energy metabolism may occur in AD, one of them, represented by the EC, is the result of primary and early alteration of mitochondria; the other one is probably the result, at least in part, of decreased functional input and is represented by hypometabolism in the FC in AD patients aged 86 or younger. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

PubMed

Haïli, Nawel; Planchard, Noelya; Arnal, Nadège; Quadrado, Martine; Vrielynck, Nathalie; Dahan, Jennifer; des Francs-Small, Catherine Colas; Mireau, Hakim

2016-01-01

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria. © 2016 American Society of Plant Biologists. All Rights Reserved.

Elucidating the spread of the emerging canid nematode Angiostrongylus vasorum between Palaearctic and Nearctic ecozones.

PubMed

Jefferies, R; Shaw, S E; Willesen, J; Viney, M E; Morgan, E R

2010-05-01

Angiostrongylus vasorum is an emerging parasite that is currently distributed through Western Europe and parts of South America. An isolated population is also present in Newfoundland, Canada. This presents a risk of onward spread into North America, but its origin is unknown. To ascertain the phylogeographic relationships and genetic diversity of A. vasorum within the western Palaearctic and eastern Nearctic ecozones, a total of 143 adult and larval nematode specimens were collected from foxes (Vulpes vulpes) and dogs (Canis lupus familiaris) in Canada, Denmark, France, Germany, Ireland, the Netherlands, Portugal and the United Kingdom, and a coyote (Canis latrans) in Canada. DNA was extracted and the second internal transcribed spacer and two mitochondrial loci were amplified and sequenced. Multiple haplotypes (n=35) based on combined mitochondrial sequences (1078bp) of the partial cytochrome oxidase subunit I (COI), large subunit ribosomal RNA (rrnL) and the complete nicotinamide adenine dinucleotide dehydrogenase 3 (NADH3) sequences, were observed throughout the Palaearctic countries sampled; however, only a single haplotype was observed for the Canadian A. vasorum population. The likely origin of A. vasorum in Newfoundland is therefore inferred to be within the western Palaearctic. There was no evidence of genetic segregation of parasites in dogs, foxes and coyotes, supporting the hypothesis that transmission occurs between wild and domestic canids. The transmission dynamics and population structure of this nematode are further discussed. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.

PubMed

Skelly, P J; Clark-Walker, G D

1990-04-01

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms.

Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.

PubMed Central

Skelly, P J; Clark-Walker, G D

1990-01-01

Saccharomyces cerevisiae mitochondrial DNA deletion mutants have been used to examine whether base-biased intergenic regions of the genome influence mitochondrial biogenesis. One strain (delta 5.0) lacks a 5-kilobase (kb) segment extending from the proline tRNA gene to the small rRNA gene that includes ori1, while a second strain (delta 3.7) is missing a 3.7-kb region between the genes for ATPase subunit 6 and glutamic acid tRNA that encompasses ori7 plus ori2. Growth of these strains on both fermentable and nonfermentable substrates does not differ from growth of the wild-type strain, indicating that the deletable regions of the genome do not play a direct role in the expression of mitochondrial genes. Examination of whether the 5- or 3.7-kb regions influence mitochondrial DNA transmission was undertaken by crossing strains and examining mitochondrial genotypes in zygotic colonies. In a cross between strain delta 5.0, harboring three active ori elements (ori2, ori3, and ori5), and strain delta 3.7, containing only two active ori elements (ori3 and ori5), there is a preferential recovery of the genome containing two active ori elements (37% of progeny) over that containing three active elements (20%). This unexpected result, suggesting that active ori elements do not influence transmission of respiratory-competent genomes, is interpreted to reflect a preferential conversion of the delta 5.0 genome to the wild type (41% of progeny). Supporting evidence for conversion over biased transmission is shown by preferential recovery of a nonparental genome in the progeny of a heterozygous cross in which both parental molecules can be identified by size polymorphisms. Images PMID:2181277

Speciation in the large-spored Metschnikowia clade and establishment of a new species, Metschnikowia borealis comb. nov.

PubMed

Marinoni, Gaëlle; Lachance, Marc-André

2004-03-01

The reproductive boundaries among species in the large-spored Metschnikowia clade were studied by prototrophic recombinant selection, electrophoretic karyotyping, mitochondrial DNA restriction analysis, and DNA sequence analysis. Inviable ascospores arose from crosses between the two varieties of Metschnikowia continentalis, indicating that they should be recognized as separate species. Prototrophic recombinants were recovered from crosses between auxotrophic mutants of Metschnikowia borealis, M. continentalis, Metschnikowia lochheadii, Metschnikowia sp. UWO(PS)00-154.1, and Candida ipomoeae, showing that some genetic exchange is possible in spite of the sterility of the asci formed in interspecific crosses. Metschnikowia hawaiiensis, although capable of ascus formation when its h(-) mating type is crossed with the h(+) mating type of the other species, did not give rise to recombinants. In the other species, some recombinants acquired the ability to form asci directly from single cells. These often contained the chromosomes of both parents, suggesting formation of allodiploid hybrids. Other recombinants behaved as haploids and were similar to one parent except for having inherited the selectable wild-type allele from the other parent. In most, but not all cases, inheritance of the mitochondrial genome was uniparental and correlated with the inheritance of the nuclear chromosome complement. In some cases, what appeared to be a recombinant mitochondrial genome was observed. Phylogenies derived from the sequences of various DNA regions were not congruent, indicating that hybridization may have taken place in nature as the large-spored species diverged from their common ancestor. Further evidence that C. ipomoeae arose from a natural recombination event was obtained, but a pair of Metschnikowia species that might represent derived forms of the parents could not be identified conclusively. C. ipomoeae and most of its closely related Metschnikowia species contained a group-II intron in the mitochondrial small-subunit ribosomal gene. The intron was absent in M. borealis, M. hawaiiensis, and other species in the genus Metschnikowia.

Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida).

PubMed

Cermáková, Petra; Verner, Zdenek; Man, Petr; Lukes, Julius; Horváth, Anton

2007-06-01

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.

Phylogeny of Anophelinae (Diptera: Culicidae) Based on Nuclear Ribosomal and Mitochondrial DNA Sequences

DTIC Science & Technology

2002-01-01

numerous animal clades, including arthropods (Giribet & Ribera , 1998, 2000). The mitochondrial cytochrome oxidase subunits I and II have proven useful as...16S and 28S, D2 rRNA. Insect Molecular Biology, 6, 273-284. Giribet, G. & Ribera , C. (1998) The position of arthropods in animal kingdom: a search...for a reliable outgroup for internal arthropod phylogeny. Molecular Phylogenetics and Evolution, 9, 481-488. Giribet, G. & Ribera , C. (2000) A review

Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy.

PubMed

Chadderton, Naomi; Palfi, Arpad; Millington-Ward, Sophia; Gobbo, Oliverio; Overlack, Nora; Carrigan, Matthew; O'Reilly, Mary; Campbell, Matthew; Ehrhardt, Carsten; Wolfrum, Uwe; Humphries, Peter; Kenna, Paul F; Farrar, G Jane

2013-01-01

Leber hereditary optic neuropathy (LHON) is a mitochondrially inherited form of visual dysfunction caused by mutations in several genes encoding subunits of the mitochondrial respiratory NADH-ubiquinone oxidoreductase complex (complex I). Development of gene therapies for LHON has been impeded by genetic heterogeneity and the need to deliver therapies to the mitochondria of retinal ganglion cells (RGCs), the cells primarily affected in LHON. The therapy under development entails intraocular injection of a nuclear yeast gene NADH-quinone oxidoreductase (NDI1) that encodes a single subunit complex I equivalent and as such is mutation independent. NDI1 is imported into mitochondria due to an endogenous mitochondrial localisation signal. Intravitreal injection represents a clinically relevant route of delivery to RGCs not previously used for NDI1. In this study, recombinant adenoassociated virus (AAV) serotype 2 expressing NDI1 (AAV-NDI1) was shown to protect RGCs in a rotenone-induced murine model of LHON. AAV-NDI1 significantly reduced RGC death by 1.5-fold and optic nerve atrophy by 1.4-fold. This led to a significant preservation of retinal function as assessed by manganese enhanced magnetic resonance imaging and optokinetic responses. Intraocular injection of AAV-NDI1 overcomes many barriers previously associated with developing therapies for LHON and holds great therapeutic promise for a mitochondrial disorder for which there are no effective therapies.

Identification of Fasciola species based on mitochondrial and nuclear DNA reveals the co-existence of intermediate Fasciola and Fasciola gigantica in Thailand.

PubMed

Wannasan, Anchalee; Khositharattanakool, Pathamet; Chaiwong, Prasong; Piangjai, Somsak; Uparanukraw, Pichart; Morakote, Nimit

2014-11-01

Molecular techniques were used to identify Fasciola species collected from Chiang Mai Thailand. Morphometrically, 65 stained and 45 fresh worms collected from cattle suggested the possible occurrence of both F. gigantica and F. hepatica. Twenty-two worms comprising 15 from cattle and 7 from human patients, were identified subsequently based on three genetic markers: mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS2). All of them presented the F. gigantica type in maternally inherited mitochondrial sequences (nad1 and cox1), with six types in each sequence (FgNDI-CM1 to FgNDI-CM6 and FgCOI-CM1 to FgCOI-CM6, respectively). Remarkably, the predominant nad1 type, FgNDI-CM6, was identical to that of aspermic Fasciola sp. formerly reported from Thailand, Japan, Korea, China, Vietnam, and Myanmar. ITS2 sequences were analyzed successfully in 20 worms. Fifteen worms showed the F. gigantica type and five (including one worm from a patient) had mixed ITS2 sequences of both F. gigantica and F. hepatica in the same worms, with additional heterogeneity within both ITS2 types. This study revealed the intermediate form of Fasciola coexisting with F. gigantica for the first time in Thailand.

Reactive oxygen species mediates homocysteine-induced mitochondrial biogenesis in human endothelial cells: Modulation by antioxidants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-de-Arce, Karen; Departamento de Biologia Celular y Molecular, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago; Foncea, Rocio

2005-12-16

It has been proposed that homocysteine (Hcy)-induces endothelial dysfunction and atherosclerosis by generation of reactive oxygen species (ROS). A previous report has shown that Hcy promotes mitochondrial damage. Considering that oxidative stress can affect mitochondrial biogenesis, we hypothesized that Hcy-induced ROS in endothelial cells may lead to increased mitochondrial biogenesis. We found that Hcy-induced ROS (1.85-fold), leading to a NF-{kappa}B activation and increase the formation of 3-nitrotyrosine. Furthermore, expression of the mitochondrial biogenesis factors, nuclear respiratory factor-1 and mitochondrial transcription factor A, was significantly elevated in Hcy-treated cells. These changes were accompanied by increase in mitochondrial mass and higher mRNAmore » and protein expression of the subunit III of cytochrome c oxidase. These effects were significantly prevented by pretreatment with the antioxidants, catechin and trolox. Taken together, our results suggest that ROS is an important mediator of mitochondrial biogenesis induced by Hcy, and that modulation of oxidative stress by antioxidants may protect against the adverse vascular effects of Hcy.« less

Inhibition of oxidative metabolism leads to p53 genetic inactivation and transformation in neural stem cells

PubMed Central

Bartesaghi, Stefano; Graziano, Vincenzo; Galavotti, Sara; Henriquez, Nick V.; Betts, Joanne; Saxena, Jayeta; Minieri, Valentina; A, Deli; Karlsson, Anna; Martins, L. Miguel; Capasso, Melania; Nicotera, Pierluigi; Brandner, Sebastian; De Laurenzi, Vincenzo; Salomoni, Paolo

2015-01-01

Alterations of mitochondrial metabolism and genomic instability have been implicated in tumorigenesis in multiple tissues. High-grade glioma (HGG), one of the most lethal human neoplasms, displays genetic modifications of Krebs cycle components as well as electron transport chain (ETC) alterations. Furthermore, the p53 tumor suppressor, which has emerged as a key regulator of mitochondrial respiration at the expense of glycolysis, is genetically inactivated in a large proportion of HGG cases. Therefore, it is becoming evident that genetic modifications can affect cell metabolism in HGG; however, it is currently unclear whether mitochondrial metabolism alterations could vice versa promote genomic instability as a mechanism for neoplastic transformation. Here, we show that, in neural progenitor/stem cells (NPCs), which can act as HGG cell of origin, inhibition of mitochondrial metabolism leads to p53 genetic inactivation. Impairment of respiration via inhibition of complex I or decreased mitochondrial DNA copy number leads to p53 genetic loss and a glycolytic switch. p53 genetic inactivation in ETC-impaired neural stem cells is caused by increased reactive oxygen species and associated oxidative DNA damage. ETC-impaired cells display a marked growth advantage in the presence or absence of oncogenic RAS, and form undifferentiated tumors when transplanted into the mouse brain. Finally, p53 mutations correlated with alterations in ETC subunit composition and activity in primary glioma-initiating neural stem cells. Together, these findings provide previously unidentified insights into the relationship between mitochondria, genomic stability, and tumor suppressive control, with implications for our understanding of brain cancer pathogenesis. PMID:25583481

The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum retain functionally overlapping mitochondria from two evolutionarily distinct lineages

PubMed Central

Imanian, Behzad; Keeling, Patrick J

2007-01-01

Background The dinoflagellates Durinskia baltica and Kryptoperidinium foliaceum are distinguished by the presence of a tertiary plastid derived from a diatom endosymbiont. The diatom is fully integrated with the host cell cycle and is so altered in structure as to be difficult to recognize it as a diatom, and yet it retains a number of features normally lost in tertiary and secondary endosymbionts, most notably mitochondria. The dinoflagellate host is also reported to retain mitochondrion-like structures, making these cells unique in retaining two evolutionarily distinct mitochondria. This redundancy raises the question of whether the organelles share any functions in common or have distributed functions between them. Results We show that both host and endosymbiont mitochondrial genomes encode genes for electron transport proteins. We have characterized cytochrome c oxidase 1 (cox1), cytochrome oxidase 2 (cox2), cytochrome oxidase 3 (cox3), cytochrome b (cob), and large subunit of ribosomal RNA (LSUrRNA) of endosymbiont mitochondrial ancestry, and cox1 and cob of host mitochondrial ancestry. We show that all genes are transcribed and that those ascribed to the host mitochondrial genome are extensively edited at the RNA level, as expected for a dinoflagellate mitochondrion-encoded gene. We also found evidence for extensive recombination in the host mitochondrial genes and that recombination products are also transcribed, as expected for a dinoflagellate. Conclusion Durinskia baltica and K. foliaceum retain two mitochondria from evolutionarily distinct lineages, and the functions of these organelles are at least partially overlapping, since both express genes for proteins in electron transport. PMID:17892581

Low genetic diversity of Pneumocystis jirovecii among Cuban population based on two-locus mitochondrial typing.

PubMed

de Armas, Yaxsier; Friaza, Vicente; Capó, Virginia; Durand-Joly, Isabelle; Govín, Anamays; de la Horra, Carmen; Dei-Cas, Eduardo; Calderón, Enrique J

2012-05-01

Genotypes of two different loci of the Pneumocystis jirovecii mitochondrial gene were studied in specimens from a total of 75 Pneumocystis pneumonia patients in Spain, France and Cuba. A new genotype of the mitochondrial small subunit rRNA gene of P. jirovecii (160A/196T) was identified, which was revealed to be the most common in these three countries, especially in Cuba where its proportion reached 93.8%. Our data imply that the new genotype might be circulating worldwide and also suggests that the distribution of P. jirovecii genotypes could be narrower in islands such as Cuba.

Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

PubMed

Sato, Shigeto; Koike, Masato; Funayama, Manabu; Ezaki, Junji; Fukuda, Takahiro; Ueno, Takashi; Uchiyama, Yasuo; Hattori, Nobutaka

2016-12-01

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Genetic differentiation of the mitochondrial cytochrome oxidase C subunit I gene in genus Paramecium (Protista, Ciliophora).

PubMed

Zhao, Yan; Gentekaki, Eleni; Yi, Zhenzhen; Lin, Xiaofeng

2013-01-01

The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses. We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure. Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.

Genetic Differentiation of the Mitochondrial Cytochrome Oxidase c Subunit I Gene in Genus Paramecium (Protista, Ciliophora)

PubMed Central

Zhao, Yan; Gentekaki, Eleni; Yi, Zhenzhen; Lin, Xiaofeng

2013-01-01

Background The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses. Methodology/Principal findings We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure. Conclusions Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp. PMID:24204730

Molecular characterization of Taenia multiceps isolates from Gansu Province, China by sequencing of mitochondrial cytochrome C oxidase subunit 1.

PubMed

Li, Wen Hui; Jia, Wan Zhong; Qu, Zi Gang; Xie, Zhi Zhou; Luo, Jian Xun; Yin, Hong; Sun, Xiao Lin; Blaga, Radu; Fu, Bao Quan

2013-04-01

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species.

Molecular Characterization of Taenia multiceps Isolates from Gansu Province, China by Sequencing of Mitochondrial Cytochrome C Oxidase Subunit 1

PubMed Central

Li, Wen Hui; Jia, Wan Zhong; Qu, Zi Gang; Xie, Zhi Zhou; Luo, Jian Xun; Yin, Hong; Sun, Xiao Lin; Blaga, Radu

2013-01-01

A total of 16 Taenia multiceps isolates collected from naturally infected sheep or goats in Gansu Province, China were characterized by sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. The complete cox1 gene was amplified for individual T. multiceps isolates by PCR, ligated to pMD18T vector, and sequenced. Sequence analysis indicated that out of 16 T. multiceps isolates 10 unique cox1 gene sequences of 1,623 bp were obtained with sequence variation of 0.12-0.68%. The results showed that the cox1 gene sequences were highly conserved among the examined T. multiceps isolates. However, they were quite different from those of the other Taenia species. Phylogenetic analysis based on complete cox1 gene sequences revealed that T. multiceps isolates were composed of 3 genotypes and distinguished from the other Taenia species. PMID:23710087

Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

PubMed

Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep

2015-06-01

Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

Altered Mitochondria, Protein Synthesis Machinery, and Purine Metabolism Are Molecular Contributors to the Pathogenesis of Creutzfeldt-Jakob Disease.

PubMed

Ansoleaga, Belén; Garcia-Esparcia, Paula; Llorens, Franc; Hernández-Ortega, Karina; Carmona Tech, Margarita; Antonio Del Rio, José; Zerr, Inga; Ferrer, Isidro

2016-06-12

Neuron loss, synaptic decline, and spongiform change are the hallmarks of sporadic Creutzfeldt-Jakob disease (sCJD), and may be related to deficiencies in mitochondria, energy metabolism, and protein synthesis. To investigate these relationships, we determined the expression levels of genes encoding subunits of the 5 protein complexes of the electron transport chain, proteins involved in energy metabolism, nucleolar and ribosomal proteins, and enzymes of purine metabolism in frontal cortex samples from 15 cases of sCJD MM1 and age-matched controls. We also assessed the protein expression levels of subunits of the respiratory chain, initiation and elongation translation factors of protein synthesis, and localization of selected mitochondrial components. We identified marked, generalized alterations of mRNA and protein expression of most subunits of all 5 mitochondrial respiratory chain complexes in sCJD cases. Expression of molecules involved in protein synthesis and purine metabolism were also altered in sCJD. These findings point to altered mRNA and protein expression of components of mitochondria, protein synthesis machinery, and purine metabolism as components of the pathogenesis of CJD. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Mitochondrial DNA Variant in COX1 Subunit Significantly Alters Energy Metabolism of Geographically Divergent Wild Isolates in Caenorhabditis elegans

PubMed Central

Dingley, Stephen D.; Polyak, Erzsebet; Ostrovsky, Julian; Srinivasan, Satish; Lee, Icksoo; Rosenfeld, Amy B.; Tsukikawa, Mai; Xiao, Rui; Selak, Mary A.; Coon, Joshua J.; Hebert, Alexander S.; Grimsrud, Paul A.; Kwon, Young Joon; Pagliarini, David J.; Gai, Xiaowu; Schurr, Theodore G.; Hüttemann, Maik; Nakamaru-Ogiso, Eiko; Falk, Marni J.

2014-01-01

Mitochondrial DNA (mtDNA) sequence variation can influence the penetrance of complex diseases and climatic adaptation. While studies in geographically defined human populations suggest that mtDNA mutations become fixed when they have conferred metabolic capabilities optimally suited for a specific environment, it has been challenging to definitively assign adaptive functions to specific mtDNA sequence variants in mammals. We investigated whether mtDNA genome variation functionally influences Caenorhabditis elegans wild isolates of distinct mtDNA lineages and geographic origins. We found that, relative to N2 (England) wild-type nematodes, CB4856 wild isolates from a warmer native climate (Hawaii) had a unique p.A12S amino acid substitution in the mtDNA-encoded COX1 core catalytic subunit of mitochondrial complex IV (CIV). Relative to N2, CB4856 worms grown at 20 °C had significantly increased CIV enzyme activity, mitochondrial matrix oxidant burden, and sensitivity to oxidative stress but had significantly reduced lifespan and mitochondrial membrane potential. Interestingly, mitochondrial membrane potential was significantly increased in CB4856 grown at its native temperature of 25 °C. A transmitochondrial cybrid worm strain, chpIR (M, CB4856 > N2), was bred as homoplasmic for the CB4856 mtDNA genome in the N2 nuclear background. The cybrid strain also displayed significantly increased CIV activity, demonstrating that this difference results from the mtDNA-encoded p.A12S variant. However, chpIR (M, CB4856 > N2) worms had significantly reduced median and maximal lifespan relative to CB4856, which may relate to their nuclear– mtDNA genome mismatch. Overall, these data suggest that C. elegans wild isolates of varying geographic origins may adapt to environmental challenges through mtDNA variation to modulate critical aspects of mitochondrial energy metabolism. PMID:24534730

A protocol for isolating insect mitochondrial genomes: a case study of NUMT in Melipona flavolineata (Hymenoptera: Apidae).

PubMed

Françoso, Elaine; Gomes, Fernando; Arias, Maria Cristina

2016-07-01

Nuclear mitochondrial DNA insertions (NUMTs) are mitochondrial DNA sequences that have been transferred into the nucleus and are recognized by the presence of indels and stop codons. Although NUMTs have been identified in a diverse range of species, their discovery was frequently accidental. Here, our initial goal was to develop and standardize a simple method for isolating NUMTs from the nuclear genome of a single bee. Subsequently, we tested our new protocol by determining whether the indels and stop codons of the cytochrome c oxidase subunit I (COI) sequence of Melipona flavolineata are of nuclear origin. The new protocol successfully demonstrated the presence of a COI NUMT. In addition to NUMT investigations, the protocol described here will also be very useful for studying mitochondrial mutations related to diseases and for sequencing complete mitochondrial genomes with high read coverage by Next-Generation technology.

mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome.

PubMed

Johnson, Simon C; Yanos, Melana E; Kayser, Ernst-Bernhard; Quintana, Albert; Sangesland, Maya; Castanza, Anthony; Uhde, Lauren; Hui, Jessica; Wall, Valerie Z; Gagnidze, Arni; Oh, Kelly; Wasko, Brian M; Ramos, Fresnida J; Palmiter, Richard D; Rabinovitch, Peter S; Morgan, Philip G; Sedensky, Margaret M; Kaeberlein, Matt

2013-12-20

Mitochondrial dysfunction contributes to numerous health problems, including neurological and muscular degeneration, cardiomyopathies, cancer, diabetes, and pathologies of aging. Severe mitochondrial defects can result in childhood disorders such as Leigh syndrome, for which there are no effective therapies. We found that rapamycin, a specific inhibitor of the mechanistic target of rapamycin (mTOR) signaling pathway, robustly enhances survival and attenuates disease progression in a mouse model of Leigh syndrome. Administration of rapamycin to these mice, which are deficient in the mitochondrial respiratory chain subunit Ndufs4 [NADH dehydrogenase (ubiquinone) Fe-S protein 4], delays onset of neurological symptoms, reduces neuroinflammation, and prevents brain lesions. Although the precise mechanism of rescue remains to be determined, rapamycin induces a metabolic shift toward amino acid catabolism and away from glycolysis, alleviating the buildup of glycolytic intermediates. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.

Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells.

PubMed

Santo-Domingo, Jaime; Chareyron, Isabelle; Broenimann, Charlotte; Lassueur, Steve; Wiederkehr, Andreas

2017-08-15

Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed, respiration, mitonuclear protein imbalance or down-stream signaling may be altered. Copyright © 2017 Elsevier Inc. All rights reserved.

Population Structure of Phytophthora nicotianae Reveals Host-Specific Lineages on Brinjal, Ridge Gourd, and Tomato in South India.

PubMed

Chowdappa, P; Kumar, B J Nirmal; Kumar, S P Mohan; Madhura, S; Bhargavi, B Reddi; Lakshmi, M Jyothi

2016-12-01

Severe outbreaks of Phytophthora fruit rot on brinjal, ridge gourd, and tomato have been observed since 2011 in Andhra Pradesh, Karnataka, Telangana, and Tamil Nadu states of India. Therefore, 76 Phytophthora nicotianae isolates, recovered from brinjal (17), ridge gourd (40), and tomato (19) from different localities in these states during the June to December cropping season of 2012 and 2013, were characterized based on phenotypic and genotypic analyses and aggressiveness on brinjal, tomato, and ridge gourd. All brinjal and ridge gourd isolates were A2, while tomato isolates were both A1 (13) and A2 (6). All isolates were metalaxyl sensitive. In addition, isolates were genotyped for three mitochondrial (ribosomal protein L5-small subunit ribosomal RNA [rpl5-rns], small subunit ribosomal RNA-cytochrome c oxidase subunit 2 [rns-cox2], and cox2+spacer) and three nuclear loci (hypothetical protein [hyp], scp-like extracellular protein [scp], and beta-tubulin [β-tub]). All regions were polymorphic but nuclear regions were more variable than mitochondrial regions. The network analysis of genotypes using the combined dataset of three nuclear regions revealed a host-specific association. However, the network generated using mitochondrial regions limited such host-specific groupings only to brinjal isolates. P. nicotianae isolates were highly aggressive and produced significantly (P ≤ 0.01) larger lesions on their respective host of origin than on other hosts. The results indicate significant genetic variation in the population of P. nicotianae, leading to identification of host-specific lineages responsible for severe outbreaks on brinjal, ridge gourd, and tomato.

Light induces translocation of NF-κB p65 to the mitochondria and suppresses expression of cytochrome c oxidase subunit III (COX III) in the rat retina

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomita, Hiroshi, E-mail: htomita@iwate-u.ac.jp; Soft-Path Engineering Research Center; Clinical Research, Innovation and Education Center, Tohoku University Hospital, 1-1 Seiryo, Aoba, Sendai, Miyagi 980-8574

2016-05-13

The transcription factor nuclear factor kappaB (NF-κB) plays various roles in cell survival, apoptosis, and inflammation. In the rat retina, NF-κB activity increases after exposure to damaging light, resulting in degeneration of photoreceptors. Here, we report that in dark-adapted rats exposed for 6 h to bright white light, the p65 subunit of retinal NF-κB translocates to the mitochondria, an event associated with a decrease in expression of cytochrome c oxidase subunit III (COX III). However, sustained exposure for 12 h depleted p65 from the mitochondria, and enhanced COX III expression. Treatment with the protective antioxidant PBN prior to light exposure prevents p65more » depletion in the mitochondria and COX III upregulation during prolonged exposure, and apoptosis in photoreceptor cells. These results indicate that COX III expression is sensitive to the abundance of NF-κB p65 in the mitochondria, which, in turn, is affected by exposure to damaging light. - Highlights: • Damaging light exposure of the retina induces NF-κB p65 mitochondrial translocation. • NF-κB p65 mitochondrial translocation is associated with the decrease of COX III expression. • Prolonged light exposure depletes mitochondrial p65 resulting in the increase in COX III expression. • NF-κB p65 and COX III expression play an important role in the light-induced photoreceptor degeneration.« less

Mechanism of protein biosynthesis in mammalian mitochondria.

PubMed

Christian, Brooke E; Spremulli, Linda L

2012-01-01

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. Copyright © 2011 Elsevier B.V. All rights reserved.

Biomarker Signatures of Mitochondrial NDUFS3 in Invasive Breast Carcinoma

PubMed Central

Suhane, Sonal; Berel, Dror; Ramanujan, V Krishnan

2011-01-01

We present evidence for potential biomarker utility of a mitochondrial complex I subunit, (NDUFS3) in discriminating normal and highly invasive breast carcinoma specimens obtained from clinical patients. Besides being a robust indicator of breast cancer aggressiveness, NDUFS3 also shows clear signatures of a hypoxia/necrosis marker in invasive ductal carcinoma specimens. Statistically significant positive correlation was observed between nuclear grade and NDUFS3 expression level in the tumor specimens analyzed. We support these findings with a plausible mechanism involving mitochondrial complex I assembly defects and/or redox buffering induced mitochondrial dysfunction during the process of cancer cell transformation. From a clinical standpoint, this novel observation adds value in augmenting the current receptor-based biomarkers for better accuracy in diagnosis and predicting survival rate in patients with breast carcinoma. PMID:21867691

Expression and function of Kv7.4 channels in rat cardiac mitochondria: possible targets for cardioprotection.

PubMed

Testai, Lara; Barrese, Vincenzo; Soldovieri, Maria Virginia; Ambrosino, Paolo; Martelli, Alma; Vinciguerra, Iolanda; Miceli, Francesco; Greenwood, Iain Andrew; Curtis, Michael John; Breschi, Maria Cristina; Sisalli, Maria Josè; Scorziello, Antonella; Canduela, Miren Josune; Grandes, Pedro; Calderone, Vincenzo; Taglialatela, Maurizio

2016-05-01

Plasmalemmal Kv7.1 (KCNQ1) channels are critical players in cardiac excitability; however, little is known on the functional role of additional Kv7 family members (Kv7.2-5) in cardiac cells. In this work, the expression, function, cellular and subcellular localization, and potential cardioprotective role against anoxic-ischaemic cardiac injury of Kv7.4 channels have been investigated. Expression of Kv7.1 and Kv7.4 transcripts was found in rat heart tissue by quantitative polymerase chain reaction. Western blots detected Kv7.4 subunits in mitochondria from Kv7.4-transfected cells, H9c2 cardiomyoblasts, freshly isolated adult cardiomyocytes, and whole hearts. Immunofluorescence experiments revealed that Kv7.4 subunits co-localized with mitochondrial markers in cardiac cells, with ∼ 30-40% of cardiac mitochondria being labelled by Kv7.4 antibodies, a result also confirmed by immunogold electron microscopy experiments. In isolated cardiac (but not liver) mitochondria, retigabine (1-30 µM) and flupirtine (30 µM), two selective Kv7 activators, increased Tl(+) influx, depolarized the membrane potential, and inhibited calcium uptake; all these effects were antagonized by the Kv7 blocker XE991. In intact H9c2 cells, reducing Kv7.4 expression by RNA interference blunted retigabine-induced mitochondrial membrane depolarization; in these cells, retigabine decreased mitochondrial Ca(2+) levels and increased radical oxygen species production, both effects prevented by XE991. Finally, retigabine reduced cellular damage in H9c2 cells exposed to anoxia/re-oxygenation and largely prevented the functional and morphological changes triggered by global ischaemia/reperfusion (I/R) in Langendorff-perfused rat hearts. Kv7.4 channels are present and functional in cardiac mitochondria; their activation exerts a significant cardioprotective role, making them potential therapeutic targets against I/R-induced cardiac injury. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

PubMed

Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

2012-01-01

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis.

PubMed

Cugno, Graziano; Parreira, José R; Ferlizza, Enea; Hernández-Castellano, Lorenzo E; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan; Campos, Alexandre M O; Almeida, André M

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer's incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15-20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.

The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis

PubMed Central

Cugno, Graziano; Parreira, José R.; Ferlizza, Enea; Hernández-Castellano, Lorenzo E.; Carneiro, Mariana; Renaut, Jenny; Castro, Noemí; Arguello, Anastasio; Capote, Juan

2016-01-01

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss. PMID:27031334

Chaperones of F[subscript 1]-ATPase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ludlam, Anthony; Brunzelle, Joseph; Pribyl, Thomas

2009-09-25

Mitochondrial F{sub 1}-ATPase contains a hexamer of alternating {alpha} and {beta} subunits. The assembly of this structure requires two specialized chaperones, Atp11p and Atp12p, that bind transiently to {beta} and {alpha}. In the absence of Atp11p and Atp12p, the hexamer is not formed, and {alpha} and {beta} precipitate as large insoluble aggregates. An early model for the mechanism of chaperone-mediated F{sub 1} assembly (Wang, Z. G., Sheluho, D., Gatti, D. L., and Ackerman, S. H. (2000) EMBO J. 19, 1486--1493) hypothesized that the chaperones themselves look very much like the {alpha} and {beta} subunits, and proposed an exchange of Atp11pmore » for {alpha} and of Atp12p for {beta}; the driving force for the exchange was expected to be a higher affinity of {alpha} and {beta} for each other than for the respective chaperone partners. One important feature of this model was the prediction that as long as Atp11p is bound to {beta} and Atp12p is bound to {alpha}, the two F{sub 1} subunits cannot interact at either the catalytic site or the noncatalytic site interface. Here we present the structures of Atp11p from Candida glabrata and Atp12p from Paracoccus denitrificans, and we show that some features of the Wang model are correct, namely that binding of the chaperones to {alpha} and {beta} prevents further interactions between these F1 subunits. However, Atp11p and Atp12p do not resemble {alpha} or {beta}, and it is instead the F{sub 1} {gamma} subunit that initiates the release of the chaperones from {alpha} and {beta} and their further assembly into the mature complex.« less

The Mitochondrial Genomes of the Zoonotic Canine Filarial Parasites Dirofilaria (Nochtiella) repens and Candidatus Dirofilaria (Nochtiella) Honkongensis Provide Evidence for Presence of Cryptic Species

PubMed Central

Yilmaz, Esra; Fritzenwanker, Moritz; Pantchev, Nikola; Lendner, Mathias; Wongkamchai, Sirichit; Otranto, Domenico; Kroidl, Inge; Dennebaum, Martin; Le, Thanh Hoa; Anh Le, Tran; Ramünke, Sabrina; Schaper, Roland; von Samson-Himmelstjerna, Georg; Poppert, Sven; Krücken, Jürgen

2016-01-01

Background Cutaneous dirofilariosis is a canine mosquito-borne zoonosis that can cause larva migrans disease in humans. Dirofilaria repens is considered an emerging pathogen occurring with high prevalence in Mediterranean areas and many parts of tropical Asia. In Hong Kong, a second species, Candidatus Dirofilaria hongkongensis, has been reported. The present study aimed to compare mitochondrial genomes from these parasites and to obtain population genetic information. Methods and Findings Complete mitochondrial genomes were obtained by PCR and Sanger sequencing or ILLUMINA sequencing for four worms. Cytochrome oxidase subunit 1 sequences identified three as D. repens (all from Europe) and one as C. D. hongkongensis (from India). Mitochondrial genomes have the same organization as in other spirurid nematodes but a higher preference for thymine in the coding strand. Phylogenetic analysis was in contradiction to current taxonomy of the Onchocercidae but in agreement with a recent multi-locus phylogenetic analysis using both mitochondrial and nuclear markers. D. repens and C. D. hongkongensis sequences clustered together and were the common sister group to Dirofilaria immitis. Analysis of a 2.5 kb mitochondrial genome fragment from macrofilaria or canine blood samples from Europe (42), Thailand (2), India (1) and Vietnam (1) revealed only small genetic differences in the D. repens samples including all European and the Vietnam sample. The Indian C. D. hongkongensis and the two Thai samples formed separate clusters and differences were comparatively large. Conclusion Genetic differences between Dirofilaria spp. causing cutaneous disease can be considerable whereas D. repens itself was genetically quite homogenous. C. D. hongkongensis was identified for the first time from the Indian subcontinent. The full mitochondrial genome sequence strengthens the hypothesis that it represents an independent species and the Thai samples might represent another cryptic species, Candidatus Dirofilaria sp. ‘Thailand II’, or a quite divergent population of C. D. hongkongensis. PMID:27727270

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

PubMed Central

Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

2010-01-01

Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane. PMID:20056103

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

PubMed

Sommer, Natascha; Hüttemann, Maik; Pak, Oleg; Scheibe, Susan; Knoepp, Fenja; Sinkler, Christopher; Malczyk, Monika; Gierhardt, Mareike; Esfandiary, Azadeh; Kraut, Simone; Jonas, Felix; Veith, Christine; Aras, Siddhesh; Sydykov, Akylbek; Alebrahimdehkordi, Nasim; Giehl, Klaudia; Hecker, Matthias; Brandes, Ralf P; Seeger, Werner; Grimminger, Friedrich; Ghofrani, Hossein A; Schermuly, Ralph T; Grossman, Lawrence I; Weissmann, Norbert

2017-08-04

Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. Isolated ventilated and perfused lungs from Cox4i2 -/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2 -/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2 -/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2 -/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2 -/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen-sensing processes in the lung and possibly also in other organs. © 2017 American Heart Association, Inc.

Composition of the mitochondrial electron transport chain in acanthamoeba castellanii: structural and evolutionary insights.

PubMed

Gawryluk, Ryan M R; Chisholm, Kenneth A; Pinto, Devanand M; Gray, Michael W

2012-11-01

The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of 'supernumerary' subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits. © 2012 Elsevier B.V. All rights reserved.

Mechanism of Protein Biosynthesis in Mammalian Mitochondria

PubMed Central

Christian, Brooke E.; Spremulli, Linda L.

2011-01-01

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. PMID:22172991

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome.

PubMed

Schäfer, B; Merlos-Lange, A M; Anderl, C; Welser, F; Zimmer, M; Wolf, K

1991-01-01

In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain anar-14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.

Crystallization of Mitochondrial Respiratory Complex II from Chicken Heart: a Membrane Protein Complex Diffracting to 2.0 Å.

PubMed Central

Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward

2006-01-01

Synopsis A multi-subunit mitochondrial membrane protein complex involved in the Krebs Cycle and respiratory chain has been crystallized in a form suitable for near-atomic resolution structure determination. A procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Å with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites. PMID:15805592

ATP Synthase Diseases of Mitochondrial Genetic Origin

PubMed Central

Dautant, Alain; Meier, Thomas; Hahn, Alexander; Tribouillard-Tanvier, Déborah; di Rago, Jean-Paul; Kucharczyk, Roza

2018-01-01

Devastating human neuromuscular disorders have been associated to defects in the ATP synthase. This enzyme is found in the inner mitochondrial membrane and catalyzes the last step in oxidative phosphorylation, which provides aerobic eukaryotes with ATP. With the advent of structures of complete ATP synthases, and the availability of genetically approachable systems such as the yeast Saccharomyces cerevisiae, we can begin to understand these molecular machines and their associated defects at the molecular level. In this review, we describe what is known about the clinical syndromes induced by 58 different mutations found in the mitochondrial genes encoding membrane subunits 8 and a of ATP synthase, and evaluate their functional consequences with respect to recently described cryo-EM structures. PMID:29670542

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria

PubMed Central

Karicheva, Olga Z.; Kolesnikova, Olga A.; Schirtz, Tom; Vysokikh, Mikhail Y.; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A.; Martin, Robert P.; Entelis, Nina; Tarassov, Ivan

2011-01-01

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNALeu(UUR). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNALeu(UUR) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders. PMID:21724600

Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria.

PubMed

Karicheva, Olga Z; Kolesnikova, Olga A; Schirtz, Tom; Vysokikh, Mikhail Y; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A; Martin, Robert P; Entelis, Nina; Tarassov, Ivan

2011-10-01

Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.

Mitochondrial functions modulate neuroendocrine, metabolic, inflammatory, and transcriptional responses to acute psychological stress

PubMed Central

Picard, Martin; McManus, Meagan J.; Gray, Jason D.; Nasca, Carla; Moffat, Cynthia; Kopinski, Piotr K.; Seifert, Erin L.; McEwen, Bruce S.; Wallace, Douglas C.

2015-01-01

The experience of psychological stress triggers neuroendocrine, inflammatory, metabolic, and transcriptional perturbations that ultimately predispose to disease. However, the subcellular determinants of this integrated, multisystemic stress response have not been defined. Central to stress adaptation is cellular energetics, involving mitochondrial energy production and oxidative stress. We therefore hypothesized that abnormal mitochondrial functions would differentially modulate the organism’s multisystemic response to psychological stress. By mutating or deleting mitochondrial genes encoded in the mtDNA [NADH dehydrogenase 6 (ND6) and cytochrome c oxidase subunit I (COI)] or nuclear DNA [adenine nucleotide translocator 1 (ANT1) and nicotinamide nucleotide transhydrogenase (NNT)], we selectively impaired mitochondrial respiratory chain function, energy exchange, and mitochondrial redox balance in mice. The resulting impact on physiological reactivity and recovery from restraint stress were then characterized. We show that mitochondrial dysfunctions altered the hypothalamic–pituitary–adrenal axis, sympathetic adrenal–medullary activation and catecholamine levels, the inflammatory cytokine IL-6, circulating metabolites, and hippocampal gene expression responses to stress. Each mitochondrial defect generated a distinct whole-body stress-response signature. These results demonstrate the role of mitochondrial energetics and redox balance as modulators of key pathophysiological perturbations previously linked to disease. This work establishes mitochondria as stress-response modulators, with implications for understanding the mechanisms of stress pathophysiology and mitochondrial diseases. PMID:26627253

Targeted transgenic overexpression of mitochondrial thymidine kinase (TK2) alters mitochondrial DNA (mtDNA) and mitochondrial polypeptide abundance: transgenic TK2, mtDNA, and antiretrovirals.

PubMed

Hosseini, Seyed H; Kohler, James J; Haase, Chad P; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-03-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-gamma. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity.

Targeted Transgenic Overexpression of Mitochondrial Thymidine Kinase (TK2) Alters Mitochondrial DNA (mtDNA) and Mitochondrial Polypeptide Abundance

PubMed Central

Hosseini, Seyed H.; Kohler, James J.; Haase, Chad P.; Tioleco, Nina; Stuart, Tami; Keebaugh, Erin; Ludaway, Tomika; Russ, Rodney; Green, Elgin; Long, Robert; Wang, Liya; Eriksson, Staffan; Lewis, William

2007-01-01

Mitochondrial toxicity limits nucleoside reverse transcriptase inhibitors (NRTIs) for acquired immune deficiency syndrome. NRTI triphosphates, the active moieties, inhibit human immunodeficiency virus reverse transcriptase and eukaryotic mitochondrial DNA polymerase pol-γ. NRTI phosphorylation seems to correlate with mitochondrial toxicity, but experimental evidence is lacking. Transgenic mice (TGs) with cardiac overexpression of thymidine kinase isoforms (mitochondrial TK2 and cytoplasmic TK1) were used to study NRTI mitochondrial toxicity. Echocardiography and nuclear magnetic resonance imaging defined cardiac performance and structure. TK gene copy and enzyme activity, mitochondrial (mt) DNA and polypeptide abundance, succinate dehydrogenase and cytochrome oxidase histochemistry, and electron microscopy correlated with transgenesis, mitochondrial structure, and biogenesis. Antiretroviral combinations simulated therapy. Untreated hTK1 or TK2 TGs exhibited normal left ventricle mass. In TK2 TGs, cardiac TK2 gene copy doubled, activity increased 300-fold, and mtDNA abundance doubled. Abundance of the 17-kd subunit of complex I, succinate dehydrogenase histochemical activity, and cristae density increased. NRTIs increased left ventricle mass 20% in TK2 TGs. TK activity increased 3 logs in hTK1 TGs, but no cardiac phenotype resulted. NRTIs abrogated functional effects of transgenically increased TK2 activity but had no effect on TK2 mtDNA abundance. Thus, NRTI mitochondrial phosphorylation by TK2 is integral to clinical NRTI mitochondrial toxicity. PMID:17322372

Unique presentation of LHON/MELAS overlap syndrome caused by m.13046T>C in MTND5.

PubMed

Kolarova, Hana; Liskova, Petra; Tesarova, Marketa; Kucerova Vidrova, Vendula; Forgac, Martin; Zamecnik, Josef; Hansikova, Hana; Honzik, Tomas

2016-12-01

Leber hereditary optic neuropathy (LHON) and mitochondrial encephalopathy, myopathy, lactic acidosis and stroke-like episodes (MELAS) syndromes are mitochondrially inherited disorders characterized by acute visual failure and variable multiorgan system presentation, respectively. A 12-year-old girl with otherwise unremarkable medical history presented with abrupt, painless loss of vision. Over the next few months, she developed moderate sensorineural hearing loss, vertigo, migraines, anhedonia and thyroiditis. Ocular examination confirmed bilateral optic nerve atrophy. Metabolic workup documented elevated cerebrospinal fluid lactate. Initial genetic analyses excluded the three most common LHON mutations. Subsequently, Sanger sequencing of the entire mitochondrial DNA (mtDNA) genome was performed. Whole mtDNA sequencing revealed a pathogenic heteroplasmic mutation m.13046T>C in MTND5 encoding the ND5 subunit of complex I. This particular variant has previously been described in a single case report of MELAS/Leigh syndrome (subacute necrotizing encephalopathy). Based on the constellation of clinical symptoms in our patient, we diagnose the condition as LHON/MELAS overlap syndrome. We describe a unique presentation of LHON/MELAS overlap syndrome resulting from a m.13046T>C mutation in a 12-year-old girl. In patients with sudden vision loss in which three of the most prevalent LHON mitochondrial mutations have been ruled out, molecular genetic examination should be extended to other mtDNA-encoded subunits of MTND5 complex I. Furthermore, atypical clinical presentations must be considered, even in well-described phenotypes.

Absence of Complex I Is Associated with Diminished Respiratory Chain Function in European Mistletoe.

PubMed

Maclean, Andrew E; Hertle, Alexander P; Ligas, Joanna; Bock, Ralph; Balk, Janneke; Meyer, Etienne H

2018-05-21

Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genes not seen in any other multicellular eukaryotes. In particular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of the respiratory complexes and mitochondrial functions. Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I. Copyright © 2018 Elsevier Ltd. All rights reserved.

Isolated cytochrome c oxidase deficiency in G93A SOD1 mice overexpressing CCS protein.

PubMed

Son, Marjatta; Leary, Scot C; Romain, Nadine; Pierrel, Fabien; Winge, Dennis R; Haller, Ronald G; Elliott, Jeffrey L

2008-05-02

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Tom7 modulates the dynamics of the mitochondrial outer membrane translocase and plays a pathway-related role in protein import.

PubMed Central

Hönlinger, A; Bömer, U; Alconada, A; Eckerskorn, C; Lottspeich, F; Dietmeier, K; Pfanner, N

1996-01-01

The preprotein translocase of the outer mitochondrial membrane is a multi-subunit complex with receptors and a general import pore. We report the molecular identification of Tom7, a small subunit of the translocase that behaves as an integral membrane protein. The deletion of TOM7 inhibited the mitochondrial import of the outer membrane protein porin, whereas the import of preproteins destined for the mitochondrial interior was impaired only slightly. However, protein import into the mitochondrial interior was strongly inhibited when it occurred in two steps: preprotein accumulation at the outer membrane in the absence of a membrane potential and subsequent further import after the re-establishment of a membrane potential. The delay of protein import into tom7delta mitochondria seemed to occur after the binding of preproteins to the outer membrane receptor sites. A lack of Tom7 stabilized the interaction between the receptors Tom20 and Tom22 and the import pore component Tom40. This indicated that Tom7 exerts a destabilizing effect on part of the outer membrane translocase, whereas Tom6 stabilizes the interaction between the receptors and the import pore. Synthetic growth defects of the double mutants tom7delta tom20delta and tom7delta tom6delta provided genetic evidence for the functional relationship of Tom7 with Tom20 and Tom6. These results suggest that (i) Tom7 plays a role in sorting and accumulation of the preproteins at the outer membrane, and (ii) Tom7 and Tom6 perform complementary functions in modulating the dynamics of the outer membrane translocase. Images PMID:8641278

Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.

PubMed

Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T; Bailowitz, Zachary; De Filippis, Elena A; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J

2010-10-01

The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. NADH- and FADH(2)-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-DL-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

Mitochondrial ATPase Subunit 6 and Cytochrome B Gene Variations in Obese Turkish Children

PubMed Central

Demir, Durkadın; Türkkahraman, Doğa; Samur, Anıl Aktaş; Lüleci, Güven; Akçurin, Sema; M. Alper, Özgül

2014-01-01

Objective: Due to the importance of energy metabolism in mitochondria, mitochondrial genome variations are evaluated in energy-related diseases such as obesity. To date, several nuclear genes were found to be related to obesity. Our aim in this study was to investigate the presence of polymorphisms in mitochondrial ATPase subunit 6 (mt-ATP6) and cytochrome b (mt-CytB) genes that may be associated with childhood obesity. Methods: The mt-ATP6 and mt-CytB genes were amplified and entirely sequenced in a series of 100 obese and in an equal number of healthy Turkish children aged between 6-14 years. Results: A total of 118 synonymous and nonsynonymous variations were detected in the obese and control groups. Only two previously reported synonymous substitutions (mt.8614T>C and mt.8994G>A) in the mt-ATP6 gene were found to be significantly higher in the obese group compared to the control group (p<0.05). In the mt-ATP6 gene, one novel nonsynonymous substitution (mt.8726C>T) and one novel synonymous substitution (mt.9108A>T) were found. In the mt-CytB gene, one nonsynonymous substitution (mt.14880T>C) and two synonymous substitutions (mt.14891C>T and mt.15091C>T) were novel substitutions. Conclusion: Two synonymous substitutions (mt.8614T>C and mt.8994G>A) in the mt-ATP6 gene may be associated with childhood obesity. Our study provides the first data about mitochondrial genome variations in a Turkish obese population and also the first in obese children. More cases should be screened in obese groups in order to understand the effects of mitochondrial polymorphisms in the development of obesity. PMID:25541891

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

PubMed

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

New anamorphic yeast species: Candida infanticola sp. nov., Candida polysorbophila sp. nov., Candida transvaalensis sp. nov. and Trigonopsis californica sp. nov.

PubMed

Kurtzman, Cletus P

2007-08-01

Three new species of Candida and a new species of Trigonopsis are described based on their recognition from phylogenetic analysis of gene sequences from large subunit ribosomal RNA, ITS1/ITS2 rRNA, mitochondrial small subunit rRNA and cytochrome oxidase II. Candida infanticola sp. nov. (type strain NRRL Y-17858, CBS 7922) was isolated from the ear of an infant in Germany and is closely related to Candida sorbophila. Candida polysorbophila sp. nov. (type strain NRRL Y-27161, CBS 7317) is a member of the Zygoascus clade and was isolated in South Africa as a contaminant from an emulsion of white oil and polysorbate. Candida transvaalensis sp. nov. (type strain NRRL Y-27140, CBS 6663) was obtained from forest litter, the Transvaal, South Africa, and forms an isolated clade with Candida santjacobensis. Trigonopsis californica sp. nov. (type strain NRRL Y-27307, CBS 10351) represents a contaminant from wine in California, and forms a well-supported clade with Trigonopsis cantarellii, Trigonopsis variabilis and Trigonopsis vinaria.

Two circular chromosomes of unequal copy number make up the mitochondrial genome of the rotifer Brachionus plicatilis.

PubMed

Suga, Koushirou; Mark Welch, David B; Tanaka, Yukari; Sakakura, Yoshitaka; Hagiwara, Atsushi

2008-06-01

The monogonont rotifer Brachionus plicatilis is an emerging model system for a diverse array of questions in limnological ecosystem dynamics, the evolution of sexual recombination, cryptic speciation, and the phylogeny of basal metazoans. We sequenced the complete mitochondrial genome of B. plicatilis sensu strictu NH1L and found that it is composed of 2 circular chromosomes, designated mtDNA-I (11,153 bp) and mtDNA-II (12,672 bp). Hybridization to DNA isolated from mitochondria demonstrated that mtDNA-I is present at 4 times the copy number of mtDNA-II. The only nucleotide similarity between the 2 chromosomes is a 4.9-kbp region of 99.5% identity including a transfer RNA (tRNA) gene and an extensive noncoding region that contains putative D-loop and control sequence. The mtDNA-I chromosome encodes 4 proteins (ATP6, COB, NAD1, and NAD2), 13 tRNAs, and the large and small subunit ribosomal RNAs; mtDNA-II encodes 8 proteins (COX1-3, NAD3-6, and NAD4L) and 9 tRNAs. Gene order is not conserved between B. plicatilis and its closest relative with a sequenced mitochondrial genome, the acanthocephalan Leptorhynchoides thecatus, or other sequenced mitochondrial genomes. Polymerase chain reaction assays and Southern hybridization to DNA from 18 strains of Brachionus suggest that the 2-chromosome structure has been stable for millions of years. The novel organization of the B. plicatilis mitochondrial genome into 2 nearly equal chromosomes of 4-fold different copy number may provide insight into the evolution of metazoan mitochondria and the phylogenetics of rotifers and other basal animal phyla.

Analysis of the Mitochondrial Genome in Hypomyces aurantius Reveals a Novel Twintron Complex in Fungi.

PubMed

Deng, Youjin; Zhang, Qihui; Ming, Ray; Lin, Longji; Lin, Xiangzhi; Lin, Yiying; Li, Xiao; Xie, Baogui; Wen, Zhiqiang

2016-06-30

Hypomyces aurantius is a mycoparasite that causes cobweb disease, a most serious disease of cultivated mushrooms. Intra-species identification is vital for disease control, however the lack of genomic data makes development of molecular markers challenging. Small size, high copy number, and high mutation rate of fungal mitochondrial genome makes it a good candidate for intra and inter species differentiation. In this study, the mitochondrial genome of H. H.a0001 was determined from genomic DNA using Illumina sequencing. The roughly 72 kb genome shows all major features found in other Hypocreales: 14 common protein genes, large and small subunit rRNAs genes and 27 tRNAs genes. Gene arrangement comparison showed conserved gene orders in Hypocreales mitochondria are relatively conserved, with the exception of Acremonium chrysogenum and Acremonium implicatum. Mitochondrial genome comparison also revealed that intron length primarily contributes to mitogenome size variation. Seventeen introns were detected in six conserved genes: five in cox1, four in rnl, three in cob, two each in atp6 and cox3, and one in cox2. Four introns were found to contain two introns or open reading frames: cox3-i2 is a twintron containing two group IA type introns; cox2-i1 is a group IB intron encoding two homing endonucleases; and cox1-i4 and cox1-i3 both contain two open reading frame (ORFs). Analyses combining secondary intronic structures, insertion sites, and similarities of homing endonuclease genes reveal two group IA introns arranged side by side within cox3-i2. Mitochondrial data for H. aurantius provides the basis for further studies relating to population genetics and species identification.

Analysis of the Mitochondrial Genome in Hypomyces aurantius Reveals a Novel Twintron Complex in Fungi

PubMed Central

Deng, Youjin; Zhang, Qihui; Ming, Ray; Lin, Longji; Lin, Xiangzhi; Lin, Yiying; Li, Xiao; Xie, Baogui; Wen, Zhiqiang

2016-01-01

Hypomyces aurantius is a mycoparasite that causes cobweb disease, a most serious disease of cultivated mushrooms. Intra-species identification is vital for disease control, however the lack of genomic data makes development of molecular markers challenging. Small size, high copy number, and high mutation rate of fungal mitochondrial genome makes it a good candidate for intra and inter species differentiation. In this study, the mitochondrial genome of H. H.a0001 was determined from genomic DNA using Illumina sequencing. The roughly 72 kb genome shows all major features found in other Hypocreales: 14 common protein genes, large and small subunit rRNAs genes and 27 tRNAs genes. Gene arrangement comparison showed conserved gene orders in Hypocreales mitochondria are relatively conserved, with the exception of Acremonium chrysogenum and Acremonium implicatum. Mitochondrial genome comparison also revealed that intron length primarily contributes to mitogenome size variation. Seventeen introns were detected in six conserved genes: five in cox1, four in rnl, three in cob, two each in atp6 and cox3, and one in cox2. Four introns were found to contain two introns or open reading frames: cox3-i2 is a twintron containing two group IA type introns; cox2-i1 is a group IB intron encoding two homing endonucleases; and cox1-i4 and cox1-i3 both contain two open reading frame (ORFs). Analyses combining secondary intronic structures, insertion sites, and similarities of homing endonuclease genes reveal two group IA introns arranged side by side within cox3-i2. Mitochondrial data for H. aurantius provides the basis for further studies relating to population genetics and species identification. PMID:27376282

Molecular insights into the m-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane.

PubMed

Lee, Seoeun; Lee, Hunsang; Yoo, Suji; Kim, Hyun

2017-12-08

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m -AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m -AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m -AAA protease subunit. Furthermore, m -AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m -AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m -AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

[Molecular and functional diversity of ATP-sensitive K+ channels: the pathophysiological roles and potential drug targets].

PubMed

Nakaya, Haruaki; Miki, Takashi; Seino, Susumu; Yamada, Katsuya; Inagaki, Nobuya; Suzuki, Masashi; Sato, Toshiaki; Yamada, Mitsuhiko; Matsushita, Kenji; Kurachi, Yoshihisa; Arita, Makoto

2003-09-01

ATP-sensitive K(+) (K(ATP)) channels comprise the pore-forming subunit (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). K(ATP) channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 or Kir6.2, functional roles of K(ATP) channels in various organs have been clarified. Kir6.1(-/-) mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2(-/-) mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K(ATP) channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2(-/-) mice lacking the sarcolemmal K(ATP) channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal K(ATP) channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial K(ATP) channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K(+) channel openers activating mitochondrial K(ATP) channels, decreased the mitochondrial membrane potential, thereby preventing the Ca(2+) overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K(+) channel openers and the activating effects on sarcolemmal K(ATP) channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of K(ATP) channels, there would be considerable differences in the tissue selectivity of K(ATP) channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout mice indicate that K(ATP) channels are involved in the mechanisms of the protection against metabolic stress. Further clarification of physiological as well as pathophysiological roles of K(ATP) channels may lead to a new therapeutic strategy to improve the quality of life.

The first genetically confirmed case of Dioctophyme renale (Nematoda: Dioctophymatida) in a patient with a subcutaneous nodule.

PubMed

Tokiwa, Toshihiro; Ueda, Wataru; Takatsuka, Satoshi; Okawa, Kiyotaka; Onodera, Masayuki; Ohta, Nobuo; Akao, Nobuaki

2014-02-01

We describe a nematode larva in a subcutaneous nodule excised from a 44-year-old Chinese male who had been living in Japan for 15 years. Morphological features suggested that the worm was a dioctophimatid nematode. PCR amplification and sequencing of small subunit ribosomal DNA and mitochondrial cytochrome subunit c oxidase genes allowed us to identify the larva as the giant kidney worm, Dioctophyme renale (Goeze, 1972). This is the first molecularly confirmed human case of a dermal D. renale infection. © 2013.

Accumulation of sphingolipid activator proteins (SAPs) A and D in granular osmiophilic deposits in miniature Schnauzer dogs with ceroid-lipofuscinosis.

PubMed

Palmer, D N; Tyynelä, J; van Mil, H C; Westlake, V J; Jolly, R D

1997-03-01

The neuronal ceroid-lipofuscinoses (NCL, Batten disease) are fatal inherited neurodegenerative diseases of children characterized by retinal and brain atrophy and the accumulation of electron-dense storage bodies in cells. Mutations in different genes underlie different major forms. The infantile disease (CLN-1, McKusick 256730) is distinguished by the storage of the sphingolipid activator proteins (SAPs) A and D in distinctive granular osmiophilic deposits (GRODs). This contrasts with the other major forms, where subunit c of mitochondrial ATP synthase is stored in various multilamellar profiles. Ceroid-lipofuscinoses also occur in dogs, including a form in miniature Schnauzers with distinctive granular osmiophilic deposit-like storage bodies. Antisera to SAPs A and D reacted to these storage bodies in situ. The presence of SAP D was confirmed by Western blotting and of SAP A by protein sequencing. Neither subunit c of mitochondrial ATP synthase nor of vacuolar ATPase is stored. This suggests that there are two families of ceroid-lipofuscinoses, the subunit c-storing forms, and those in which SAPs A and D, and perhaps other proteins, accumulate. Further work is required to determine whether other forms with granular osmiophilic deposits belong to the latter class and the genetic relationships between them and the human infantile disease.

The mitochondrial electron transfer flavoprotein complex is essential for survival of Arabidopsis in extended darkness.

PubMed

Ishizaki, Kimitsune; Schauer, Nicolas; Larson, Tony R; Graham, Ian A; Fernie, Alisdair R; Leaver, Christopher J

2006-09-01

In mammals, the electron transfer flavoprotein (ETF) is a heterodimeric protein composed of two subunits, alpha and beta, that is responsible for the oxidation of at least nine mitochondrial matrix flavoprotein dehydrogenases. Electrons accepted by ETF are further transferred to the main respiratory chain via the ETF ubiquinone oxide reductase (ETFQO). Sequence analysis of the unique Arabidopsis homologues of two subunits of ETF revealed their high similarity to both subunits of the mammalian ETF. Yeast two-hybrid experiments showed that the Arabidopsis ETFalpha and ETFbeta can form a heteromeric protein. Isolation and characterization of two independent T-DNA insertional Arabidopsis mutants of the ETFbeta gene revealed accelerated senescence and early death compared to wild-type during extended darkness. Furthermore in contrast to wild-type, the etfb mutants demonstrated a significant accumulation of several amino acids, isovaleryl CoA and phytanoyl CoA during dark-induced carbohydrate deprivation. These phenotypic characteristics of etfb mutants are broadly similar to those that we observed previously in Arabidopsis etfqo mutants, suggesting functional association between ETF and ETFQO in Arabidopsis, and confirming the essential roles of the ETF/ETFQO electron transfer complex in the catabolism of leucine and involvement in the chlorophyll degradation pathway activated during dark-induced carbohydrate deprivation.

Sequence variability in three mitochondrial genes among four roundworm species from wild animals in China.

PubMed

Chang, Qiao-Cheng; Gao, Jun-Feng; Sheng, Zhong-Hua; Lou, Yan; Zheng, Xu; Wang, Chun-Ren

2015-02-01

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely portions of cytochrome c oxidase subunit 1 (pcox1), NADH dehydrogenase subunit 1 (pnad1) and NADH dehydrogenase subunit 4 (pnad4), for Toxocara canis. Baylisacaris transfuga. Ascaris suum and Parascaris equorum from Canis lupus. Ursus thibetanus. Sus scrofa and Equus burchelli in China were examined. The lengths of the sequences of pcox1, pnad1 and pnad4 were 711 bp, 648 bp and 666 bp, respectively. No intra-species differences were detected in pcox1 for the four examined ascarid species, in pnad1 for T. canis. A. suum and P. equorum, and in pnad4 for B. transfuga and P. equorum. Sequence differences in pnad4 for six roundworm samples of T. canis and P. equorum were 0-0.1% and 0-0.3%, respectively, and were 0-0.3% in pnad1 for six roundworm samples isolate of B. transfuga. The inter-specific sequence differences among four species were 8.7-12.4% for pcox1, 13.9-17.7% for pnad1, and 14.0-25.7% for pnad4. Phylogenetic analyses suggested that the three mtDNA fragments could be used to identify ascarid species in families Ascaridiae and Toxocaridae.

Roles of Neuroglobin Binding to Mitochondrial Complex III Subunit Cytochrome c1 in Oxygen-Glucose Deprivation-Induced Neurotoxicity in Primary Neurons.

PubMed

Yu, Zhanyang; Zhang, Yu; Liu, Ning; Yuan, Jing; Lin, Li; Zhuge, Qichuan; Xiao, Jian; Wang, Xiaoying

2016-07-01

Neuroglobin (Ngb) is a tissue globin specifically expressed in brain neurons. Recent studies by our laboratory and others have demonstrated that Ngb is protective against stroke and related neurological disorders, but the mechanisms remain poorly understood. We previously identified cytochrome c1 (Cyc1) as an Ngb-interacting molecule by yeast two-hybrid screening. Cyc1 is a subunit of mitochondria complex III, which is a component of mitochondrial respiratory chain and a major source of reactive oxygen species (ROS) production under both physiological and pathological conditions. In this study, we for the first time defined Ngb-Cyc1 binding, and investigated its roles in oxygen-glucose deprivation (OGD)/reoxygenation-induced neurotoxicity and ROS production in primary neurons. Immunocytochemistry and co-immunoprecipitation validated Ngb-Cyc1 binding, which was significantly increased by OGD and Ngb overexpression. We found 4 h OGD with/without 4 h reoxygenation significantly increased complex III activity, but this activity elevation was significantly attenuated in three groups of neurons: Ngb overexpression, specific complex III inhibitor stigmatellin, or stigmatellin plus Ngb overexpression, whereas there was no significant differences between these three groups, suggesting Ngb-Cyc1 binding may function in suppressing OGD-mediated complex III activity elevation. Importantly, these three groups of neurons also showed significant decreases in OGD-induced superoxide anion generation and neurotoxicity. These results suggest that Ngb can bind to mitochondrial complex III subunit Cyc1, leading to suppression of OGD-mediated complex III activity and subsequent ROS production elevation, and eventually reduction of OGD-induced neurotoxicity. This molecular signaling cascade may be at least part of the mechanisms of Ngb neuroprotection against OGD-induced neurotoxicity.

The p.M292T NDUFS2 mutation causes complex I-deficient Leigh syndrome in multiple families.

PubMed

Tuppen, Helen A L; Hogan, Vanessa E; He, Langping; Blakely, Emma L; Worgan, Lisa; Al-Dosary, Mazhor; Saretzki, Gabriele; Alston, Charlotte L; Morris, Andrew A; Clarke, Michael; Jones, Simon; Devlin, Anita M; Mansour, Sahar; Chrzanowska-Lightowlers, Zofia M A; Thorburn, David R; McFarland, Robert; Taylor, Robert W

2010-10-01

Isolated complex I deficiency is the most frequently observed oxidative phosphorylation defect in children with mitochondrial disease, leading to a diverse range of clinical presentations, including Leigh syndrome. For most patients the genetic cause of the biochemical defect remains unknown due to incomplete understanding of the complex I assembly process. Nonetheless, a plethora of pathogenic mutations have been described to date in the seven mitochondrial-encoded subunits of complex I as well as in 12 of the nuclear-encoded subunits and in six assembly factors. Whilst several mitochondrial DNA mutations are recurrent, the majority of these mutations are reported in single families. We have sequenced core structural and functional nuclear-encoded subunits of complex I in a cohort of 34 paediatric patients with isolated complex I deficiency, identifying pathogenic mutations in 6 patients. These included a novel homozygous NDUFS1 mutation in an Asian child with Leigh syndrome, a previously identified NDUFS8 mutation (c.236C>T, p.P79L) in a second Asian child with Leigh-like syndrome and six novel, compound heterozygous NDUFS2 mutations in four white Caucasian patients with Leigh or Leigh-like syndrome. Three of these children harboured an identical NDUFS2 mutation (c.875T>C, p.M292T), which was also identified in conjunction with a novel NDUFS2 splice site mutation (c.866+4A>G) in a fourth Caucasian child who presented to a different diagnostic centre, with microsatellite and single nucleotide polymorphism analyses indicating that this was due to an ancient common founder event. Our results confirm that NDUFS2 is a mutational hotspot in Caucasian children with isolated complex I deficiency and recommend the routine diagnostic investigation of this gene in patients with Leigh or Leigh-like phenotypes.

Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive KATP channel in rat brain nonsynaptosomal mitochondria.

PubMed

Brustovetsky, Tatiana; Shalbuyeva, Natalia; Brustovetsky, Nickolay

2005-10-01

Pharmacological modulation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) sensitive to diazoxide and 5-hydroxydecanoate (5-HD) represents an attractive strategy to protect cells against ischaemia/reperfusion- and stroke-related injury. To re-evaluate a functional role for the mitoKATP in brain, we used Percoll-gradient-purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (DeltaPsi) and depolarization-induced K+ efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K+ inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP-sensitive K+ channel of the plasma membrane (cellKATP), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5-hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoKATP were applied to show the role of the channel in regulating matrix volume, respiration, and DeltaPsi and K+ fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoKATP activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoKATP, similar to the cellKATP, in brain mitochondria. On the other hand, the depolarization-evoked release of K+ suppressed by ATP could be re-activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K+ efflux similarly to ATP. These observations implicate the ANT in ATP-sensitive K+ transport in brain mitochondria.

Differential mitochondrial DNA and gene expression in inherited retinal dysplasia in miniature Schnauzer dogs.

PubMed

Appleyard, Greg D; Forsyth, George W; Kiehlbauch, Laura M; Sigfrid, Kristen N; Hanik, Heather L J; Quon, Anita; Loewen, Matthew E; Grahn, Bruce H

2006-05-01

To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers. Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy. RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense. Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

Thyrotropin-releasing hormone controls mitochondrial biology in human epidermis.

PubMed

Knuever, Jana; Poeggeler, Burkhard; Gáspár, Erzsébet; Klinger, Matthias; Hellwig-Burgel, Thomas; Hardenbicker, Celine; Tóth, Balázs I; Bíró, Tamás; Paus, Ralf

2012-03-01

Mitochondrial capacity and metabolic potential are under the control of hormones, such as thyroid hormones. The most proximal regulator of the hypothalamic-pituitary-thyroid (HPT) axis, TRH, is the key hypothalamic integrator of energy metabolism via its impact on thyroid hormone secretion. Here, we asked whether TRH directly modulates mitochondrial functions in normal, TRH-receptor-positive human epidermis. Organ-cultured human skin was treated with TRH (5-100 ng/ml) for 12-48 h. TRH significantly increased epidermal immunoreactivity for the mitochondria-selective subunit I of respiratory chain complex IV (MTCO1). This resulted from an increased MTCO1 transcription and protein synthesis and a stimulation of mitochondrial biogenesis as demonstrated by transmission electron microscopy and TRH-enhanced mitochondrial DNA synthesis. TRH also significantly stimulated the transcription of several other mitochondrial key genes (TFAM, HSP60, and BMAL1), including the master regulator of mitochondrial biogenesis (PGC-1α). TRH significantly enhanced mitochondrial complex I and IV enzyme activity and enhanced the oxygen consumption of human skin samples, which shows that the stimulated mitochondria are fully vital because the main source for cellular oxygen consumption is mitochondrial endoxidation. These findings identify TRH as a potent, novel neuroendocrine stimulator of mitochondrial activity and biogenesis in human epidermal keratinocytes in situ. Thus, human epidermis offers an excellent model for dissecting neuroendocrine controls of human mitochondrial biology under physiologically relevant conditions and for exploring corresponding clinical applications.

DNA barcoding as a tool for coral reef conservation

NASA Astrophysics Data System (ADS)

Neigel, J.; Domingo, A.; Stake, J.

2007-09-01

DNA Barcoding (DBC) is a method for taxonomic identification of animals that is based entirely on the 5' portion of the mitochondrial gene, cytochrome oxidase subunit I ( COI-5). It can be especially useful for identification of larval forms or incomplete specimens lacking diagnostic morphological characters. DBC can also facilitate the discovery of species and in defining “molecular taxonomic units” in problematic groups. However, DBC is not a panacea for coral reef taxonomy. In two of the most ecologically important groups on coral reefs, the Anthozoa and Porifera, COI-5 sequences have diverged too little to be diagnostic for all species. Other problems for DBC include paraphyly in mitochondrial gene trees and lack of differentiation between hybrids and their maternal ancestors. DBC also depends on the availability of databases of COI-5 sequences, which are still in early stages of development. A global effort to barcode all fish species has demonstrated the importance of large-scale coordination and is yielding promising results. Whether or not COI-5 by itself is sufficient for species assignments has become a contentious question; it is generally advantageous to use sequences from multiple loci.

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley

PubMed Central

de Ponzzes-Gomes, Camila M.P.B.S.; de Mélo, Dângelly L.F.M.; Santana, Caroline A.; Pereira, Giuliano E.; Mendonça, Michelle O.C.; Gomes, Fátima C.O.; Oliveira, Evelyn S.; Barbosa, Antonio M.; Trindade, Rita C.; Rosa, Carlos A.

2014-01-01

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 105 cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production. PMID:25242923

Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley.

PubMed

de Ponzzes-Gomes, Camila M P B S; de Mélo, Dângelly L F M; Santana, Caroline A; Pereira, Giuliano E; Mendonça, Michelle O C; Gomes, Fátima C O; Oliveira, Evelyn S; Barbosa, Antonio M; Trindade, Rita C; Rosa, Carlos A

2014-01-01

The aims of this work was to characterise indigenous Saccharomyces cerevisiae strains in the naturally fermented juice of grape varieties Cabernet Sauvignon, Grenache, Tempranillo, Sauvignon Blanc and Verdejo used in the São Francisco River Valley, northeastern Brazil. In this study, 155 S. cerevisiae and 60 non-Saccharomyces yeasts were isolated and identified using physiological tests and sequencing of the D1/D2 domains of the large subunit of the rRNA gene. Among the non-Saccharomyces species, Rhodotorula mucilaginosa was the most common species, followed by Pichia kudriavzevii, Candida parapsilosis, Meyerozyma guilliermondii, Wickerhamomyces anomalus, Kloeckera apis, P. manshurica, C. orthopsilosis and C. zemplinina. The population counts of these yeasts ranged among 1.0 to 19 × 10(5) cfu/mL. A total of 155 isolates of S. cerevisiae were compared by mitochondrial DNA restriction analysis, and five molecular mitochondrial DNA restriction profiles were detected. Indigenous strains of S. cerevisiae isolated from grapes of the São Francisco Valley can be further tested as potential starters for wine production.

MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity

PubMed Central

Patron, Maria; Checchetto, Vanessa; Raffaello, Anna; Teardo, Enrico; Vecellio Reane, Denis; Mantoan, Maura; Granatiero, Veronica; Szabò, Ildikò; De Stefani, Diego; Rizzuto, Rosario

2014-01-01

Summary Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca2+ concentrations, preventing deleterious Ca2+ cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca2+] in situ and allowing tight physiological control. At low [Ca2+], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca2+], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca2+ signals generated in the cytoplasm. PMID:24560927

Identifying the true oysters (Bivalvia: Ostreidae) with mitochondrial phylogeny and distance-based DNA barcoding.

PubMed

Liu, Jun; Li, Qi; Kong, Lingfeng; Yu, Hong; Zheng, Xiaodong

2011-09-01

Oysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographic distribution, are a challenging group for taxonomists and phylogenetics. As a useful tool for molecular species identification, DNA barcoding might offer significant potential for oyster identification and taxonomy. This study used two mitochondrial fragments, cytochrome c oxidase I (COI) and the large ribosomal subunit (16S rDNA), to assess whether oyster species could be identified by phylogeny and distance-based DNA barcoding techniques. Relationships among species were estimated by the phylogenetic analyses of both genes, and then pairwise inter- and intraspecific genetic divergences were assessed. Species forming well-differentiated clades in the molecular phylogenies were identical for both genes even when the closely related species were included. Intraspecific variability of 16S rDNA overlapped with interspecific divergence. However, average intra- and interspecific genetic divergences for COI were 0-1.4% (maximum 2.2%) and 2.6-32.2% (minimum 2.2%), respectively, indicating the existence of a barcoding gap. These results confirm the efficacy of species identification in oysters via DNA barcodes and phylogenetic analysis. © 2011 Blackwell Publishing Ltd.

A Krebs Cycle Component Limits Caspase Activation Rate through Mitochondrial Surface Restriction of CRL Activation.

PubMed

Aram, Lior; Braun, Tslil; Braverman, Carmel; Kaplan, Yosef; Ravid, Liat; Levin-Zaidman, Smadar; Arama, Eli

2016-04-04

How cells avoid excessive caspase activity and unwanted cell death during apoptotic caspase-mediated removal of large cellular structures is poorly understood. We investigate caspase-mediated extrusion of spermatid cytoplasmic contents in Drosophila during spermatid individualization. We show that a Krebs cycle component, the ATP-specific form of the succinyl-CoA synthetase β subunit (A-Sβ), binds to and activates the Cullin-3-based ubiquitin ligase (CRL3) complex required for caspase activation in spermatids. In vitro and in vivo evidence suggests that this interaction occurs on the mitochondrial surface, thereby limiting the source of CRL3 complex activation to the vicinity of this organelle and reducing the potential rate of caspase activation by at least 60%. Domain swapping between A-Sβ and the GTP-specific SCSβ (G-Sβ), which functions redundantly in the Krebs cycle, show that the metabolic and structural roles of A-Sβ in spermatids can be uncoupled, highlighting a moonlighting function of this Krebs cycle component in CRL activation. Copyright © 2016 Elsevier Inc. All rights reserved.

Cold ischemia contributes to the development of chronic rejection and mitochondrial injury after cardiac transplantation.

PubMed

Schneeberger, Stefan; Amberger, Albert; Mandl, Julia; Hautz, Theresa; Renz, Oliver; Obrist, Peter; Meusburger, Hugo; Brandacher, Gerald; Mark, Walter; Strobl, Daniela; Troppmair, Jakob; Pratschke, Johann; Margreiter, Raimund; Kuznetsov, Andrey V

2010-12-01

Chronic rejection (CR) remains an unsolved hurdle for long-term heart transplant survival. The effect of cold ischemia (CI) on progression of CR and the mechanisms resulting in functional deficit were investigated by studying gene expression, mitochondrial function, and enzymatic activity. Allogeneic (Lew→F344) and syngeneic (Lew→Lew) heart transplantations were performed with or without 10 h of CI. After evaluation of myocardial contraction, hearts were excised at 2, 10, 40, and 60 days for investigation of vasculopathy, gene expression, enzymatic activities, and mitochondrial respiration. Gene expression studies identified a gene cluster coding for subunits of the mitochondrial electron transport chain regulated in response to CI and CR. Myocardial performance, mitochondrial function, and mitochondrial marker enzyme activities declined in all allografts with time after transplantation. These declines were more rapid and severe in CI allografts (CR-CI) and correlated well with progression of vasculopathy and fibrosis. Mitochondria related gene expression and mitochondrial function are substantially compromised with the progression of CR and show that CI impacts on progression, gene profile, and mitochondrial function of CR. Monitoring mitochondrial function and enzyme activity might allow for earlier detection of CR and cardiac allograft dysfunction. © 2010 The Authors. Journal compilation © 2010 European Society for Organ Transplantation.

Mitochondrial telomerase reverse transcriptase binds to and protects mitochondrial DNA and function from damage.

PubMed

Haendeler, Judith; Dröse, Stefan; Büchner, Nicole; Jakob, Sascha; Altschmied, Joachim; Goy, Christine; Spyridopoulos, Ioakim; Zeiher, Andreas M; Brandt, Ulrich; Dimmeler, Stefanie

2009-06-01

The enzyme telomerase and its catalytic subunit the telomerase reverse transcriptase (TERT) are important for maintenance of telomere length in the nucleus. Recent studies provided evidence for a mitochondrial localization of TERT. Therefore, we investigated the exact localization of TERT within the mitochondria and its function. Here, we demonstrate that TERT is localized in the matrix of the mitochondria. TERT binds to mitochondrial DNA at the coding regions for ND1 and ND2. Binding of TERT to mitochondrial DNA protects against ethidium bromide-induced damage. TERT increases overall respiratory chain activity, which is most pronounced at complex I and dependent on the reverse transcriptase activity of the enzyme. Moreover, mitochondrial reactive oxygen species are increased after genetic ablation of TERT by shRNA. Mitochondrially targeted TERT and not wild-type TERT revealed the most prominent protective effect on H(2)O(2)-induced apoptosis. Lung fibroblasts from 6-month-old TERT(-/-) mice (F2 generation) showed increased sensitivity toward UVB radiation and heart mitochondria exhibited significantly reduced respiratory chain activity already under basal conditions, demonstrating the protective function of TERT in vivo. Mitochondrial TERT exerts a novel protective function by binding to mitochondrial DNA, increasing respiratory chain activity and protecting against oxidative stress-induced damage.

AMP-Activated Protein Kinase Deficiency Rescues Paraquat-Induced Cardiac Contractile Dysfunction Through an Autophagy-Dependent Mechanism

PubMed Central

Wang, Qiurong; Yang, Lifang; Hua, Yinan; Nair, Sreejayan; Xu, Xihui; Ren, Jun

2014-01-01

Aim: Paraquat, a quaternary nitrogen herbicide, is a highly toxic prooxidant resulting in multi-organ failure including the heart although the underlying mechanism still remains elusive. This study was designed to examine the role of the cellular fuel sensor AMP-activated protein kinase (AMPK) in paraquat-induced cardiac contractile and mitochondrial injury. Results: Wild-type and transgenic mice with overexpression of a mutant AMPK α2 subunit (kinase dead, KD), with reduced activity in both α1 and α2 subunits, were administered with paraquat (45 mg/kg) for 48 h. Paraquat elicited cardiac mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic diameter and reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca2+ handling, reduced cell survival, and overt mitochondrial damage (loss in mitochondrial membrane potential). In addition, paraquat treatment promoted phosphorylation of AMPK and autophagy. Interestingly, deficiency in AMPK attenuated paraquat-induced cardiac contractile and intracellular Ca2+ derangement. The beneficial effect of AMPK inhibition was associated with inhibition of the AMPK-TSC-mTOR-ULK1 signaling cascade. In vitro study revealed that inhibitors for AMPK and autophagy attenuated paraquat-induced cardiomyocyte contractile dysfunction. Conclusion: Taken together, our findings revealed that AMPK may mediate paraquat-induced myocardial anomalies possibly by regulating the AMPK/mTOR-dependent autophagy. PMID:25092649

Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability.

PubMed

Joardar, Vinita; Abrams, Natalie F; Hostetler, Jessica; Paukstelis, Paul J; Pakala, Suchitra; Pakala, Suman B; Zafar, Nikhat; Abolude, Olukemi O; Payne, Gary; Andrianopoulos, Alex; Denning, David W; Nierman, William C

2012-12-12

The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25-36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing.

Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans.

PubMed

Nakano, Tadao; Okamoto, Munehiro; Ikeda, Yatsukaho; Hasegawa, Hideo

2006-12-01

Sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, nuclear internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA), and 5S rDNA of Enterobius vermicularis from captive chimpanzees in five zoos/institutions in Japan were analyzed and compared with those of pinworm eggs from humans in Japan. Three major types of variants appearing in both CO1 and ITS2 sequences, but showing no apparent connection, were observed among materials collected from the chimpanzees. Each one of them was also observed in pinworms in humans. Sequences of 5S rDNA were identical in the materials from chimpanzees and humans. Phylogenetic analysis of CO1 gene revealed three clusters with high bootstrap value, suggesting considerable divergence, presumably correlated with human evolution, has occurred in the human pinworms. The synonymy of E. gregorii with E. vermicularis is supported by the molecular evidence.

A Mitochondrial ATP synthase Subunit Interacts with TOR Signaling to Modulate Protein Homeostasis and Lifespan in Drosophila

PubMed Central

Sun, Xiaoping; Wheeler, Charles T.; Yolitz, Jason; Laslo, Mara; Alberico, Thomas; Sun, Yaning; Song, Qisheng; Zou, Sige

2014-01-01

SUMMARY Diet composition is a critical determinant of lifespan and nutrient imbalance is detrimental health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. We investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extended lifespan in females fed low carbohydrate-to-protein (C:P) diets, but not the high C:P ratio diet. This extension was associated with increased resistance to oxidative stress, transcriptional changes in metabolism, proteostasis and immune genes, reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and MAPK signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. Our data reveal a link among diet, mitochondria, MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging. PMID:25220459

Structure and variation of the mitochondrial genome of fishes.

PubMed

Satoh, Takashi P; Miya, Masaki; Mabuchi, Kohji; Nishida, Mutsumi

2016-09-07

The mitochondrial (mt) genome has been used as an effective tool for phylogenetic and population genetic analyses in vertebrates. However, the structure and variability of the vertebrate mt genome are not well understood. A potential strategy for improving our understanding is to conduct a comprehensive comparative study of large mt genome data. The aim of this study was to characterize the structure and variability of the fish mt genome through comparative analysis of large datasets. An analysis of the secondary structure of proteins for 250 fish species (248 ray-finned and 2 cartilaginous fishes) illustrated that cytochrome c oxidase subunits (COI, COII, and COIII) and a cytochrome bc1 complex subunit (Cyt b) had substantial amino acid conservation. Among the four proteins, COI was the most conserved, as more than half of all amino acid sites were invariable among the 250 species. Our models identified 43 and 58 stems within 12S rRNA and 16S rRNA, respectively, with larger numbers than proposed previously for vertebrates. The models also identified 149 and 319 invariable sites in 12S rRNA and 16S rRNA, respectively, in all fishes. In particular, the present result verified that a region corresponding to the peptidyl transferase center in prokaryotic 23S rRNA, which is homologous to mt 16S rRNA, is also conserved in fish mt 16S rRNA. Concerning the gene order, we found 35 variations (in 32 families) that deviated from the common gene order in vertebrates. These gene rearrangements were mostly observed in the area spanning the ND5 gene to the control region as well as two tRNA gene cluster regions (IQM and WANCY regions). Although many of such gene rearrangements were unique to a specific taxon, some were shared polyphyletically between distantly related species. Through a large-scale comparative analysis of 250 fish species mt genomes, we elucidated various structural aspects of the fish mt genome and the encoded genes. The present results will be important for understanding functions of the mt genome and developing programs for nucleotide sequence analysis. This study demonstrated the significance of extensive comparisons for understanding the structure of the mt genome.

Low abundance of the matrix arm of complex I in mitochondria predicts longevity in mice

PubMed Central

Miwa, Satomi; Jow, Howsun; Baty, Karen; Johnson, Amy; Czapiewski, Rafal; Saretzki, Gabriele; Treumann, Achim; von Zglinicki, Thomas

2014-01-01

Mitochondrial function is an important determinant of the ageing process; however, the mitochondrial properties that enable longevity are not well understood. Here we show that optimal assembly of mitochondrial complex I predicts longevity in mice. Using an unbiased high-coverage high-confidence approach, we demonstrate that electron transport chain proteins, especially the matrix arm subunits of complex I, are decreased in young long-living mice, which is associated with improved complex I assembly, higher complex I-linked state 3 oxygen consumption rates and decreased superoxide production, whereas the opposite is seen in old mice. Disruption of complex I assembly reduces oxidative metabolism with concomitant increase in mitochondrial superoxide production. This is rescued by knockdown of the mitochondrial chaperone, prohibitin. Disrupted complex I assembly causes premature senescence in primary cells. We propose that lower abundance of free catalytic complex I components supports complex I assembly, efficacy of substrate utilization and minimal ROS production, enabling enhanced longevity. PMID:24815183

Topology of subunits of the mammalian cytochrome c oxidase: Relationship to the assembly of the enzyme complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu-Zhong Zhang; Ewart, G.; Capaldi, R.A.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane({sup 35}S)sulfonate and sodium methyl 4-({sup 3}H)formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibodymore » (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C-domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage produce from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.« less

Assembly of β-barrel proteins in the mitochondrial outer membrane.

PubMed

Höhr, Alexandra I C; Straub, Sebastian P; Warscheid, Bettina; Becker, Thomas; Wiedemann, Nils

2015-01-01

Mitochondria evolved through endosymbiosis of a Gram-negative progenitor with a host cell to generate eukaryotes. Therefore, the outer membrane of mitochondria and Gram-negative bacteria contain pore proteins with β-barrel topology. After synthesis in the cytosol, β-barrel precursor proteins are first transported into the mitochondrial intermembrane space. Folding and membrane integration of β-barrel proteins depend on the mitochondrial sorting and assembly machinery (SAM) located in the outer membrane, which is related to the β-barrel assembly machinery (BAM) in bacteria. The SAM complex recognizes β-barrel proteins by a β-signal in the C-terminal β-strand that is required to initiate β-barrel protein insertion into the outer membrane. In addition, the SAM complex is crucial to form membrane contacts with the inner mitochondrial membrane by interacting with the mitochondrial contact site and cristae organizing system (MICOS) and shares a subunit with the endoplasmic reticulum-mitochondria encounter structure (ERMES) that links the outer mitochondrial membrane to the endoplasmic reticulum (ER). Copyright © 2014 Elsevier B.V. All rights reserved.

Nonneutral mitochondrial DNA variation in humans and chimpanzees

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nachman, M.W.; Aquadro, C.F.; Brown, W.M.

1996-03-01

We sequenced the NADH dehydrogenase subunit 3 (ND3) gene from a sample of 61 humans, five common chimpanzees, and one gorilla to test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution. Within humans and within chimpanzees, the ratio of replacement to silent nucleotide substitutions was higher than observed in comparisons between species, contrary to neutral expectations. To test the generality of this result, we reanalyzed published human RFLP data from the entire mitochondrial genome. Gains of restriction sites relative to a known human mtDNA sequence were used to infer unambiguous nucleotide substitutions.more » We also compared the complete mtDNA sequences of three humans. Both the RFLP data and the sequence data reveal a higher ratio of replacement to silent nucleotide substitutions within humans than is seen between species. This pattern is observed at most or all human mitochondrial genes and is inconsistent with a strictly neutral model. These data suggest that many mitochondrial protein polymorphisms are slightly deleterious, consistent with studies of human mitochondrial diseases. 59 refs., 2 figs., 8 tabs.« less

Lon in maintaining mitochondrial and endoplasmic reticulum homeostasis.

PubMed

Yang, Jieyeqi; Chen, Wenying; Zhang, Boyang; Tian, Fengli; Zhou, Zheng; Liao, Xin; Li, Chen; Zhang, Yi; Han, Yanyan; Wang, Yan; Li, Yuzhe; Wang, Guo-Qing; Shen, Xiao Li

2018-06-01

As a vital member of AAA+ (ATPase associated with diverse cellular activities) protein superfamily, Lon, a homo-hexameric ring-shaped protein complex with a serine-lysine catalytic dyad, is highly conserved throughout almost all prokaryotic and eukaryotic organisms. Lon protease (LONP) plays an important role in maintaining mitoproteostasis through selectively recognizing and degrading oxidatively modified mitoproteins within mitochondrial matrix, such as oxidized aconitase, phosphorylated mitochondrial transcription factor A, etc. Furthermore, the up-regulated LONP increased mitochondrial ROS generation to promote cell survival, cell proliferation, epithelial-mesenchymal transition, and cell migration, which was attributed to the up-regulation of NADH:ubiquinone oxidoreductase core subunit S8 via interaction with chaperone Lon under hypoxic or oxidative stress in tumorigenesis. In addition, Lon also participated in protein kinase RNA (PKR)-like endoplasmic reticulum kinase signaling pathway under endoplasmic reticulum (ER) stress. In short, Lon, as a pivotal stress-responsive protein that involved in the crosstalks among mitochondria, ER and nucleus, participated in multifarious important cellular processes crucial for cell survival, such as the mitochondrial protein quality control system, the mitochondrial unfolded protein response, the mtDNA maintenance, and the ER unfolded protein response.

Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

PubMed Central

Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

2014-01-01

The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

Recombinant Buckwheat Trypsin Inhibitor Induces Mitophagy by Directly Targeting Mitochondria and Causes Mitochondrial Dysfunction in Hep G2 Cells.

PubMed

Wang, Zhuanhua; Li, Shanshan; Ren, Rong; Li, Jiao; Cui, Xiaodong

2015-09-09

Mitochondria are essential targets for cancer chemotherapy and other disease treatments. Recombinant buckwheat trypsin inhibitor (rBTI), a member of the potato type I proteinase inhibitor family, was derived from tartary buckwheat extracts. Our results showed that rBTI directly targeted mitochondria and induced mitochondrial fragmentation and mitophagy. This occurs through enhanced depolarization of the mitochondrial membrane potential, increasing reactive oxygen species (ROS) generation associated with the rise of the superoxide dismutase and catalase activity and glutathione peroxidase (GSH) content, and changes in the GSH/oxidized glutathione ratio. Mild and transient ROS induced by rBTI were shown to be important signaling molecules required to induce Hep G2 mitophagy to remove dysfunctional mitochondria. Furthermore, rBTI could directly induce mitochondrial fragmentation. It was also noted that rBTI highly increased colocalization of mitochondria in treated cells compared to nontreated cells. Tom 20, a subunit of the translocase of the mitochondrial outer membrane complex responsible for recognizing mitochondrial presequences, may be the direct target of rBTI.

Insulin and IGF-1 improve mitochondrial function in a PI-3K/Akt-dependent manner and reduce mitochondrial generation of reactive oxygen species in Huntington's disease knock-in striatal cells.

PubMed

Ribeiro, Márcio; Rosenstock, Tatiana R; Oliveira, Ana M; Oliveira, Catarina R; Rego, A Cristina

2014-09-01

Oxidative stress and mitochondrial dysfunction have been described in Huntington's disease, a disorder caused by expression of mutant huntingtin (mHtt). IGF-1 was previously shown to protect HD cells, whereas insulin prevented neuronal oxidative stress. In this work we analyzed the role of insulin and IGF-1 in striatal cells derived from HD knock-in mice on mitochondrial production of reactive oxygen species (ROS) and related antioxidant and signaling pathways influencing mitochondrial function. Insulin and IGF-1 decreased mitochondrial ROS induced by mHtt and normalized mitochondrial SOD activity, without affecting intracellular glutathione levels. IGF-1 and insulin promoted Akt phosphorylation without changing the nuclear levels of phosphorylated Nrf2 or Nrf2/ARE activity. Insulin and IGF-1 treatment also decreased mitochondrial Drp1 phosphorylation, suggesting reduced mitochondrial fragmentation, and ameliorated mitochondrial function in HD cells in a PI-3K/Akt-dependent manner. This was accompanied by increased total and phosphorylated Akt, Tfam, and mitochondrial-encoded cytochrome c oxidase II, as well as Tom20 and Tom40 in mitochondria of insulin- and IGF-1-treated mutant striatal cells. Concomitantly, insulin/IGF-1-treated mutant cells showed reduced apoptotic features. Hence, insulin and IGF-1 improve mitochondrial function and reduce mitochondrial ROS caused by mHtt by activating the PI-3K/Akt signaling pathway, in a process independent of Nrf2 transcriptional activity, but involving enhanced mitochondrial levels of Akt and mitochondrial-encoded complex IV subunit. Copyright © 2014 Elsevier Inc. All rights reserved.

Targeted impairment of thymidine kinase 2 expression in cells induces mitochondrial DNA depletion and reveals molecular mechanisms of compensation of mitochondrial respiratory activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villarroya, Joan, E-mail: joanvillarroya@gmail.com; Institut de Recerca l'Hospital de la Santa Creu i Sant Pau, Barcelona; Lara, Mari-Carmen

Highlights: {yields} We impaired TK2 expression in Ost TK1{sup -} cells via siRNA-mediated interference (TK2{sup -}). {yields} TK2 impairment caused severe mitochondrial DNA (mtDNA) depletion in quiescent cells. {yields} Despite mtDNA depletion, TK2{sup -} cells show high cytochrome oxidase activity. {yields} Depletion of mtDNA occurs without imbalance in the mitochondrial dNTP pool. {yields} Nuclear-encoded ENT1, DNA-pol {gamma}, TFAM and TP gene expression is lowered in TK2{sup -} cells. -- Abstract: The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed themore » first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene (TK2) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1{sup -} cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase {gamma}, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.« less

Impact of mutations within the [Fe-S] cluster or the lipoic acid biosynthesis pathways on mitochondrial protein expression profiles in fibroblasts from patients.

PubMed

Lebigot, E; Gaignard, P; Dorboz, I; Slama, A; Rio, M; de Lonlay, P; Héron, B; Sabourdy, F; Boespflug-Tanguy, O; Cardoso, A; Habarou, F; Ottolenghi, C; Thérond, P; Bouton, C; Golinelli-Cohen, M P; Boutron, A

2017-11-01

Lipoic acid (LA) is the cofactor of the E2 subunit of mitochondrial ketoacid dehydrogenases and plays a major role in oxidative decarboxylation. De novo LA biosynthesis is dependent on LIAS activity together with LIPT1 and LIPT2. LIAS is an iron‑sulfur (Fe-S) cluster-containing mitochondrial protein, like mitochondrial aconitase (mt-aco) and some subunits of respiratory chain (RC) complexes I, II and III. All of them harbor at least one [Fe-S] cluster and their activity is dependent on the mitochondrial [Fe-S] cluster (ISC) assembly machinery. Disorders in the ISC machinery affect numerous Fe-S proteins and lead to a heterogeneous group of diseases with a wide variety of clinical symptoms and combined enzymatic defects. Here, we present the biochemical profiles of several key mitochondrial [Fe-S]-containing proteins in fibroblasts from 13 patients carrying mutations in genes encoding proteins involved in either the lipoic acid (LIPT1 and LIPT2) or mitochondrial ISC biogenesis (FDX1L, ISCA2, IBA57, NFU1, BOLA3) pathway. Ten of them are new patients described for the first time. We confirm that the fibroblast is a good cellular model to study these deficiencies, except for patients presenting mutations in FDX1L and a muscular clinical phenotype. We find that oxidative phosphorylation can be affected by LA defects in LIPT1 and LIPT2 patients due to excessive oxidative stress or to another mechanism connecting LA and respiratory chain activity. We confirm that NFU1, BOLA3, ISCA2 and IBA57 operate in the maturation of [4Fe-4S] clusters and not in [2Fe-2S] protein maturation. Our work suggests a functional difference between IBA57 and other proteins involved in maturation of [Fe-S] proteins. IBA57 seems to require BOLA3, NFU1 and ISCA2 for its stability and NFU1 requires BOLA3. Finally, our study establishes different biochemical profiles for patients according to their mutated protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Complete sequence and gene organization of the mitochondrial genome of Asio flammeus (Strigiformes, strigidae).

PubMed

Zhang, Yanan; Song, Tao; Pan, Tao; Sun, Xiaonan; Sun, Zhonglou; Qian, Lifu; Zhang, Baowei

2016-07-01

The complete sequence of the mitochondrial genome was determined for Asio flammeus, which is distributed widely in geography. The length of the complete mitochondrial genome was 18,966 bp, containing 2 rRNA genes, 22 tRNA genes, 13 protein-coding genes (PCGs), and 1 non-coding region (D-loop). All the genes were distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes which were encoded on the L-strand. The D-loop of A. flammeus contained many tandem repeats of varying lengths and repeat numbers. The molecular-based phylogeny showed that our species acted as the sister group to A. capensis and the supported Asio was the monophyletic group.

Inactivation of Genes Encoding Subunits of the Peripheral and Membrane Arms of Neurospora Mitochondrial Complex I and Effects on Enzyme Assembly

PubMed Central

Duarte, M.; Sousa, R.; Videira, A.

1995-01-01

We have isolated and characterized the nuclear genes encoding the 12.3-kD subunit of the membrane arm and the 29.9-kD subunit of the peripheral arm of complex I from Neurospora crassa. The former gene was known to be located in linkage group I and the latter is now assigned to linkage group IV of the fungal genome. The genes were separately transformed into different N. crassa strains and transformants with duplicated DNA sequences were isolated. Selected transformants were then mated with other strains to generate repeat-induced point mutations in both copies of the genes present in the nucleus of the parental transformant. From the progeny of the crosses, we were then able to recover two individual mutants lacking the 12.3- and 29.9-kD proteins in their mitochondria, mutants nuo12.3 and nuo29.9, respectively. Several other subunits of complex I are present in the mutant organelles, although with altered stoichiometries as compared with those in the wild-type strain. Based on the analysis of Triton-solubilized mitochondrial complexes in sucrose gradients, neither mutant is able to fully assemble complex I. Our results indicate that mutant nuo12.3 separately assembles the peripheral arm and most of the membrane arm of the enzyme. Mutant nuo29.9 seems to accumulate the membrane arm of complex I and being devoid of the peripheral part. This implicates the 29.9-kD protein in an early step of complex I assembly. PMID:7768434

Complete Mitochondrial Genome Sequences of Chinese Indigenous Sheep with Different Tail Types and an Analysis of Phylogenetic Evolution in Domestic Sheep.

PubMed

Fan, Hongying; Zhao, Fuping; Zhu, Caiye; Li, Fadi; Liu, Jidong; Zhang, Li; Wei, Caihong; Du, Lixin

2016-05-01

China has a long history of sheep (Ovis aries [O. aries]) breeding and an abundance of sheep genetic resources. Knowledge of the complete O. aries mitogenome should facilitate the study of the evolutionary history of the species. Therefore, the complete mitogenome of O. aries was sequenced and annotated. In order to characterize the mitogenomes of 3 Chinese sheep breeds (Altay sheep [AL], Shandong large-tailed sheep [SD], and small-tailed Hulun Buir sheep [sHL]), 19 sets of primers were employed to amplify contiguous, overlapping segments of the complete mitochondrial DNA (mtDNA) sequence of each breed. The sizes of the complete mitochondrial genomes of the sHL, AL, and SD breeds were 16,617 bp, 16,613 bp, and 16,613 bp, respectively. The mitochondrial genomes were deposited in the GenBank database with accession numbers KP702285 (AL sheep), KP981378 (SD sheep), and KP981380 (sHL sheep) respectively. The organization of the 3 analyzed sheep mitochondrial genomes was similar, with each consisting of 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes, and 1 control region (D-loop). The NADH dehydrogenase subunit 6 (ND6) and 8 tRNA genes were encoded on the light strand, whereas the rest of the mitochondrial genes were encoded on the heavy strand. The nucleotide skewness of the coding strands of the 3 analyzed mitogenomes was biased toward A and T. We constructed a phylogenetic tree using the complete mitogenomes of each type of sheep to allow us to understand the genetic relationships between Chinese breeds of O. aries and those developed and utilized in other countries. Our findings provide important information regarding the O. aries mitogenome and the evolutionary history of O. aries inside and outside China. In addition, our results provide a foundation for further exploration of the taxonomic status of O. aries.

Complete Mitochondrial Genome Sequences of Chinese Indigenous Sheep with Different Tail Types and an Analysis of Phylogenetic Evolution in Domestic Sheep

PubMed Central

Fan, Hongying; Zhao, Fuping; Zhu, Caiye; Li, Fadi; Liu, Jidong; Zhang, Li; Wei, Caihong; Du, Lixin

2016-01-01

China has a long history of sheep (Ovis aries [O. aries]) breeding and an abundance of sheep genetic resources. Knowledge of the complete O. aries mitogenome should facilitate the study of the evolutionary history of the species. Therefore, the complete mitogenome of O. aries was sequenced and annotated. In order to characterize the mitogenomes of 3 Chinese sheep breeds (Altay sheep [AL], Shandong large-tailed sheep [SD], and small-tailed Hulun Buir sheep [sHL]), 19 sets of primers were employed to amplify contiguous, overlapping segments of the complete mitochondrial DNA (mtDNA) sequence of each breed. The sizes of the complete mitochondrial genomes of the sHL, AL, and SD breeds were 16,617 bp, 16,613 bp, and 16,613 bp, respectively. The mitochondrial genomes were deposited in the GenBank database with accession numbers KP702285 (AL sheep), KP981378 (SD sheep), and KP981380 (sHL sheep) respectively. The organization of the 3 analyzed sheep mitochondrial genomes was similar, with each consisting of 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA), 13 protein-coding genes, and 1 control region (D-loop). The NADH dehydrogenase subunit 6 (ND6) and 8 tRNA genes were encoded on the light strand, whereas the rest of the mitochondrial genes were encoded on the heavy strand. The nucleotide skewness of the coding strands of the 3 analyzed mitogenomes was biased toward A and T. We constructed a phylogenetic tree using the complete mitogenomes of each type of sheep to allow us to understand the genetic relationships between Chinese breeds of O. aries and those developed and utilized in other countries. Our findings provide important information regarding the O. aries mitogenome and the evolutionary history of O. aries inside and outside China. In addition, our results provide a foundation for further exploration of the taxonomic status of O. aries. PMID:26954183

The complete sequences and gene organisation of the mitochondrial genomes of the heterodont bivalves Acanthocardia tuberculata and Hiatella arctica – and the first record for a putative Atpase subunit 8 gene in marine bivalves

PubMed Central

Dreyer, Hermann; Steiner, Gerhard

2006-01-01

Background Mitochondrial (mt) gene arrangement is highly variable among molluscs and especially among bivalves. Of the 30 complete molluscan mt-genomes published to date, only one is of a heterodont bivalve, although this is the most diverse taxon in terms of species numbers. We determined the complete sequence of the mitochondrial genomes of Acanthocardia tuberculata and Hiatella arctica, (Mollusca, Bivalvia, Heterodonta) and describe their gene contents and genome organisations to assess the variability of these features among the Bivalvia and their value for phylogenetic inference. Results The size of the mt-genome in Acanthocardia tuberculata is 16.104 basepairs (bp), and in Hiatella arctica 18.244 bp. The Acanthocardia mt-genome contains 12 of the typical protein coding genes, lacking the Atpase subunit 8 (atp8) gene, as all published marine bivalves. In contrast, a complete atp8 gene is present in Hiatella arctica. In addition, we found a putative truncated atp8 gene when re-annotating the mt-genome of Venerupis philippinarum. Both mt-genomes reported here encode all genes on the same strand and have an additional trnM. In Acanthocardia several large non-coding regions are present. One of these contains 3.5 nearly identical copies of a 167 bp motive. In Hiatella, the 3' end of the NADH dehydrogenase subunit (nad)6 gene is duplicated together with the adjacent non-coding region. The gene arrangement of Hiatella is markedly different from all other known molluscan mt-genomes, that of Acanthocardia shows few identities with the Venerupis philippinarum. Phylogenetic analyses on amino acid and nucleotide levels robustly support the Heterodonta and the sister group relationship of Acanthocardia and Venerupis. Monophyletic Bivalvia are resolved only by a Bayesian inference of the nucleotide data set. In all other analyses the two unionid species, being to only ones with genes located on both strands, do not group with the remaining bivalves. Conclusion The two mt-genomes reported here add to and underline the high variability of gene order and presence of duplications in bivalve and molluscan taxa. Some genomic traits like the loss of the atp8 gene or the encoding of all genes on the same strand are homoplastic among the Bivalvia. These characters, gene order, and the nucleotide sequence data show considerable potential of resolving phylogenetic patterns at lower taxonomic levels. PMID:16948842

Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

PubMed

Jia, Yuzhi; Chang, Hsiang-Chun; Schipma, Matthew J; Liu, Jing; Shete, Varsha; Liu, Ning; Sato, Tatsuya; Thorp, Edward B; Barger, Philip M; Zhu, Yi-Jun; Viswakarma, Navin; Kanwar, Yashpal S; Ardehali, Hossein; Thimmapaya, Bayar; Reddy, Janardan K

2016-01-01

Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart. Collectively, these observations suggest that Med1 plays a critical role in the maintenance of heart function impacting on multiple metabolic, compensatory and reparative pathways with a likely therapeutic potential in the management of heart failure.

Historical and contemporary demography of United States populations of Western Corn Rootworm (Diabrotica virgifera virgifera)

EPA Science Inventory

Western corn rootworm (Diabrotica virgifera virgifera; WCR) was sampled across much of its U.S. range for population genetic analyses. We assayed sequence variation at the mitochondrial cytochrome oxidase subunit I (COI) locus and allelic variation at eleven microsatellite loci. ...

Treatment strategies for inherited optic neuropathies: past, present and future

PubMed Central

Yu-Wai-Man, P; Votruba, M; Moore, A T; Chinnery, P F

2014-01-01

Bilateral visual loss secondary to inherited optic neuropathies is an important cause of registrable blindness among children and young adults. The two prototypal disorders seen in clinical practice are Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (DOA). About 90% of LHON cases are due to one of three mitochondrial DNA (mtDNA) point mutations: m.3460G>A, m.11778G>A, and m.14484T>C, which affect critical complex I subunits of the mitochondrial respiratory chain. The majority of patients with DOA harbour pathogenic mutations within OPA1, a nuclear gene that codes for a multifunctional inner mitochondrial membrane protein. Despite their contrasting genetic basis, LHON and DOA share overlapping pathological and clinical features that serve to highlight the striking tissue-specific vulnerability of the retinal ganglion cell (RGC) layer to disturbed mitochondrial function. In addition to severe visual loss secondary to progressive optic nerve degeneration, a subgroup of patients will also develop a more aggressive syndromic phenotype marked by significant neurological deficits. The management of LHON and DOA remains largely supportive, but major advances in our understanding of the mechanisms underpinning RGC loss in these two disorders are paving the way for novel forms of treatment aimed at halting or reversing visual deterioration at different stages of the disease process. In addition to neuroprotective strategies for rescuing RGCs from irreversible cell death, innovative in vitro fertilisation techniques are providing the tantalising prospect of preventing the germline transmission of pathogenic mtDNA mutations, eradicating in so doing the risk of disease in future generations. PMID:24603424

Molecular gene organisation and secondary structure of the mitochondrial large subunit ribosomal RNA from the cultivated Basidiomycota Agrocybe aegerita: a 13 kb gene possessing six unusual nucleotide extensions and eight introns.

PubMed

Gonzalez, P; Barroso, G; Labarère, J

1999-04-01

The complete gene sequence and secondary structure of the mitochondrial LSU rRNA from the cultivated Basidiomycota Agrocybe aegerita was derived by chromosome walking. The A.aegerita LSU rRNA gene (13 526 nt) represents, to date, the longest described, due to the highest number of introns (eight) and the occurrence of six long nucleotidic extensions. Seven introns belong to group I, while the intronic sequence i5 constitutes the first typical group II intron reported in a fungal mitochondrial LSU rDNA. As with most fungal LSU rDNA introns reported to date, four introns (i5-i8) are distributed in domain V associated with the peptidyl-transferase activity. One intron (i1) is located in domain I, and three (i2-i4) in domain II. The introns i2-i8 possess homologies with other fungal, algal or protozoan introns located at the same position in LSU rDNAs. One of them (i6) is located at the same insertion site as most Ascomycota or algae LSU introns, suggesting a possible inheritance from a common ancestor. On the contrary, intron i1 is located at a so-far unreported insertion site. Among the six unusual nucleotide extensions, five are located in domain I and one in domain V. This is the first report of a mitochondrial LSU rRNA gene sequence and secondary structure for the whole Basidiomycota division.

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia.

PubMed

Moberg, Per; Nilsson, Stefan; Ståhl, Annelie; Eriksson, Anna-Carin; Glaser, Elzbieta; Mäler, Lena

2004-03-05

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.

A novel deficiency of mitochondrial ATPase of nuclear origin.

PubMed

Houstek, J; Klement, P; Floryk, D; Antonická, H; Hermanská, J; Kalous, M; Hansíková, H; Hout'ková, H; Chowdhury, S K; Rosipal, T; Kmoch, S; Stratilová, L; Zeman, J

1999-10-01

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

An mtDNA mutation accelerates liver aging by interfering with the ROS response and mitochondrial life cycle.

PubMed

Niemann, Jan; Johne, Cindy; Schröder, Susanne; Koch, Franziska; Ibrahim, Saleh M; Schultz, Julia; Tiedge, Markus; Baltrusch, Simone

2017-01-01

Mitochondrial dysfunction affects liver metabolism, but it remains unclear whether this interferes with normal liver aging. We investigated several mitochondrial pathways in hepatocytes and liver tissue from a conplastic mouse strain compared with the control C57BL/6NTac strain over 18 months of life. The C57BL/6NTac-mtNODLtJ mice differed from C57BL/6NTac mice by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase. Young C57BL/6NTac-mtNODLtJ mice showed reduced mitochondrial metabolism but similar reactive oxygen species (ROS) production to C57BL/6NTac mice. Whereas ROS increased almost equally up to 9 months in both strains, different mitochondrial adaptation strategies resulted in decreasing ROS in advanced age in C57BL/6NTac mice, but persistent ROS production in C57BL/6NTac-mtNODLtJ mice. Only the conplastic strain developed elongated mitochondrial networks with artificial loop structures, depressed autophagy, high mitochondrial respiration and up-regulated antioxidative response. Our results indicate that mtDNA mutations accelerate liver ballooning degeneration and carry a serious risk of premature organ aging. Copyright © 2016 Elsevier Inc. All rights reserved.

Frequent heteroplasmy and recombination in the mitochondrial genomes of the basidiomycete mushroom Thelephora ganbajun.

PubMed

Wang, Pengfei; Sha, Tao; Zhang, Yunrun; Cao, Yang; Mi, Fei; Liu, Cunli; Yang, Dan; Tang, Xiaozhao; He, Xiaoxia; Dong, Jianyong; Wu, Jinyan; Yoell, Shanze; Yoell, Liam; Zhang, Ke-Qin; Zhang, Ying; Xu, Jianping

2017-05-09

In the majority of sexual eukaryotes, the mitochondrial genomes are inherited uniparentally. As a result, individual organisms are homoplasmic, containing mitochondrial DNA (mtDNA) from a single parent. Here we analyzed the mitochondrial genotypes in Clade I of the gourmet mushroom Thelephora ganbajun from its broad geographic distribution range. A total of 299 isolates from 28 geographic locations were sequenced at three mitochondrial loci: the mitochondrial small ribosomal RNA gene, and the cytochrome c oxidase subunits I (COX1) and III (COX3) genes. Quantitative PCR analyses showed that the strains had about 60-160 copies of mitochondrial genomes per cell. Interestingly, while no evidence of heteroplasmy was found at the 12S rRNA gene, 262 of the 299 isolates had clear evidence of heterogeneity at either the COX1 (261 isolates) or COX3 (12 isolates) gene fragments. The COX1 heteroplasmy was characterized by two types of introns residing at different sites of the same region and at different frequencies among the isolates. Allelic association analyses of the observed mitochondrial polymorphic nucleotide sites suggest that mtDNA recombination is common in natural populations of this fungus. Our results contrast the prevailing view that heteroplasmy, if exists, is only transient in basidiomycete fungi.

Maple syrup urine disease: The E1{beta} gene of human branched-chain {alpha}-ketoacid dehydrogenase complex has 11 rather than 10 exons, and the 3{prime} UTR in one of the two E1{beta} mRNAs arises from intronic sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, J.L.; Chuang, D.T.; Cox, R.P.

1996-06-01

Maple syrup urine disease (MSUD) or branched-chain ketoaciduria is caused by a deficiency in the mitochondrial branched-chain {alpha}-ketoacid dehydrogenase (BCKAD) complex. The clinical manifestations are characterized by accumulation of branched chain amino and {alpha}-ketoacids, which leads to severe cerebral edema with seizures, ketoacidosis, and mental retardation. The BCKAD complex comprises three catalytic components, i.e., a decarboxylase (E1) consisting of two E1{alpha} (M{sub r} = 46,000) and two E1{Beta} (M{sub r} = 37,500) subunits, a transacylase (E2) that contains 24 lipoic acid-bearing subunits, and a dehydrogenase (E3), which is a homodimeric flavoprotein. MSUD is genetically heterogeneous, since mutations in the E1{alpha}more » subunit (type IA MSUD), the E1{Beta} subunit (type IB), the E2 subunit (type II) and the E3 subunit (type III) have been described. The functional consequences of certain mutations in the BCKAD complex have been studied. 23 refs., 3 figs.« less

Evolutionary implications of mitochondrial genetic variation: mitochondrial genetic effects on OXPHOS respiration and mitochondrial quantity change with age and sex in fruit flies.

PubMed

Wolff, J N; Pichaud, N; Camus, M F; Côté, G; Blier, P U; Dowling, D K

2016-04-01

The ancient acquisition of the mitochondrion into the ancestor of modern-day eukaryotes is thought to have been pivotal in facilitating the evolution of complex life. Mitochondria retain their own diminutive genome, with mitochondrial genes encoding core subunits involved in oxidative phosphorylation. Traditionally, it was assumed that there was little scope for genetic variation to accumulate and be maintained within the mitochondrial genome. However, in the past decade, mitochondrial genetic variation has been routinely tied to the expression of life-history traits such as fertility, development and longevity. To examine whether these broad-scale effects on life-history trait expression might ultimately find their root in mitochondrially mediated effects on core bioenergetic function, we measured the effects of genetic variation across twelve different mitochondrial haplotypes on respiratory capacity and mitochondrial quantity in the fruit fly, Drosophila melanogaster. We used strains of flies that differed only in their mitochondrial haplotype, and tested each sex separately at two different adult ages. Mitochondrial haplotypes affected both respiratory capacity and mitochondrial quantity. However, these effects were highly context-dependent, with the genetic effects contingent on both the sex and the age of the flies. These sex- and age-specific genetic effects are likely to resonate across the entire organismal life-history, providing insights into how mitochondrial genetic variation may contribute to sex-specific trajectories of life-history evolution. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.

Localized Cerebral Energy Failure in DNA Polymerase Gamma-Associated Encephalopathy Syndromes

ERIC Educational Resources Information Center

Tzoulis, Charalampos; Neckelmann, Gesche; Mork, Sverre J.; Engelsen, Bernt E.; Viscomi, Carlo; Moen, Gunnar; Ersland, Lars; Zeviani, Massimo; Bindoff, Laurence A.

2010-01-01

Mutations in the catalytic subunit of the mitochondrial DNA-polymerase gamma cause a wide spectrum of clinical disease ranging from infantile hepato-encephalopathy to juvenile/adult-onset spinocerebellar ataxia and late onset progressive external ophthalmoplegia. Several of these syndromes are associated with an encephalopathy that…

Phylogenetic relationships and taxonomic revision of Paranoplocephala Lühe, 1910 sensu lato (Cestoda, Cyclophyllidea, Anoplocephalidae)

USDA-ARS?s Scientific Manuscript database

An extensive phylogenetic analysis and genus-level taxonomic revision of Paranoplocephala Lühe, 1910 -like cestodes (Cyclophyllidea, Anoplocephalidae) are presented. The phylogenetic analysis is based on DNA sequences of two partial mitochondrial genes, i.e. cytochrome c oxidase subunit 1 (cox1) and...

Tryptophan 334 Oxidation in Bovine Cytochrome c Oxidase Subunit I Involves Free Radical Migration

PubMed Central

Lemma-Gray, Patrizia; Weintraub, Susan T.; Carroll, Christopher A.; Musatov, Andrej; Robinson, Neal C.

2007-01-01

A single tryptophan (W334(I)) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W334(I) is converted to hydroxytryptophan as identified by HPLC-ESI/MS/MS analysis of peptides derived from the three SDS-PAGE purified subunits (total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively). W334(I) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W48(IV) and W19(VIIc), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et. al., 2004, Biochemistry 43, 1003–1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan. PMID:17239857

The metabolite α-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR.

PubMed

Chin, Randall M; Fu, Xudong; Pai, Melody Y; Vergnes, Laurent; Hwang, Heejun; Deng, Gang; Diep, Simon; Lomenick, Brett; Meli, Vijaykumar S; Monsalve, Gabriela C; Hu, Eileen; Whelan, Stephen A; Wang, Jennifer X; Jung, Gwanghyun; Solis, Gregory M; Fazlollahi, Farbod; Kaweeteerawat, Chitrada; Quach, Austin; Nili, Mahta; Krall, Abby S; Godwin, Hilary A; Chang, Helena R; Faull, Kym F; Guo, Feng; Jiang, Meisheng; Trauger, Sunia A; Saghatelian, Alan; Braas, Daniel; Christofk, Heather R; Clarke, Catherine F; Teitell, Michael A; Petrascheck, Michael; Reue, Karen; Jung, Michael E; Frand, Alison R; Huang, Jing

2014-06-19

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

A phylogenetic analysis of armored scale insects (Hemiptera: Diaspididae), based upon nuclear, mitochondrial, and endosymbiont gene sequences.

PubMed

Andersen, Jeremy C; Wu, Jin; Gruwell, Matthew E; Gwiazdowski, Rodger; Santana, Sharlene E; Feliciano, Natalie M; Morse, Geoffrey E; Normark, Benjamin B

2010-12-01

Armored scale insects (Hemiptera: Diaspididae) are among the most invasive insects in the world. They have unusual genetic systems, including diverse types of paternal genome elimination (PGE) and parthenogenesis. Intimate relationships with their host plants and bacterial endosymbionts make them potentially important subjects for the study of co-evolution. Here, we expand upon recent phylogenetic work (Morse and Normark, 2006) by analyzing armored scale and endosymbiont DNA sequences from 125 species of armored scale insect, represented by 253 samples and eight outgroup species. We used fragments of four different gene regions: the nuclear protein-coding gene Elongation Factor 1α (EF1α), the large ribosomal subunit (28S) rDNA, a mitochondrial region spanning parts of cytochrome oxidase I (COI) and cytochrome oxidase II (COII), and the small ribosomal subunit (16S) rDNA from the primary bacterial endosymbiont Uzinura diaspidicola. Maximum likelihood, and Bayesian analyses were performed producing highly congruent topological results. A comparison of two datasets, one with and one without missing data, found that missing data had little effect on topology. Our results broadly corroborate several major features of the existing classification, although we do not find any of the subfamilies, tribes or subtribes to be monophyletic as currently constituted. Using ancestral state reconstruction we estimate that the ancestral armored scale had the late PGE sex system, and it may as well have been pupillarial, though results differed between reconstruction methods. These results highlight the need for a complete revision of this family, and provide the groundwork for future taxonomic work in armored scale insects. Copyright © 2010 Elsevier Inc. All rights reserved.

Molecular analyses reveal two geographic and genetic lineages for tapeworms, Taenia solium and Taenia saginata, from Ecuador using mitochondrial DNA.

PubMed

Solano, Danilo; Navarro, Juan Carlos; León-Reyes, Antonio; Benítez-Ortiz, Washington; Rodríguez-Hidalgo, Richar

2016-12-01

Tapeworms Taenia solium and Taenia saginata are the causative agents of taeniasis/cysticercosis. These are diseases with high medical and veterinary importance due to their impact on public health and rural economy in tropical countries. The re-emergence of T. solium as a result of human migration, the economic burden affecting livestock industry, and the large variability of symptoms in several human cysticercosis, encourage studies on genetic diversity, and the identification of these parasites with molecular phylogenetic tools. Samples collected from the Ecuadorian provinces: Loja, Guayas, Manabí, Tungurahua (South), and Imbabura, Pichincha (North) from 2000 to 2012 were performed under Maximum Parsimony analyses and haplotype networks using partial sequences of mitochondrial DNA, cytochrome oxidase subunit I (COI) and NADH subunit I (NDI), from Genbank and own sequences of Taenia solium and Taenia saginata from Ecuador. Both species have shown reciprocal monophyly, which confirms its molecular taxonomic identity. The COI and NDI genes results suggest phylogenetic structure for both parasite species from south and north of Ecuador. In T. solium, both genes gene revealed greater geographic structure, whereas in T. saginata, the variability for both genes was low. In conclusion, COI haplotype networks of T. solium suggest two geographical events in the introduction of this species in Ecuador (African and Asian lineages) and occurring sympatric, probably through the most common routes of maritime trade between the XV-XIX centuries. Moreover, the evidence of two NDI geographical lineages in T. solium from the north (province of Imbabura) and the south (province of Loja) of Ecuador derivate from a common Indian ancestor open new approaches for studies on genetic populations and eco-epidemiology. Copyright © 2016 Elsevier Inc. All rights reserved.

Subunit association of gamma-glutamyltranspeptidase of Escherichia coli K-12.

PubMed

Hashimoto, W; Suzuki, H; Nohara, S; Tachi, H; Yamamoto, K; Kumagai, H

1995-12-01

gamma-Glutamyltranspeptidase [EC 2.3.2.2] of Escherichia coli K-12 consists of one large subunit and one small subunit, which can be separated from each other by high-performance liquid chromatography. Using ion spray mass spectrometry, the masses of the large and the small subunit were determined to be 39,207 and 20,015, respectively. The large subunit exhibited no gamma-glutamyltranspeptidase activity and the small subunit had little enzymatic activity, but a mixture of the two subunits showed partial recovery of the enzymatic activity. The results of native-polyacrylamide gel electrophoresis suggested that they could partially recombine, and that the recombined dimer exhibited enzymatic activity. The gene of gamma-glutamyltranspeptidase encoded a signal peptide, and the large and small subunits in a single open reading frame in that order. Two kinds of plasmid were constructed encoding the signal peptide and either the large or the small subunit. A gamma-glutamyltranspeptidase-less mutant of E. coli K-12 was transformed with each plasmid or with both of them. The strain harboring the plasmid encoding each subunit produced a small amount of the corresponding subunit protein in the periplasmic space but exhibited no enzymatic activity. The strain transformed with both plasmids together exhibited the enzymatic activity, but its specific activity was approximately 3% of that of a strain harboring a plasmid encoding the intact structural gene. These results indicate that a portion of the separated large and small subunits can be reconstituted in vitro and exhibit the enzymatic activity, and that the expressed large and small subunits independently are able to associate in vivo and be folded into an active structure, though the specific activity of the associated subunits was much lower than that of native enzyme. This suggests that the synthesis of gamma-glutamyltranspeptidase in a single precursor polypeptide and subsequent processing are more effective to construct the intact structure of gamma-glutamyltranspeptidase than the association of the separated large and small subunits.

Intraspecific Variation of Eysarcoris guttigerus (Hemiptera: Pentatomidae) in Japanese Southwest Population Based on Mitochondrial DNA

PubMed Central

Yamaji, Takuya; Ishikawa, Tadashi; Nomura, Masashi

2016-01-01

The white-spotted globular bug Eysarcoris guttigerus (Thunberg) (Hemiptera: Pentatomidae) is widely distributed in East Asia and the Pacific region. In Japan, the species is found in grassy or composite weeds in the western area of the main islands and Ryukyu Islands of Japan. One notable characteristic of the Eysarcoris genus is the two white spots on the scutellum. This is not the case with the Ishigaki Island population, however, which sports red spots instead of white, suggesting that intraspecific variation exists in the species. Therefore, we investigated intraspecific variation in E. guttigerus using mitochondrial NADH dehydrogenase subunit 2 (ND2), cytochrome oxidase subunit 1 (CO1), cytochrome b (Cytb), tRNA-Serine (tRNAser), NADH dehydrogenase subunit 1 (ND1), and 16S ribosomal RNA (16SrRNA) genes from 13 populations of Japan. The obtained maximum likelihood phylogenetic tree was divided into three groups—Group 1: Mainland, Group 2: Central Ryukyu Islands (Okinawa-Amamioshima Islands), and Group 3: South Ryukyu Islands (Ishigaki Island). The Ishigaki population was significantly separated from the other populations with consistent differences in spot color. The estimated period of divergence between the Ishigaki population and the other populations was consistent with the period of formation of the Kerama Gap in the Ryukyu arc. Thus, the process of formation of the Kerama Gap may have influenced the intraspecific variation of E. guttigerus. PMID:26798143

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jack Preiss

Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit canmore » be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from the catalytic ancestor. Previous results showed that Asp145 in the small subunit of the wild-type is essential for catalysis, whereas the homologous Asp160 in the Large WT subunit is not. However, in this study, mutation D160N or D160E in the LK44R/T54K subunit abolished the activity, which shows the ancestral essential role of this residue and confirms that the catalysis of SmallD145NLarge K44R/T54K occurs in the L(b) subunit. A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits. The tree showed that plant Small and Large subunits can be divided into two and four distinct groups, respectively. The two main groups of S subunits are from dicot and monocot plants, whereas Large subunit groups correlate better with their documented tissue expression. The first Large-subunit group is generally expressed in photosynthetic tissues and comprises Large subunits from dicots and monocots. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. Thus few as two mutations turned the L subunit from Solanum tuberosum catalytic, showing that L and S subunits share a common catalytic ancestor, rather than a non-catalytic one. The L subunit evolved to have a regulatory role, lost catalytic residues more than 130 million years ago before monocots and dicots diverged, and preserved, possibly as a byproduct, the active site domain.« less

Dissecting tumor metabolic heterogeneity: Telomerase and large cell size metabolically define a sub-population of stem-like, mitochondrial-rich, cancer cells

PubMed Central

Lamb, Rebecca; Ozsvari, Bela; Bonuccelli, Gloria; Smith, Duncan L.; Pestell, Richard G.; Martinez-Outschoorn, Ubaldo E.; Clarke, Robert B.; Sotgia, Federica; Lisanti, Michael P.

2015-01-01

Tumor cell metabolic heterogeneity is thought to contribute to tumor recurrence, distant metastasis and chemo-resistance in cancer patients, driving poor clinical outcome. To better understand tumor metabolic heterogeneity, here we used the MCF7 breast cancer line as a model system to metabolically fractionate a cancer cell population. First, MCF7 cells were stably transfected with an hTERT-promoter construct driving GFP expression, as a surrogate marker of telomerase transcriptional activity. To enrich for immortal stem-like cancer cells, MCF7 cells expressing the highest levels of GFP (top 5%) were then isolated by FACS analysis. Notably, hTERT-GFP(+) MCF7 cells were significantly more efficient at forming mammospheres (i.e., stem cell activity) and showed increased mitochondrial mass and mitochondrial functional activity, all relative to hTERT-GFP(−) cells. Unbiased proteomics analysis of hTERT-GFP(+) MCF7 cells directly demonstrated the over-expression of 33 key mitochondrial proteins, 17 glycolytic enzymes, 34 ribosome-related proteins and 17 EMT markers, consistent with an anabolic cancer stem-like phenotype. Interestingly, MT-CO2 (cytochrome c oxidase subunit 2; Complex IV) expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-GFP(+) MCF7 cells. Importantly, most of these candidate biomarkers were transcriptionally over-expressed in human breast cancer epithelial cells in vivo. Similar results were obtained using cell size (forward/side scatter) to fractionate MCF7 cells. Larger stem-like cells also showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, this simple and rapid approach for the enrichment of immortal anabolic stem-like cancer cells will allow us and others to develop new prognostic biomarkers and novel anti-cancer therapies, by specifically and selectively targeting this metabolic sub-population of aggressive cancer cells. Based on our proteomics and functional analysis, FDA-approved inhibitors of protein synthesis and/or mitochondrial biogenesis, may represent novel treatment options for targeting these anabolic stem-like cancer cells. PMID:26323205

The complete mitogenome of the Australian tadpole shrimp Triops australiensis (Spencer & Hall, 1895) (Crustacea: Branchiopoda: Notostraca).

PubMed

Gan, Han Ming; Tan, Mun Hua; Lee, Yin Peng; Austin, Christopher M

2016-05-01

The mitochondrial genome sequence of the Australian tadpole shrimp, Triops australiensis is presented (GenBank Accession Number: NC_024439) and compared with other Triops species. Triops australiensis has a mitochondrial genome of 15,125 base pairs consisting of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a non-coding AT-rich region. The T. australiensis mitogenome is composed of 36.4% A, 16.1% C, 12.3% G and 35.1% T. The mitogenome gene order conforms to the primitive arrangement for Branchiopod crustaceans, which is also conserved within the Pancrustacean.

The complete mitochondrial genome of black-footed ferret, Mustela nigripes (Mustela, Mustelinae).

PubMed

Zhao, Ren-Bin; Zhou, Chao-Yang; Lu, Zhi-Xiang; Hu, Peng; Liu, Jian-Qiong; Tan, Wei-Wei; Yang, Tong-Hua

2016-05-01

In this study, the complete mitochondrial genome sequence of black-footed ferret, Mustela nigripes, is determined for the first time. This mitogenome is 16,556 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The overall base composition is A (32.9%), C (26.1%), G (13.8%), and T (27.2%), so the percentage of A and T (60.1%) is higher than that of G and C. Most of the genes are encoded on H-strand, except for the ND6 subunit gene and six tRNA genes. The complete mitochondrial genome sequence reported here would be useful for further phylogenetic analysis and conservation genetic studies in M. nigripes.

Fine structure of OXI1, the mitochondrial gene coding for subunit II of yeast cytochrome c oxidase.

PubMed

Weiss-Brummer, B; Guba, R; Haid, A; Schweyen, R J

1979-12-01

Genetic and biochemical studies have been performed with 110 mutants which are defective in cytochrome a·a3 and map in the regions on mit DNA previously designated OXI1 and OXI2. With 88 mutations allocated to OXI1 fine structure mapping was achieved by the analysis of rho (-) deletions. The order of six groups of mutational sites (A 1, A2, B 1, B2, C 1, C2) thus determined was confirmed by oxi i x oxi j recombination analysis.Analysis of mitochondrially translated polypeptides of oxil mutants by SDS-polyacrylamide electrophoresis reveals three classes of mutant patterns: i) similar to wild-tpye (19 mutants); ii) lacking SU II of cytochrome c oxidase (53 mutants); iii) lacking this subunit and exhibiting a single new polypeptide of lower Mr (16 mutants). Mutations of each of these classes are scattered over the OXI1 region without any detectable clustering; this is consistent with the assumption that all oxil mutations studied are within the same gene.New polypeptides observed in oxil mutants of class iii) vary in Mr in the range from 10,500 to 33,000. Those of Mr 17,000 to 33,000 are shown to be antigenically related to subunit II of cytochrome c oxidase. Colinearity is established between the series of new polypeptides of Mr values increasing from 10,500 to 31,500 and the order of the respective mutational sites on the map, e.g. mutations mapping in A 1 generate the smallest and mutations mapping in C2 the largest mutant fragments.From these data we conclude that i) all mutations allocated to the OXI1 region are in the same gene; ii) this gene codes for subunit II of cytochrome c oxidase; iii) the direction of translation is from CAP to 0X12. Out of 19 mutants allocated to OXI2 three exhibit a new polypeptide; these and all the other oxi2 mutants lack subunit III of cytochrome oxidase. This result provides preliminary evidence that the OXI2 region harbours the structural gene for this subunit III.

Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism.

PubMed

Shirakihara, Yasuo; Shiratori, Aya; Tanikawa, Hiromi; Nakasako, Masayoshi; Yoshida, Masasuke; Suzuki, Toshiharu

2015-08-01

F1-ATPase (F1) is the catalytic sector in F(o)F1-ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β-subunits undergo nucleotide occupancy-dependent and concerted open-close conformational changes that are accompanied by rotation of the γ-subunit. Bacterial and chloroplast F1 are inhibited by their own ε-subunit. In the ε-inhibited Escherichia coli F1 structure, the ε-subunit stabilizes the overall conformation (half-closed, closed, open) of the β-subunits by inserting its C-terminal helix into the α3β3 cavity. The structure of ε-inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε-subunit, but the thermophilic ε-subunit stabilizes another unique overall conformation (open, closed, open) of the β-subunits. The ε-C-terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε-C-terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε-inhibition differently. For inhibition, the ε-subunit contacts the second catches of some of the β- and α-subunits, the N- and C-terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β- and α- second catches in ε-inhibition-specific positions, and prevent rotation of the γ-subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1. © 2015 FEBS.

Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction partners of phosphorylated STAT5 in adipocytes and adipose tissue.

PubMed

Richard, Allison J; Hang, Hardy; Stephens, Jacqueline M

2017-12-01

STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene c ytokine- i nducible SH 2-containing protein ( cish ). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Isomeric forms of specifically. beta. -subunit labeled mitochondrial F/sub 1/-adenosinetriphosphatase with different properties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, J.H.; Wu, J.C.; Joshi, V.

1986-05-01

Treatment of the mitochondrial F/sub 1/-ATPase (MF/sub 1/) containing 1 specific 7-(4-nitro-2,1,3-(/sup 14/C)benzoxadiazolyl)-label (NBD) per enzyme molecule with acetylcysteine (AC) shows that the ratio r of specific ATPase activity of (O-NBD)/sub n/MF/sub 1/ to that of the control MF/sub 1/ increases linearly with the number of labels removed by AC from r < 0.1 to r > 0.9 and that dr/dn approx. = -1 as expected from specific labeling of an essential Tyr in the catalytic ..beta..' subunit. The r value of this labeled enzyme can also be increased 10-fold by LiCl-induced rearrangement of its subunits without removing any ofmore » the label. Similar treatment of the rearranged (O-NBD)/sub n/MF/sub 1/ shows that only a fraction of its radioactive labels can be removed at the normal rate by AC with dr/dn approx. = -1. The remaining labels have little inhibitory effect and are removed at much slower rates by AC with dr/dn approx. = 0. If the reaction with the rearranged (O-NBD)/sub n/MF/sub 1/ is terminated by gel-filtration when most of the labels on ..beta..' have been removed, an isomeric form of the covalently labeled enzyme is obtained with n > 0.5 but r approx. = 1, indicating that its labels are on the subunits (..beta..'') which do not catalyze directly. Incubation of O-..beta..'-NBD-MF/sub 1/ and O-BETA''-NBD-MF/sub 1/ at pH 8.95 gives N-..beta..'-NBD-MF/sub 1/ and N-..beta..''-NBD-MF/sub 1/ respectively with different fluorescence quenching characteristics.« less

Human mitochondrial NDUFS3 protein bearing Leigh syndrome mutation is more prone to aggregation than its wild-type.

PubMed

Jaokar, Tulika M; Patil, Deepak P; Shouche, Yogesh S; Gaikwad, Sushama M; Suresh, C G

2013-12-01

NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCl induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Mosaic Origins of a Complex Chimeric Mitochondrial Gene in Silene vulgaris

PubMed Central

Storchova, Helena; Müller, Karel; Lau, Steffen; Olson, Matthew S.

2012-01-01

Chimeric genes are significant sources of evolutionary innovation that are normally created when portions of two or more protein coding regions fuse to form a new open reading frame. In plant mitochondria astonishingly high numbers of different novel chimeric genes have been reported, where they are generated through processes of rearrangement and recombination. Nonetheless, because most studies do not find or report nucleotide variation within the same chimeric gene, evolution after the origination of these chimeric genes remains unstudied. Here we identify two alleles of a complex chimera in Silene vulgaris that are divergent in nucleotide sequence, genomic position relative to other mitochondrial genes, and expression patterns. Structural patterns suggest a history partially influenced by gene conversion between the chimeric gene and functional copies of subunit 1 of the mitochondrial ATP synthase gene (atp1). We identified small repeat structures within the chimeras that are likely recombination sites allowing generation of the chimera. These results establish the potential for chimeric gene divergence in different plant mitochondrial lineages within the same species. This result contrasts with the absence of diversity within mitochondrial chimeras found in crop species. PMID:22383961

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture.

PubMed

Neuenfeldt, Anne; Lorber, Bernard; Ennifar, Eric; Gaudry, Agnès; Sauter, Claude; Sissler, Marie; Florentz, Catherine

2013-02-01

In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs.

Anti-cancer analogues ME-143 and ME-344 exert toxicity by directly inhibiting mitochondrial NADH: ubiquinone oxidoreductase (Complex I).

PubMed

Lim, Sze Chern; Carey, Kirstyn T; McKenzie, Matthew

2015-01-01

Isoflavonoids have been shown to inhibit tumor proliferation and metastasis by activating cell death pathways. As such, they have been widely studied as potential therapies for cancer prevention. The second generation synthetic isoflavan analogues ME-143 and ME-344 also exhibit anti-cancer effects, however their specific molecular targets have not been completely defined. To identify these targets, we examined the effects of ME-143 and ME-344 on cellular metabolism and found that they are potent inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) complex I (NADH: ubiquinone oxidoreductase) activity. In isolated HEK293T mitochondria, ME-143 and ME-344 reduced complex I activity to 14.3% and 28.6% of control values respectively. In addition to the inhibition of complex I, ME-344 also significantly inhibited mitochondrial complex III (ubiquinol: ferricytochrome-c oxidoreductase) activity by 10.8%. This inhibition of complex I activity (and to a lesser extent complex III activity) was associated with a reduction in mitochondrial oxygen consumption. In permeabilized HEK293T cells, ME-143 and ME-344 significantly reduced the maximum ADP-stimulated respiration rate to 62.3% and 70.0% of control levels respectively in the presence of complex I-linked substrates. Conversely, complex II-linked respiration was unaffected by either drug. We also observed that the inhibition of complex I-linked respiration caused the dissipation of the mitochondrial membrane potential (ΔΨm). Blue native (BN-PAGE) analysis revealed that prolonged loss of ΔΨm results in the destabilization of the native OXPHOS complexes. In particular, treatment of 143B osteosarcoma, HeLa and HEK293T human embryonic kidney cells with ME-344 for 4 h resulted in reduced steady-state levels of mature complex I. Degradation of the complex I subunit NDUFA9, as well as the complex IV (ferrocytochrome c: oxygen oxidoreductase) subunit COXIV, was also evident. The identification of OXPHOS complex I as a target of ME-143 and ME-344 advances our understanding of how these drugs induce cell death by disrupting mitochondrial metabolism, and will direct future work to maximize the anti-cancer capacity of these and other isoflavone-based compounds.

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

USDA-ARS?s Scientific Manuscript database

Six DNA regions were evaluated in a multi-national, multi-laboratory consortium as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it...

Two founding events identified in Asian citrus psyllid populations collected in the Americas

USDA-ARS?s Scientific Manuscript database

A phylogeographic analysis inferred from the partial mitochondrial cytochrome oxidase subunit I gene (COI) (433-bp) was performed with 22 populations (n=121) of Diaphorina citri collected in the Americas. Eight populations (n=46) from four countries in South America and 14 populations (n=75) from f...

Structure-function insights into direct lipid transfer between membranes by Mmm1-Mdm12 of ERMES.

PubMed

Kawano, Shin; Tamura, Yasushi; Kojima, Rieko; Bala, Siqin; Asai, Eri; Michel, Agnès H; Kornmann, Benoît; Riezman, Isabelle; Riezman, Howard; Sakae, Yoshitake; Okamoto, Yuko; Endo, Toshiya

2018-03-05

The endoplasmic reticulum (ER)-mitochondrial encounter structure (ERMES) physically links the membranes of the ER and mitochondria in yeast. Although the ER and mitochondria cooperate to synthesize glycerophospholipids, whether ERMES directly facilitates the lipid exchange between the two organelles remains controversial. Here, we compared the x-ray structures of an ERMES subunit Mdm12 from Kluyveromyces lactis with that of Mdm12 from Saccharomyces cerevisiae and found that both Mdm12 proteins possess a hydrophobic pocket for phospholipid binding. However in vitro lipid transfer assays showed that Mdm12 alone or an Mmm1 (another ERMES subunit) fusion protein exhibited only a weak lipid transfer activity between liposomes. In contrast, Mdm12 in a complex with Mmm1 mediated efficient lipid transfer between liposomes. Mutations in Mmm1 or Mdm12 impaired the lipid transfer activities of the Mdm12-Mmm1 complex and furthermore caused defective phosphatidylserine transport from the ER to mitochondrial membranes via ERMES in vitro. Therefore, the Mmm1-Mdm12 complex functions as a minimal unit that mediates lipid transfer between membranes. © 2018 Kawano et al.

Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting.

PubMed

Dung, Vuu My; Suong, Dang Ngoc Anh; Okamaoto, Yuji; Hiramatsu, Yu; Thao, Dang Thi Phuong; Yoshida, Hideki; Takashima, Hiroshi; Yamaguchi, Masamitsu

2018-05-15

Pyruvate dehydrogenase complex deficiency (PDCD) is a common primary cause of defects in mitochondrial function and also can lead to peripheral neuropathy. Pyruvate dehydrogenase E1 component subunit beta (PDHB) is a subunit of pyruvate dehydrogenase E1, which is a well-known component of PDC. In Drosophila melanogaster, the CG11876 (dPDHB) gene is a homolog of human PDHB. In this study, we established a Drosophila model with neuron-specific knockdown of dPDHB to investigate its role in neuropathy pathogenesis. Knockdown of dPDHB in pan-neurons induced locomotor defects in both larval and adult stages, which were consistent with abnormal morphology of the motor neuron terminals at neuromuscular junctions and mitochondrial fragmentation in brains. Moreover, neuron-specific knockdown of dPDHB also shortened the lifespan of adult flies. In addition, flies with knockdown of dPDHB manifested a rough eye phenotype and aberrant photoreceptor axon targeting. These results with the Drosophila model suggest the involvement of PDHB in peripheral neuropathy. Copyright © 2018 Elsevier Inc. All rights reserved.

Dual interaction of scaffold protein Tim44 of mitochondrial import motor with channel-forming translocase subunit Tim23

PubMed Central

Ting, See-Yeun; Yan, Nicholas L; Schilke, Brenda A; Craig, Elizabeth A

2017-01-01

Proteins destined for the mitochondrial matrix are targeted to the inner membrane Tim17/23 translocon by their presequences. Inward movement is driven by the matrix-localized, Hsp70-based motor. The scaffold Tim44, interacting with the matrix face of the translocon, recruits other motor subunits and binds incoming presequence. The basis of these interactions and their functional relationships remains unclear. Using site-specific in vivo crosslinking and genetic approaches in Saccharomyces cerevisiae, we found that both domains of Tim44 interact with the major matrix-exposed loop of Tim23, with the C-terminal domain (CTD) binding Tim17 as well. Results of in vitro experiments showed that the N-terminal domain (NTD) is intrinsically disordered and binds presequence near a region important for interaction with Hsp70 and Tim23. Our data suggest a model in which the CTD serves primarily to anchor Tim44 to the translocon, whereas the NTD is a dynamic arm, interacting with multiple components to drive efficient translocation. DOI: http://dx.doi.org/10.7554/eLife.23609.001 PMID:28440746

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

PubMed

Kobayashi, Misato; Hoshinaga, Yukiko; Miura, Natsuko; Tokuda, Yuki; Shigeoka, Shigeru; Murai, Atsushi; Horio, Fumihiko

2014-01-01

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

A mitochondrial ATP synthase subunit interacts with TOR signaling to modulate protein homeostasis and lifespan in Drosophila.

PubMed

Sun, Xiaoping; Wheeler, Charles T; Yolitz, Jason; Laslo, Mara; Alberico, Thomas; Sun, Yaning; Song, Qisheng; Zou, Sige

2014-09-25

Diet composition is a critical determinant of lifespan, and nutrient imbalance is detrimental to health. However, how nutrients interact with genetic factors to modulate lifespan remains elusive. We investigated how diet composition influences mitochondrial ATP synthase subunit d (ATPsyn-d) in modulating lifespan in Drosophila. ATPsyn-d knockdown extended lifespan in females fed low carbohydrate-to-protein (C:P) diets but not the high C:P ratio diet. This extension was associated with increased resistance to oxidative stress; transcriptional changes in metabolism, proteostasis, and immune genes; reduced protein damage and aggregation, and reduced phosphorylation of S6K and ERK in TOR and mitogen-activated protein kinase (MAPK) signaling, respectively. ATPsyn-d knockdown did not extend lifespan in females with reduced TOR signaling induced genetically by Tsc2 overexpression or pharmacologically by rapamycin. Our data reveal a link among diet, mitochondria, and MAPK and TOR signaling in aging and stresses the importance of considering genetic background and diet composition in implementing interventions for promoting healthy aging. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

PubMed

Chaumont, F; Silva Filho, M de C; Thomas, D; Leterme, S; Boutry, M

1994-02-01

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

Mitochondrial DNA depletion by ethidium bromide decreases neuronal mitochondrial creatine kinase: Implications for striatal energy metabolism.

PubMed

Warren, Emily Booth; Aicher, Aidan Edward; Fessel, Joshua Patrick; Konradi, Christine

2017-01-01

Mitochondrial DNA (mtDNA), the discrete genome which encodes subunits of the mitochondrial respiratory chain, is present at highly variable copy numbers across cell types. Though severe mtDNA depletion dramatically reduces mitochondrial function, the impact of tissue-specific mtDNA reduction remains debated. Previously, our lab identified reduced mtDNA quantity in the putamen of Parkinson's Disease (PD) patients who had developed L-DOPA Induced Dyskinesia (LID), compared to PD patients who had not developed LID and healthy subjects. Here, we present the consequences of mtDNA depletion by ethidium bromide (EtBr) treatment on the bioenergetic function of primary cultured neurons, astrocytes and neuron-enriched cocultures from rat striatum. We report that EtBr inhibition of mtDNA replication and transcription consistently reduces mitochondrial oxygen consumption, and that neurons are significantly more sensitive to EtBr than astrocytes. EtBr also increases glycolytic activity in astrocytes, whereas in neurons it reduces the expression of mitochondrial creatine kinase mRNA and levels of phosphocreatine. Further, we show that mitochondrial creatine kinase mRNA is similarly downregulated in dyskinetic PD patients, compared to both non-dyskinetic PD patients and healthy subjects. Our data support a hypothesis that reduced striatal mtDNA contributes to energetic dysregulation in the dyskinetic striatum by destabilizing the energy buffering system of the phosphocreatine/creatine shuttle.

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

PubMed

Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

2017-12-08

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

Human primitive brain displays negative mitochondrial-nuclear expression correlation of respiratory genes.

PubMed

Barshad, Gilad; Blumberg, Amit; Cohen, Tal; Mishmar, Dan

2018-06-14

Oxidative phosphorylation (OXPHOS), a fundamental energy source in all human tissues, requires interactions between mitochondrial (mtDNA)- and nuclear (nDNA)-encoded protein subunits. Although such interactions are fundamental to OXPHOS, bi-genomic coregulation is poorly understood. To address this question, we analyzed ∼8500 RNA-seq experiments from 48 human body sites. Despite well-known variation in mitochondrial activity, quantity, and morphology, we found overall positive mtDNA-nDNA OXPHOS genes' co-expression across human tissues. Nevertheless, negative mtDNA-nDNA gene expression correlation was identified in the hypothalamus, basal ganglia, and amygdala (subcortical brain regions, collectively termed the "primitive" brain). Single-cell RNA-seq analysis of mouse and human brains revealed that this phenomenon is evolutionarily conserved, and both are influenced by brain cell types (involving excitatory/inhibitory neurons and nonneuronal cells) and by their spatial brain location. As the "primitive" brain is highly oxidative, we hypothesized that such negative mtDNA-nDNA co-expression likely controls for the high mtDNA transcript levels, which enforce tight OXPHOS regulation, rather than rewiring toward glycolysis. Accordingly, we found "primitive" brain-specific up-regulation of lactate dehydrogenase B ( LDHB ), which associates with high OXPHOS activity, at the expense of LDHA , which promotes glycolysis. Analyses of co-expression, DNase-seq, and ChIP-seq experiments revealed candidate RNA-binding proteins and CEBPB as the best regulatory candidates to explain these phenomena. Finally, cross-tissue expression analysis unearthed tissue-dependent splice variants and OXPHOS subunit paralogs and allowed revising the list of canonical OXPHOS transcripts. Taken together, our analysis provides a comprehensive view of mito-nuclear gene co-expression across human tissues and provides overall insights into the bi-genomic regulation of mitochondrial activities. © 2018 Barshad et al.; Published by Cold Spring Harbor Laboratory Press.

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

PubMed

Minchenko, O H; Riabovol, O O; Tsymbal, D O; Minchenko, D O; Ratushna, O O

2016-01-01

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

Mitochondrial DNA Depletion in Respiratory Chain-Deficient Parkinson Disease Neurons.

PubMed

Grünewald, Anne; Rygiel, Karolina A; Hepplewhite, Philippa D; Morris, Christopher M; Picard, Martin; Turnbull, Doug M

2016-03-01

To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI-IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single-neuron level. Multiple-label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI-IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser-capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication-associated 7S DNA employing a triplex real-time polymerase chain reaction (PCR) assay. Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single-cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription-primed mtDNA replication. Consistent with this, real-time PCR analysis revealed fewer transcription/replication-associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA-encoded factors mechanistically connected via TFAM. © 2016 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

Mitochondrial DNA Depletion in Respiratory Chain–Deficient Parkinson Disease Neurons

PubMed Central

Rygiel, Karolina A.; Hepplewhite, Philippa D.; Morris, Christopher M.; Picard, Martin; Turnbull, Doug M.

2016-01-01

Objective To determine the extent of respiratory chain abnormalities and investigate the contribution of mtDNA to the loss of respiratory chain complexes (CI–IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single‐neuron level. Methods Multiple‐label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI–IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2, and COXI) and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser‐capture microdissection, were assayed for mtDNA deletions, copy number, and presence of transcription/replication‐associated 7S DNA employing a triplex real‐time polymerase chain reaction (PCR) assay. Results Whereas mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At the single‐cell level, CI and II deficiencies were correlated in patients. The CI deficiency concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription‐primed mtDNA replication. Consistent with this, real‐time PCR analysis revealed fewer transcription/replication‐associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe CI deficiency. Interpretation Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA‐encoded factors mechanistically connected via TFAM. ANN NEUROL 2016;79:366–378 PMID:26605748

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Biogenesis of cytosolic ribosomes requires the essential iron–sulphur protein Rli1p and mitochondria

PubMed Central

Kispal, Gyula; Sipos, Katalin; Lange, Heike; Fekete, Zsuzsanna; Bedekovics, Tibor; Janáky, Tamás; Bassler, Jochen; Aguilar Netz, Daili J; Balk, Janneke; Rotte, Carmen; Lill, Roland

2005-01-01

Mitochondria perform a central function in the biogenesis of cellular iron–sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p. PMID:15660134

Oxygen and differentiation status modulate the effect of X-ray irradiation on physiology and mitochondrial proteome of human neuroblastoma cells.

PubMed

Džinić, Tamara; Hartwig, Sonja; Lehr, Stefan; Dencher, Norbert A

2016-12-01

Cytotoxic effects, including oxidative stress, of low linear energy transfer (LET)-ionizing radiation are often underestimated and studies of their mechanisms using cell culture models are widely conducted with cells cultivated at atmospheric oxygen that does not match its physiological levels in body tissues. Also, cell differentiation status plays a role in the outcome of experiments. We compared effects of 2 Gy X-ray irradiation on the physiology and mitochondrial proteome of nondifferentiated and human neuroblastoma (SH-SY5Y) cells treated with retinoic acid cultivated at 21% and 5% O 2 . Irradiation did not affect the amount of subunits of OxPhos complexes and other non-OxPhos mitochondrial proteins, except for heat shock protein 70, which was increased depending on oxygen level and differentiation status. These two factors were proven to modulate mitochondrial membrane potential and the bioenergetic status of cells. We suggest, moreover, that oxygen plays a role in the differentiation of human SH-SY5Y cells.

The mitochondrial DNA 10197 G > A mutation causes MELAS/Leigh overlap syndrome presenting with acute auditory agnosia.

PubMed

Leng, Yinglin; Liu, Yuhe; Fang, Xiaojing; Li, Yao; Yu, Lei; Yuan, Yun; Wang, Zhaoxia

2015-04-01

Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes/Leigh (MELAS/LS) overlap syndrome is a mitochondrial disorder subtype with clinical and magnetic resonance imaging (MRI) features that are characteristic of both MELAS and Leigh syndrome (LS). Here, we report an MELAS/LS case presenting with cortical deafness and seizures. Cranial MRI revealed multiple lesions involving bilateral temporal lobes, the basal ganglia and the brainstem, which conformed to neuroimaging features of both MELAS and LS. Whole mitochondrial DNA (mtDNA) sequencing and PCR-RFLP revealed a de novo heteroplasmic m.10197 G > A mutation in the NADH dehydrogenase subunit 3 gene (ND3), which was predicted to cause an alanine to threonine substitution at amino acid 47. Although the mtDNA m.10197 G > A mutation has been reported in association with LS, Leber hereditary optic neuropathy and dystonia, it has never been linked with MELAS/LS overlap syndrome. Our patient therefore expands the phenotypic spectrum of the mtDNA m.10197 G > A mutation.

Preliminary Characterization of Mitochondrial Genome of Melipona scutellaris, a Brazilian Stingless Bee

PubMed Central

Silverio, Manuella Souza; Rodovalho, Vinícius de Rezende; Bonetti, Ana Maria; de Oliveira, Guilherme Corrêa; Cuadros-Orellana, Sara; Ueira-Vieira, Carlos; Rodrigues dos Santos, Anderson

2014-01-01

Bees are manufacturers of relevant economical products and have a pollinator role fundamental to ecosystems. Traditionally, studies focused on the genus Melipona have been mostly based on behavioral, and social organization and ecological aspects. Only recently the evolutionary history of this genus has been assessed using molecular markers, including mitochondrial genes. Even though these studies have shed light on the evolutionary history of the Melipona genus, a more accurate picture may emerge when full nuclear and mitochondrial genomes of Melipona species become available. Here we present the assembly, annotation, and characterization of a draft mitochondrial genome of the Brazilian stingless bee Melipona scutellaris using Melipona bicolor as a reference organism. Using Illumina MiSeq data, we achieved the annotation of all protein coding genes, as well as the genes for the two ribosomal subunits (16S and 12S) and transfer RNA genes as well. Using the COI sequence as a DNA barcode, we found that M. cramptoni is the closest species to M. scutellaris. PMID:25019088

Preliminary characterization of mitochondrial genome of Melipona scutellaris, a Brazilian stingless bee.

PubMed

Silverio, Manuella Souza; Rodovalho, Vinícius de Rezende; Bonetti, Ana Maria; de Oliveira, Guilherme Corrêa; Cuadros-Orellana, Sara; Ueira-Vieira, Carlos; Rodrigues dos Santos, Anderson

2014-01-01

Bees are manufacturers of relevant economical products and have a pollinator role fundamental to ecosystems. Traditionally, studies focused on the genus Melipona have been mostly based on behavioral, and social organization and ecological aspects. Only recently the evolutionary history of this genus has been assessed using molecular markers, including mitochondrial genes. Even though these studies have shed light on the evolutionary history of the Melipona genus, a more accurate picture may emerge when full nuclear and mitochondrial genomes of Melipona species become available. Here we present the assembly, annotation, and characterization of a draft mitochondrial genome of the Brazilian stingless bee Melipona scutellaris using Melipona bicolor as a reference organism. Using Illumina MiSeq data, we achieved the annotation of all protein coding genes, as well as the genes for the two ribosomal subunits (16S and 12S) and transfer RNA genes as well. Using the COI sequence as a DNA barcode, we found that M. cramptoni is the closest species to M. scutellaris.

Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum.

PubMed

Ke, Hangjun; Morrisey, Joanne M; Ganesan, Suresh M; Mather, Michael W; Vaidya, Akhil B

2012-09-01

We have shown that transgenic Plasmodium falciparum parasites expressing the yeast DHODH (dihydroorotate dehydrogenase) are independent of the mtETC (mitochondrial electron transport chain), suggesting that they might not need the mitochondrial genome (mtDNA), since it only encodes three protein subunits belonging to the mtETC and fragmentary ribosomal RNA molecules. Disrupting the mitochondrial RNA polymerase (mtRNAP), which is critical for mtDNA replication and transcription, might then cause the generation of a ρ(0) parasite line lacking mtDNA. We made multiple attempts to disrupt the mtRNAP gene by double crossover recombination methods in parasite lines expressing yDHODH either episomally or integrated in the genome, but were unable to produce the desired knockout. We verified that the mtRNAP gene was accessible to recombination by successfully integrating a triple HA tag at the 3' end via single cross-over recombination. These studies suggest that mtRNAP is essential even in mtETC-independent P. falciparum parasites. Copyright © 2012 Elsevier B.V. All rights reserved.

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells.

PubMed

Dong, Lan-Feng; Kovarova, Jaromira; Bajzikova, Martina; Bezawork-Geleta, Ayenachew; Svec, David; Endaya, Berwini; Sachaphibulkij, Karishma; Coelho, Ana R; Sebkova, Natasa; Ruzickova, Anna; Tan, An S; Kluckova, Katarina; Judasova, Kristyna; Zamecnikova, Katerina; Rychtarcikova, Zuzana; Gopalan, Vinod; Andera, Ladislav; Sobol, Margarita; Yan, Bing; Pattnaik, Bijay; Bhatraju, Naveen; Truksa, Jaroslav; Stopka, Pavel; Hozak, Pavel; Lam, Alfred K; Sedlacek, Radislav; Oliveira, Paulo J; Kubista, Mikael; Agrawal, Anurag; Dvorakova-Hortova, Katerina; Rohlena, Jakub; Berridge, Michael V; Neuzil, Jiri

2017-02-15

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ 0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ 0 mouse melanoma cells into syngeneic C57BL/6N su9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ 0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer.

BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei

PubMed Central

Oppenheim, Rebecca D.; Limenitakis, Julien; Polonais, Valerie; Seeber, Frank; Barrett, Michael P.; Billker, Oliver; McConville, Malcolm J.; Soldati-Favre, Dominique

2014-01-01

While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens. PMID:25032958

[Leigh syndrome resulting from a de novo mitochondrial DNA mutation (T8993G)].

PubMed

Playán, A; Solano-Palacios, A; González de la Rosa, J B; Merino-Arribas, J M; Andreu, A L; López-Pérez, M; Montoya, J

Several degenerative neurological diseases are caused by mutations in the mitochondrial gene coding for subunit 6 of the ATPase. Thus, NARP (neurogenic weakness, ataxia, and retinitis pigmentosa) and Leigh syndromes are associated to a T8993G mutation when the percentage of mutant mitochondrial DNA is low (60 90%) or high (>90%), respectively. Leigh syndrome is also caused by a second mutation in the same position T8993C. The patient, a boy that died at 6 months, had generalized hypotonia, psychomotor delay, hepatomegaly, choreic movements and hyporreflexia. MRI showed hypodensities in the basal ganglia and brain stem as well as hyperlactacidemia. Molecular genetic analysis of the mitochondrial DNA showed that the patient had the T8993G mutation in a percentage higher than 95%. No mutated DNA was detected in blood of the proband s mother, maternal aunt and grandmother. The point mutation T8993G may occur de novo, at high levels, causing neurodegenerative diseases.

Juvenile Leigh syndrome, optic atrophy, ataxia, dystonia, and epilepsy due to T14487C mutation in the mtDNA-ND6 gene: a mitochondrial syndrome presenting from birth to adolescence.

PubMed

Leshinsky-Silver, Esther; Shuvalov, Ruslan; Inbar, Shani; Cohen, Sarit; Lev, Dorit; Lerman-Sagie, Tally

2011-04-01

An increasing number of reports describe mutations in mitochondrial DNA coding regions, especially in mitochondrial DNA- encoded nicotinamide adenine dinucleotide dehydrogenase subunit genes of the respiratory chain complex I, as causing early-onset Leigh syndrome. The authors report the molecular findings in a 24-year-old patient with juvenile-onset Leigh syndrome presenting with optic atrophy, ataxia dystonia, and epilepsy. A brain magnetic resonance imaging revealed bilateral basal ganglia and thalamic hypointensities, and a magnetic resonance spectroscopy revealed an increased lactate peak. The authors identified a T14487C change causing M63V substitution in the mitochondrial ND6 gene. The mutation was heteroplasmic in muscle and blood samples, with different mutation loads, and was absent in the patient's mother's urine and blood samples. They suggest that the T14487C mtDNA mutation should be analyzed in Leigh syndrome, presenting with optic atrophy, ataxia, dystonia, and epilepsy, regardless of age.

Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as Productive Assembly Intermediates in Arabidopsis*

PubMed Central

Li, Lei; Nelson, Clark J.; Carrie, Chris; Gawryluk, Ryan M. R.; Solheim, Cory; Gray, Michael W.; Whelan, James; Millar, A. Harvey

2013-01-01

Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabidopsis cell cultures with 15N and isolating mitochondria, we have identified CI subcomplexes through differences in 15N incorporation into their protein subunits. The 200-kDa subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII2. In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII2, indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly. PMID:23271729

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys

PubMed Central

2014-01-01

Background Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Methods Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Results Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Conclusions Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology. PMID:25034633

Complete mitochondrial genome sequences from five Eimeria species (Apicomplexa; Coccidia; Eimeriidae) infecting domestic turkeys.

PubMed

Ogedengbe, Mosun E; El-Sherry, Shiem; Whale, Julia; Barta, John R

2014-07-17

Clinical and subclinical coccidiosis is cosmopolitan and inflicts significant losses to the poultry industry globally. Seven named Eimeria species are responsible for coccidiosis in turkeys: Eimeria dispersa; Eimeria meleagrimitis; Eimeria gallopavonis; Eimeria meleagridis; Eimeria adenoeides; Eimeria innocua; and, Eimeria subrotunda. Although attempts have been made to characterize these parasites molecularly at the nuclear 18S rDNA and ITS loci, the maternally-derived and mitotically replicating mitochondrial genome may be more suited for species level molecular work; however, only limited sequence data are available for Eimeria spp. infecting turkeys. The purpose of this study was to sequence and annotate the complete mitochondrial genomes from 5 Eimeria species that commonly infect the domestic turkey (Meleagris gallopavo). Six single-oocyst derived cultures of five Eimeria species infecting turkeys were PCR-amplified and sequenced completely prior to detailed annotation. Resulting sequences were aligned and used in phylogenetic analyses (BI, ML, and MP) that included complete mitochondrial genomes from 16 Eimeria species or concatenated CDS sequences from each genome. Complete mitochondrial genome sequences were obtained for Eimeria adenoeides Guelph, 6211 bp; Eimeria dispersa Briston, 6238 bp; Eimeria meleagridis USAR97-01, 6212 bp; Eimeria meleagrimitis USMN08-01, 6165 bp; Eimeria gallopavonis Weybridge, 6215 bp; and Eimeria gallopavonis USKS06-01, 6215 bp). The order, orientation and CDS lengths of the three protein coding genes (COI, COIII and CytB) as well as rDNA fragments encoding ribosomal large and small subunit rRNA were conserved among all sequences. Pairwise sequence identities between species ranged from 88.1% to 98.2%; sequence variability was concentrated within CDS or between rDNA fragments (where indels were common). No phylogenetic reconstruction supported monophyly of Eimeria species infecting turkeys; Eimeria dispersa may have arisen via host switching from another avian host. Phylogenetic analyses suggest E. necatrix and E. tenella are related distantly to other Eimeria of chickens. Mitochondrial genomes of Eimeria species sequenced to date are highly conserved with regard to gene content and structure. Nonetheless, complete mitochondrial genome sequences and, particularly the three CDS, possess sufficient sequence variability for differentiating Eimeria species of poultry. The mitochondrial genome sequences are highly suited for molecular diagnostics and phylogenetics of coccidia and, potentially, genetic markers for molecular epidemiology.

Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging

PubMed Central

Salehpour, F.; Ghanian, Z.; Yang, C.; Zheleznova, N. N.; Kurth, T.; Dash, R. K.; Cowley, A. W.

2015-01-01

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67phox subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67phox was rendered functionally inactive by zinc finger nuclease mutation of the gene (SSp67phox-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SSp67phox-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SSp67phox-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SSp67phox rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67phox subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat. PMID:26062875

Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging.

PubMed

Salehpour, F; Ghanian, Z; Yang, C; Zheleznova, N N; Kurth, T; Dash, R K; Cowley, A W; Ranji, M

2015-08-15

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat. Copyright © 2015 the American Physiological Society.

Mutations in COA3 cause isolated complex IV deficiency associated with neuropathy, exercise intolerance, obesity, and short stature.

PubMed

Ostergaard, Elsebet; Weraarpachai, Woranontee; Ravn, Kirstine; Born, Alfred Peter; Jønson, Lars; Duno, Morten; Wibrand, Flemming; Shoubridge, Eric A; Vissing, John

2015-03-01

We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors. The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes.

PubMed

Caballero, Susana; Duchêne, Sebastian; Garavito, Manuel F; Slikas, Beth; Baker, C Scott

2015-01-01

A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the 'river dolphins', early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three 'river dolphins' (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans.

Initial Evidence for Adaptive Selection on the NADH Subunit Two of Freshwater Dolphins by Analyses of Mitochondrial Genomes

PubMed Central

Caballero, Susana; Duchêne, Sebastian; Garavito, Manuel F.; Slikas, Beth; Baker, C. Scott

2015-01-01

A small number of cetaceans have adapted to an entirely freshwater environment, having colonized rivers in Asia and South America from an ancestral origin in the marine environment. This includes the ‘river dolphins’, early divergence from the odontocete lineage, and two species of true dolphins (Family Delphinidae). Successful adaptation to the freshwater environment may have required increased demands in energy involved in processes such as the mitochondrial osmotic balance. For this reason, riverine odontocetes provide a compelling natural experiment in adaptation of mammals from marine to freshwater habitats. Here we present initial evidence of positive selection in the NADH dehydrogenase subunit 2 of riverine odontocetes by analyses of full mitochondrial genomes, using tests of selection and protein structure modeling. The codon model with highest statistical support corresponds to three discrete categories for amino acid sites, those under positive, neutral, and purifying selection. With this model we found positive selection at site 297 of the NADH dehydrogenase subunit 2 (dN/dS>1.0,) leading to a substitution of an Ala or Val from the ancestral state of Thr. A phylogenetic reconstruction of 27 cetacean mitogenomes showed that an Ala substitution has evolved at least four times in cetaceans, once or more in the three ‘river dolphins’ (Families Pontoporidae, Lipotidae and Inidae), once in the riverine Sotalia fluviatilis (but not in its marine sister taxa), once in the riverine Orcaella brevirostris from the Mekong River (but not in its marine sister taxa) and once in two other related marine dolphins. We located the position of this amino acid substitution in an alpha-helix channel in the trans-membrane domain in both the E. coli structure and Sotalia fluviatilis model. In E. coli this position is located in a helix implicated in a proton translocation channel of respiratory complex 1 and may have a similar role in the NADH dehydrogenases of cetaceans. PMID:25946045

Cytochrome c oxidase rather than cytochrome c is a major determinant of mitochondrial respiratory capacity in skeletal muscle of aged rats: role of carnitine and lipoic acid.

PubMed

Tamilselvan, Jayavelu; Sivarajan, Kumarasamy; Anusuyadevi, Muthuswamy; Panneerselvam, Chinnakkannu

2007-09-01

The release of mitochondrial cytochrome c followed by activation of caspase cascade has been reported with aging in various tissues, whereas little is known about the caspase-independent pathway involved in mitochondrial dysfunction. To determine the functional impact of cytochrome c loss on mitochondrial respiratory capacity, we monitored NADH redox transitions and oxygen consumption in isolated skeletal muscle mitochondria of 4- and 24-month-old rats in the presence and absence of exogenous cytochrome c; and assessed the efficacy of cosupplementation of carnitine and lipoic acid on age-related alteration in mitochondrial respiration. The loss of mitochondrial cytochrome c with age was accompanied with alteration in respiratory transition, which in turn was not rescued by exogenous addition of cytochrome c to isolated mitochondria. The analysis of mitochondrial and nuclear-encoded cytochrome c oxidase subunits suggests that the decreased levels of cytochrome c oxidase may be attributed for the irresponsiveness to exogenously added cytochrome c on mitochondrial respiratory transitions, possibly through reduction of upstream electron carriers. Oral supplementation of carnitine and lipoic acid to aged rats help to maintaining the mitochondrial oxidative capacity by regulating the release of cytochrome c and improves cytochrome c oxidase transcript levels. Thus, carnitine and lipoic acid supplementation prevents the loss of cytochrome c and their associated decline in cytochrome c oxidase activity; thereby, effectively attenuating any putative decrease in cellular energy and redox status with age.

Brain region- and sex-specific modulation of mitochondrial glucocorticoid receptor phosphorylation in fluoxetine treated stressed rats: effects on energy metabolism.

PubMed

Adzic, Miroslav; Lukic, Iva; Mitic, Milos; Djordjevic, Jelena; Elaković, Ivana; Djordjevic, Ana; Krstic-Demonacos, Marija; Matić, Gordana; Radojcic, Marija

2013-12-01

Antidepressants affect glucocorticoid receptor (GR) functioning partly through modulation of its phosphorylation but their effects on mitochondrial GR have remained undefined. We investigated the ability of chronic fluoxetine treatment to affect chronic stress-induced changes of mitochondrial GR and its phosphoisoforms (pGRs) in the prefrontal cortex and hippocampus of female and male rats. Since mitochondrial GR regulates oxidative phosphorylation, expression of mitochondrial-encoded subunits of cytochrome (cyt) c oxidase and its activity were also investigated. Chronic stress caused accumulation of the GR in mitochondria of female prefrontal cortex, while the changes in the hippocampus were sex-specific at the levels of pGRs. Expression of mitochondrial COXs genes corresponded to chronic stress-modulated mitochondrial GR in both tissues of both genders and to cyt c oxidase activity in females. Moreover, the metabolic parameters in stressed animals were affected by fluoxetine therapy only in the hippocampus. Namely, fluoxetine effects on mitochondrial COXs and cyt c oxidase activity in the hippocampus seem to be conveyed through pGR232 in females, while in males this likely occurs through other mechanisms. In summary, sex-specific regulation of cyt c oxidase by the stress and antidepressant treatment and its differential convergence with mitochondrial GR signaling in the prefrontal cortex and hippocampus could contribute to clarification of sex-dependent vulnerability to stress-related disorders and sex-specific clinical impact of antidepressants. Copyright © 2013 Elsevier Ltd. All rights reserved.

Novel remodeling of the mouse heart mitochondrial proteome in response to acute insulin stimulation

PubMed Central

Pedersen, Brian A; Yazdi, Puya G; Taylor, Jared F; Khattab, Omar S; Chen, Yu-Han; Chen, Yumay; Wang, Ping H

2015-01-01

Mitochondrial dysfunction contributes to the pathophysiology of diabetic cardiomyopathy. The aim of this study was to investigate the acute changes in the mitochondrial proteome in response to insulin stimulation. Cardiac mitochondria from C57BL/6 mice after insulin stimulation were analyzed using two-dimensional fluorescence difference gel electrophoresis. MALDI-TOF MS/MS was utilized to identify differences. Two enzymes involved in metabolism and four structural proteins were identified. Succinyl-CoA ligase [ADP forming] subunit beta was identified as one of the differentially regulated proteins. Upon insulin stimulation, a relatively more acidic isoform of this protein was increased by 53% and its functional activity was decreased by ∼32%. This proteomic remodeling in response to insulin stimulation may play an important role in the normal and diabetic heart. PMID:26610654

Phylogeny and evolution of the auks (subfamily Alcinae) based on mitochondrial DNA sequences

USGS Publications Warehouse

Moum, Truls; Johansen, Steinar; Erikstad, Kjell Einar; Piatt, John F.

1994-01-01

The genetic divergence and phylogeny of the auks was assessed by mitochondrial DNA sequence comparisons in a study using 19 of the 22 auk species and two outgroup representatives. We compared more than 500 nucleotides from each of two mitochondrial genes encoding 12S rRNA and the NADH dehydrogenase subunit 6. Divergence times were estimated from transversional substitutions. The dovekie (Alle alle) is related to the razorbill (Alca torda) and the murres (Uria spp). Furthermore, the Xantus's murrelet (Synthliboramphus hypoleucus) and the ancient (Synthliboramphus antiquus) and Japanese murrelets (Synthliboramphus wumizusume) are genetically distinct members of the same main lineage, whereas brachyramphine and synthliboramphine murrelets are not closely related. An early adaptive radiation of six main species groups of auks seems to trace back to Middle Miocene. Later speciation probably involved ecological differentiations and geographical isolations.

The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.

PubMed

Haid, A; Schweyen, R J; Bechmann, H; Kaudewitz, F; Solioz, M; Schatz, G

1979-03-01

Mitochondrial mutants of Saccharomyces cerevisiae defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome. The mutants mapped between OLI1 and OLI2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho- deletion mapping and recombination analysis. The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLI2 and E being closest to OLI1. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence. 1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2 leads to OLI1. The mutant closest to OLI1 accumulates an apocytochrome b which is slightly larger than that of wild type. 2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red. 3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome aa3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown. 4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).

Hereditary sensory neuropathy type 1-associated deoxysphingolipids cause neurotoxicity, acute calcium handling abnormalities and mitochondrial dysfunction in vitro.

PubMed

Wilson, Emma R; Kugathasan, Umaiyal; Abramov, Andrey Y; Clark, Alex J; Bennett, David L H; Reilly, Mary M; Greensmith, Linda; Kalmar, Bernadett

2018-05-18

Hereditary sensory neuropathy type 1 (HSN-1) is a peripheral neuropathy most frequently caused by mutations in the SPTLC1 or SPTLC2 genes, which code for two subunits of the enzyme serine palmitoyltransferase (SPT). SPT catalyzes the first step of de novo sphingolipid synthesis. Mutations in SPT result in a change in enzyme substrate specificity, which causes the production of atypical deoxysphinganine and deoxymethylsphinganine, rather than the normal enzyme product, sphinganine. Levels of these abnormal compounds are elevated in blood of HSN-1 patients and this is thought to cause the peripheral motor and sensory nerve damage that is characteristic of the disease, by a largely unresolved mechanism. In this study, we show that exogenous application of these deoxysphingoid bases causes dose- and time-dependent neurotoxicity in primary mammalian neurons, as determined by analysis of cell survival and neurite length. Acutely, deoxysphingoid base neurotoxicity manifests in abnormal Ca 2+ handling by the endoplasmic reticulum (ER) and mitochondria as well as dysregulation of cell membrane store-operated Ca 2+ channels. The changes in intracellular Ca 2+ handling are accompanied by an early loss of mitochondrial membrane potential in deoxysphingoid base-treated motor and sensory neurons. Thus, these results suggest that exogenous deoxysphingoid base application causes neuronal mitochondrial dysfunction and Ca 2+ handling deficits, which may play a critical role in the pathogenesis of HSN-1. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns.

PubMed

Azzam, Sausan; Broadwater, Laurie; Li, Shuo; Freeman, Ernest J; McDonough, Jennifer; Gregory, Roger B

2013-05-01

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights.

A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

PubMed Central

2013-01-01

Background Experimental autoimmune encephalomyelitis (EAE) is an autoimmune, inflammatory disease of the central nervous system that is widely used as a model of multiple sclerosis (MS). Mitochondrial dysfunction appears to play a role in the development of neuropathology in MS and may also play a role in disease pathology in EAE. Here, surface enhanced laser desorption ionization mass spectrometry (SELDI-MS) has been employed to obtain protein expression profiles from mitochondrially enriched fractions derived from EAE and control mouse brain. To gain insight into experimental variation, the reproducibility of sub-cellular fractionation, anion exchange fractionation as well as spot-to-spot and chip-to-chip variation using pooled samples from brain tissue was examined. Results Variability of SELDI mass spectral peak intensities indicates a coefficient of variation (CV) of 15.6% and 17.6% between spots on a given chip and between different chips, respectively. Thinly slicing tissue prior to homogenization with a rotor homogenizer showed better reproducibility (CV = 17.0%) than homogenization of blocks of brain tissue with a Teflon® pestle (CV = 27.0%). Fractionation of proteins with anion exchange beads prior to SELDI-MS analysis gave overall CV values from 16.1% to 18.6%. SELDI mass spectra of mitochondrial fractions obtained from brain tissue from EAE mice and controls displayed 39 differentially expressed proteins (p≤ 0.05) out of a total of 241 protein peaks observed in anion exchange fractions. Hierarchical clustering analysis showed that protein fractions from EAE animals with severe disability clearly segregated from controls. Several components of electron transport chain complexes (cytochrome c oxidase subunit 6b1, subunit 6C, and subunit 4; NADH dehydrogenase flavoprotein 3, alpha subcomplex subunit 2, Fe-S protein 4, and Fe-S protein 6; and ATP synthase subunit e) were identified as possible differentially expressed proteins. Myelin Basic Protein isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. Conclusions SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights. PMID:23635033

The mitochondrial genomes of Campodea fragilis and C. lubbocki(Hexapoda: Diplura): high genetic divergence in a morphologically uniformtaxon

DOE Office of Scientific and Technical Information (OSTI.GOV)

Podsiadlowski, L.; Carapelli, A.; Nardi, F.

2005-12-01

Mitochondrial genomes from two dipluran hexapods of the genus Campodea have been sequenced. Gene order is the same as in most other hexapods and crustaceans. Secondary structures of tRNAs reveal specific structural changes in tRNA-C, tRNA-R, tRNA-S1 and tRNA-S2. Comparative analyses of nucleotide and amino acid composition, as well as structural features of both ribosomal RNA subunits, reveal substantial differences among the analyzed taxa. Although the two Campodea species are morphologically highly uniform, genetic divergence is larger than expected, suggesting a long evolutionary history under stable ecological conditions.

Alterations in the mitochondrial regulatory pathways constituted by the nuclear co-factors PGC-1alpha or PGC-1beta and mitofusin 2 in skeletal muscle in type 2 diabetes.

PubMed

Zorzano, Antonio; Hernández-Alvarez, María Isabel; Palacín, Manuel; Mingrone, Geltrude

2010-01-01

Muscle mitochondrial metabolism is regulated by a number of factors, many of which are responsible for the transcription of nuclear genes encoding mitochondrial proteins such as PPARdelta, PGC-1alpha or PGC-1beta. Recent evidence indicates that proteins participating in mitochondrial dynamics also regulate mitochondrial metabolism. Thus, in cultured cells the mitochondrial fusion protein mitofusin 2 (Mfn2) stimulates respiration, substrate oxidation and the expression of subunits involved in respiratory complexes. Mitochondrial dysfunction has been reported in skeletal muscle of type 2 diabetic patients. Reduced mitochondrial mass and defective activity has been proposed to explain this dysfunction. Alterations in mitochondrial metabolism may be crucial to account for some of the pathophysiological traits that characterize type 2 diabetes. Skeletal muscle of type 2 diabetic patients shows reduced expression of PGC-1alpha, PGC-1beta, and Mfn2. In addition, a differential response to bilio-pancreatic diversion-induced weight loss in non-diabetic and type 2 diabetic patients has been reported. While non-diabetic morbidly obese subjects showed an increased expression of genes encoding Mfn2, PGC-1alpha, PGC-1beta, PPARdelta or SIRT1 in response to bariatric surgery-induced weight loss, no effect was detected in type 2 diabetic patients. These observations suggest the existence of a heritable component responsible for the abnormal control of the expression of genes encoding for modulators of mitochondrial biogenesis/metabolism, and which may participate in the development of the disease. Copyright © 2010 Elsevier B.V. All rights reserved.

Exercise training improves vascular mitochondrial function

PubMed Central

Park, Song-Young; Rossman, Matthew J.; Gifford, Jayson R.; Bharath, Leena P.; Bauersachs, Johann; Richardson, Russell S.; Abel, E. Dale; Symons, J. David

2016-01-01

Exercise training is recognized to improve cardiac and skeletal muscle mitochondrial respiratory capacity; however, the impact of chronic exercise on vascular mitochondrial respiratory function is unknown. We hypothesized that exercise training concomitantly increases both vascular mitochondrial respiratory capacity and vascular function. Arteries from both sedentary (SED) and swim-trained (EX, 5 wk) mice were compared in terms of mitochondrial respiratory function, mitochondrial content, markers of mitochondrial biogenesis, redox balance, nitric oxide (NO) signaling, and vessel function. Mitochondrial complex I and complex I + II state 3 respiration and the respiratory control ratio (complex I + II state 3 respiration/complex I state 2 respiration) were greater in vessels from EX relative to SED mice, despite similar levels of arterial citrate synthase activity and mitochondrial DNA content. Furthermore, compared with the SED mice, arteries from EX mice displayed elevated transcript levels of peroxisome proliferative activated receptor-γ coactivator-1α and the downstream targets cytochrome c oxidase subunit IV isoform 1, isocitrate dehydrogenase (Idh) 2, and Idh3a, increased manganese superoxide dismutase protein expression, increased endothelial NO synthase phosphorylation (Ser1177), and suppressed reactive oxygen species generation (all P < 0.05). Although there were no differences in EX and SED mice concerning endothelium-dependent and endothelium-independent vasorelaxation, phenylephrine-induced vasocontraction was blunted in vessels from EX compared with SED mice, and this effect was normalized by NOS inhibition. These training-induced increases in vascular mitochondrial respiratory capacity and evidence of improved redox balance, which may, at least in part, be attributable to elevated NO bioavailability, have the potential to protect against age- and disease-related challenges to arterial function. PMID:26825520

Diesel Exhaust Particulate Extracts Inhibit Transcription of Nuclear Respiratory Factor-1 and Cell Viability in Human Umbilical Vein Endothelial Cells

PubMed Central

Mattingly, Kathleen A.; Klinge, Carolyn M.

2011-01-01

Endothelial dysfunction precedes cardiovascular disease and is accompanied by mitochondrial dysfunction. Here we tested the hypothesis that diesel exhaust particulate extracts (DEPEs), prepared from a truck run at different speeds and engine loads, would inhibit genomic estrogen receptor activation of nuclear respiratory factor-1 (NRF-1) transcription in human umbilical vein endothelial cells (HUVECs). Additionally, we examined how DEPEs affect NRF-1 regulated TFAM expression and, in turn, Tfam-regulated mtDNA-encoded cytochrome c oxidase subunit I (COI, MTCO1) and NADH dehydrogenase subunit I (NDI) expression as well as cell proliferation and viability. We report that 17β-estradiol (E2), 4-hydroxytamoxifen (4-OHT), and raloxifene increased NRF-1 transcription in HUVECs in an ER-dependent manner. DEPEs inhibited NRF-1 transcription and this suppression was not ablated by concomitant treatment with E2, 4-OHT, or raloxifene, indicating that the effect was not due to inhibition of ER activity. While E2 increased HUVEC proliferation and viability, DEPEs inhibited viability but not proliferation. Resveratrol increased NRF-1 transcription in an ER-dependent manner in HUVECs, and ablated DEPE inhibition of basal NRF-1 expression. Given that NRF-1 is a key nuclear transcription factor regulating genes involved in mitochondrial activity and biogenesis, these data suggest that DEPEs may adversely affect mitochondrial function leading to endothelial dysfunction and resveratrol may block these effects. PMID:22105178

Bnip3 mediates doxorubicin-induced cardiac myocyte necrosis and mortality through changes in mitochondrial signaling

PubMed Central

Dhingra, Rimpy; Margulets, Victoria; Chowdhury, Subir Roy; Thliveris, James; Jassal, Davinder; Fernyhough, Paul; Dorn, Gerald W.; Kirshenbaum, Lorrie A.

2014-01-01

Doxorubicin (DOX) is widely used for treating human cancers, but can induce heart failure through an undefined mechanism. Herein we describe a previously unidentified signaling pathway that couples DOX-induced mitochondrial respiratory chain defects and necrotic cell death to the BH3-only protein Bcl-2-like 19kDa-interacting protein 3 (Bnip3). Cellular defects, including vacuolization and disrupted mitochondria, were observed in DOX-treated mice hearts. This coincided with mitochondrial localization of Bnip3, increased reactive oxygen species production, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, and necrosis. Interestingly, a 3.1-fold decrease in maximal mitochondrial respiration was observed in cardiac mitochondria of mice treated with DOX. In vehicle-treated control cells undergoing normal respiration, the respiratory chain complex IV subunit 1 (COX1) was tightly bound to uncoupling protein 3 (UCP3), but this complex was disrupted in cells treated with DOX. Mitochondrial dysfunction induced by DOX was accompanied by contractile failure and necrotic cell death. Conversely, shRNA directed against Bnip3 or a mutant of Bnip3 defective for mitochondrial targeting abrogated DOX-induced loss of COX1-UCP3 complexes and respiratory chain defects. Finally, Bnip3−/− mice treated with DOX displayed relatively normal mitochondrial morphology, respiration, and mortality rates comparable to those of saline-treated WT mice, supporting the idea that Bnip3 underlies the cardiotoxic effects of DOX. These findings reveal a new signaling pathway in which DOX-induced mitochondrial respiratory chain defects and necrotic cell death are mutually dependent on and obligatorily linked to Bnip3 gene activation. Interventions that antagonize Bnip3 may prove beneficial in preventing mitochondrial injury and heart failure in cancer patients undergoing chemotherapy. PMID:25489073

Naringin Ameliorates HIV-1 Nucleoside Reverse Transcriptase Inhibitors- Induced Mitochondrial Toxicity.

PubMed

Oluwafeyisetan, Adebiyi; Olubunmi, Adebiyi; Peter, Owira

2016-01-01

Mitochondrial reactive oxygen species (ROS) generation and defective oxidative phosphorylation (OXPHOS) have been proposed as possible mechanisms underlying the development of nucleoside reverse transcriptase inhibitors (NRTIs)-induced mitochondrial toxicities. Available options in managing these complications have, so far, produced controversial results, thus necessitating further research into newer agents with promise. Antioxidant and free-radical scavenging effects of naringin, a plant-derived flavonoid, have previously been demonstrated. This study was designed to investigate the effects of naringin on NRTIs-induced mitochondrial toxicity. Wistar rats were randomly divided into Zidovudine (AZT)-only (100 mg/kg body weight BW); AZT+Naringin (100+50 mg/kg BW); AZT+Vitamin E (100+100 mg/kg BW); Stavudine (d4T)- only (50 mg/kg BW); d4T+Naringin (50+50 mg/kg BW); d4T+Vitamin E (50+100 mg/kg BW) and Vehicle (3.0 mL/kg BW)-treated groups, respectively. After 56 days of oral daily dosing, rats were euthanized by halothane overdose, blood collected by cardiac puncture and livers promptly excised for further biochemical and ultrastructural analyses. </p> Results: AZT- or d4T-only caused significant mitochondrial dysfunction and mitochondrial ultrastructural damage compared to controls, while either naringin or vitamin E reversed indices of mitochondrial dysfunction evidenced by significantly reduced mitochondrial malondialdehyde (MDA) and blood lactate concentrations, increased liver manganese superoxide dismutase (MnSOD) activity and upregulate expression of mitochondrial-encoded subunit of electron transport chain (ETC) complex IV protein compared to AZT- or d4T-only treated rats. Furthermore, naringin or vitamin E, respectively, ameliorated mitochondrial damage observed in AZT- or d4T-only treated rats. Naringin ameliorated oxidative stress and NRTI-induced mitochondrial damage and might, therefore, be beneficial in managing toxicities and complications arising from NRTI use.

Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.

PubMed

Boczonadi, Veronika; Meyer, Kathrin; Gonczarowska-Jorge, Humberto; Griffin, Helen; Roos, Andreas; Bartsakoulia, Marina; Bansagi, Boglarka; Ricci, Giulia; Palinkas, Fanni; Zahedi, René P; Bruni, Francesco; Kaspar, Brian; Lochmüller, Hanns; Boycott, Kym M; Müller, Juliane S; Horvath, Rita

2018-06-15

The nuclear-encoded glycyl-tRNA synthetase gene (GARS) is essential for protein translation in both cytoplasm and mitochondria. In contrast, different genes encode the mitochondrial and cytosolic forms of most other tRNA synthetases. Dominant GARS mutations were described in inherited neuropathies, while recessive mutations cause severe childhood-onset disorders affecting skeletal muscle and heart. The downstream events explaining tissue-specific phenotype-genotype relations remained unclear. We investigated the mitochondrial function of GARS in human cell lines and in the GarsC210R mouse model. Human-induced neuronal progenitor cells (iNPCs) carrying dominant and recessive GARS mutations showed alterations of mitochondrial proteins, which were more prominent in iNPCs with dominant, neuropathy-causing mutations. Although comparative proteomic analysis of iNPCs showed significant changes in mitochondrial respiratory chain complex subunits, assembly genes, Krebs cycle enzymes and transport proteins in both recessive and dominant mutations, proteins involved in fatty acid oxidation were only altered by recessive mutations causing mitochondrial cardiomyopathy. In contrast, significant alterations of the vesicle-associated membrane protein-associated protein B (VAPB) and its downstream pathways such as mitochondrial calcium uptake and autophagy were detected in dominant GARS mutations. The role of VAPB has been supported by similar results in the GarsC210R mice. Our data suggest that altered mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) may be important disease mechanisms leading to neuropathy in this condition.

Deficiency of PHB complex impairs respiratory supercomplex formation and activates mitochondrial flashes.

PubMed

Jian, Chongshu; Xu, Fengli; Hou, Tingting; Sun, Tao; Li, Jinghang; Cheng, Heping; Wang, Xianhua

2017-08-01

Prohibitins (PHBs; prohibitin 1, PHB1 or PHB, and prohibitin 2, PHB2) are evolutionarily conserved and ubiquitously expressed mitochondrial proteins. PHBs form multimeric ring complexes acting as scaffolds in the inner mitochondrial membrane. Mitochondrial flashes (mitoflashes) are newly discovered mitochondrial signaling events that reflect electrical and chemical excitations of the organelle. Here, we investigate the possible roles of PHBs in the regulation of mitoflash signaling. Downregulation of PHBs increases mitoflash frequency by up to 5.4-fold due to elevated basal reactive oxygen species (ROS) production in the mitochondria. Mechanistically, PHB deficiency impairs the formation of mitochondrial respiratory supercomplexes (RSCs) without altering the abundance of individual respiratory complex subunits. These impairments induced by PHB deficiency are effectively rescued by co-expression of PHB1 and PHB2, indicating that the multimeric PHB complex acts as the functional unit. Furthermore, downregulating other RSC assembly factors, including SCAFI (also known as COX7A2L), RCF1a (HIGD1A), RCF1b (HIGD2A), UQCC3 and SLP2 (STOML2), all activate mitoflashes through elevating mitochondrial ROS production. Our findings identify the PHB complex as a new regulator of RSC formation and mitoflash signaling, and delineate a general relationship among RSC formation, basal ROS production and mitoflash biogenesis. © 2017. Published by The Company of Biologists Ltd.

Complete mitochondrial genome of Concholepas concholepas inferred by 454 pyrosequencing and mtDNA expression in two mollusc populations.

PubMed

Núñez-Acuña, Gustavo; Aguilar-Espinoza, Andrea; Gallardo-Escárate, Cristian

2013-03-01

Despite the great relevance of mitochondrial genome analysis in evolutionary studies, there is scarce information on how the transcripts associated with the mitogenome are expressed and their role in the genetic structuring of populations. This work reports the complete mitochondrial genome of the marine gastropod Concholepas concholepas, obtained by 454 pryosequencing, and an analysis of mitochondrial transcripts of two populations 1000 km apart along the Chilean coast. The mitochondrion of C. concholepas is 15,495 base pairs (bp) in size and contains the 37 subunits characteristic of metazoans, as well as a non-coding region of 330 bp. In silico analysis of mitochondrial gene variability showed significant differences among populations. In terms of levels of relative abundance of transcripts associated with mitochondrion in the two populations (assessed by qPCR), the genes associated with complexes III and IV of the mitochondrial genome had the highest levels of expression in the northern population while transcripts associated with the ATP synthase complex had the highest levels of expression in the southern population. Moreover, fifteen polymorphic SNPs were identified in silico between the mitogenomes of the two populations. Four of these markers implied different amino acid substitutions (non-synonymous SNPs). This work contributes novel information regarding the mitochondrial genome structure and mRNA expression levels of C. concholepas. Copyright © 2012 Elsevier Inc. All rights reserved.

Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast.

PubMed

Hori, Akiko; Yoshida, Minoru; Ling, Feng

2011-05-01

Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Fluoride decreased the sperm ATP of mice through inhabiting mitochondrial respiration.

PubMed

Sun, Zilong; Zhang, Wen; Xue, Xingchen; Zhang, Yuliang; Niu, Ruiyan; Li, Xuying; Li, Baojun; Wang, Xiaowen; Wang, Jundong

2016-02-01

Fluoride-induced low sperm motility was observed in accumulated investigations. However, the effect of fluoride exposure on ATP generation which is essential to sperm motility remains to be elucidated. In this study, 120 healthy male mice were orally administrated with 0, 25, 50, and 100 mg L(-1) NaF for 90 d. Results showed that compared with controls, fluoride ingestion significantly reduced sperm count, survival, as well as mobility and total ATP level in sperm untreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or pyruvate, which was used to establish glycolysis or mitochondrial respiration model, respectively. Data further revealed that sperm mobility and ATP level under mitochondrial respiration condition were significantly suppressed, while no statistical difference occurred in the model of glycolysis, indicating ATP derived from mitochondria was affected. Moreover, mRNA expressions of mitochondrial cytochrome b (mt-Cytb) and cytochrome c oxidase subunit 2 (mt-COX2), two important molecules in mitochondrial electron transport chain (ETC), were down-regulated in all fluoride treatment groups. Mitochondria in sperm of mice exposed to 100 mg L(-1) NaF appeared to be irregular and vacuolated. These findings suggested that decreased sperm motility induced by fluoride may result from low ATP generation due to the disturbed ETC in sperm mitochondrial. Copyright © 2015 Elsevier Ltd. All rights reserved.

The mitochondrial genome of the ethanol-metabolizing, wine cellar mold Zasmidium cellare is the smallest for a filamentous ascomycete

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodwin, Stephen; McCorison, Cassandra B.; Cavaletto, Jessica R.

Fungi in the class Dothideomycetes often live in extreme environments or have unusual physiology. One of these, the wine cellar mold Zasmidium cellare, produces thick curtains of mycelial growth in cellars with high humidity, and its ability to metabolize volatile organic compounds including alcohols, esters and formaldehyde is thought to improve air quality. It grows slowly but appears to outcompete ordinarily faster-growing species under anaerobic conditions.Whether these abilities have affected its mitochondrial genome is not known.To fill this gap, its mitochondrial genome was assembled as part of a whole- genome shotgun-sequencing project.The circular-mapping mitochondrial genome of Z. cellare, at onlymore » 23,743 bp, is the smallest yet reported for a filamentous fungus.It contains the complete set of 14 protein-coding genes seen typically in other filamentous fungi, along with genes for large and small ribosomal RNA subunits, 25 predicted tRNA genes capable of decoding all 20 amino acids, and a single open reading frame potentially coding for a protein of unknown function.The Z. cellare mitochondrial genome had genes encoded on both strands with a single change of direction, different from most other fungi but consistent with the Dothideomycetes. The high synteny among mitochondrial genomes of fungi in the Eurotiomycetes broke down almost completely in the Dothideomycetes.Only a low level of microsynteny was observed among protein-coding and tRNA genes in comparison with Mycosphaerella graminicola (synonym Zymoseptoria tritici), the only other fungus in the order Capnodiales with a sequenced mitochondrial genome, involving the three gene pairs atp8-atp9, nad2-nad3, and nad4L-nad5.However, even this low level of microsynteny did not extend to other fungi in the Dothideomycetes and Eurotiomycetes. Phylogenetic analysis of concatenated protein-coding genes confirmed the relationship between Z. cellare and M. graminicola in the Capnodiales, although conclusions were limited due to low sampling density.Other than its small size, the only unusual feature of the Z. cellare mitochondrial genome was two copies of a 110-bp sequence that were duplicated, inverted and separated by approximately 1 kb. This inverted-repeat sequence confused the assembly program but appears to have no functional significance.The small size of the Z. cellare mitochondrial genome was due to slightly smaller genes, lack of introns and non-essential genes, reduced intergenic spaces and very few ORFs relative to other fungi rather than a loss of essential genes. Whether this reduction facilitates its unusual biology remains unknown.« less

Mitochondrial NADH Fluorescence is Enhanced by Complex I Binding

PubMed Central

Blinova, Ksenia; Levine, Rodney L.; Boja, Emily S.; Griffiths, Gary L.; Shi, Zhen-Dan; Ruddy, Brian; Balaban, Robert S.

2012-01-01

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state. PMID:18702505

Mechanism of neem limonoids-induced cell death in cancer: role of oxidative phosphorylation

PubMed Central

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph; O’Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. PMID:26627937

Co-occurring Down syndrome and SUCLA2-related mitochondrial depletion syndrome.

PubMed

Couser, Natario L; Marchuk, Daniel S; Smith, Laurie D; Arreola, Alexandra; Kaiser-Rogers, Kathleen A; Muenzer, Joseph; Pandya, Arti; Gucsavas-Calikoglu, Muge; Powell, Cynthia M

2017-10-01

Mitochondrial DNA depletion syndrome 5 (MIM 612073) is a rare autosomal recessive disorder caused by homozygous or compound heterozygous pathogenic variants in the beta subunit of the succinate-CoA ligase gene located within the 13q14 band. We describe two siblings of Hispanic descent with SUCLA2-related mitochondrial depletion syndrome (encephalomyopathic form with methylmalonic aciduria); the older sibling is additionally affected with trisomy 21. SUCLA2 sequencing identified homozygous p.Arg284Cys pathogenic variants in both patients. This mutation has previously been identified in four individuals of Italian and Caucasian descent. The older sibling with concomitant disease has a more severe phenotype than what is typically described in patients with either SUCLA2-related mitochondrial depletion syndrome or Down syndrome alone. The younger sibling, who has a normal female chromosome complement, is significantly less affected compared to her brother. While the clinical and molecular findings have been reported in about 50 patients affected with a deficiency of succinate-CoA ligase caused by pathogenic variants in SUCLA2, this report describes the first known individual affected with both a mitochondrial depletion syndrome and trisomy 21. © 2017 Wiley Periodicals, Inc.

Assembly of the mitochondrial membrane system: mutations in the pho2 locus of the mitochondrial genome of Saccharomyces cerevisiae.

PubMed

Coruzzi, G; Trembath, M K; Tzagoloff, A

1978-12-01

Two mutants of Saccharomyces cerevisiae which show a loss of mitochondrial rutamycin-sensitive ATPase activity are described. Although phenotypically similar to mutants of the mitochondrial locus pho1 [F. Foury and A. Tzagoloff (1976) Eur. J. Biochem. 68, 113-119], these mutants define a second ATPase locus on the mitochondrial DNA (designated pho2), which is genetically unlinked to pho1. Analysis of recombination in crosses involving multiple antibiotic resistance markers indicates that the locus is in the segment of the genome between ery1 and oli2, very close to oli1. In fact it is proposed that the oli1 and pho2 mutations are in the same gene. Supporting evidence for this proposal includes: 1. The analysis of marker retention in petite mutants shows that the oli1 and pho2 loci were either retained or lost together in all cases. 2. Recombination frequencies of 0.05% or less are observed in crosses between the oli1 and pho2 loci. 3. When rho+ revertants are isolated from the pho2 mutants they frequently are oligomycin resistant. 4. pho2 mutants have an altered subunit 9 of the ATPase complex.

Mechanism of neem limonoids-induced cell death in cancer: Role of oxidative phosphorylation.

PubMed

Yadav, Neelu; Kumar, Sandeep; Kumar, Rahul; Srivastava, Pragya; Sun, Leimin; Rapali, Peter; Marlowe, Timothy; Schneider, Andrea; Inigo, Joseph R; O'Malley, Jordan; Londonkar, Ramesh; Gogada, Raghu; Chaudhary, Ajay K; Yadava, Nagendra; Chandra, Dhyan

2016-01-01

We have previously reported that neem limonoids (neem) induce multiple cancer cell death pathways. Here we dissect the underlying mechanisms of neem-induced apoptotic cell death in cancer. We observed that neem-induced caspase activation does not require Bax/Bak channel-mediated mitochondrial outer membrane permeabilization, permeability transition pore, and mitochondrial fragmentation. Neem enhanced mitochondrial DNA and mitochondrial biomass. While oxidative phosphorylation (OXPHOS) Complex-I activity was decreased, the activities of other OXPHOS complexes including Complex-II and -IV were unaltered. Increased reactive oxygen species (ROS) levels were associated with an increase in mitochondrial biomass and apoptosis upon neem exposure. Complex-I deficiency due to the loss of Ndufa1-encoded MWFE protein inhibited neem-induced caspase activation and apoptosis, but cell death induction was enhanced. Complex II-deficiency due to the loss of succinate dehydrogenase complex subunit C (SDHC) robustly decreased caspase activation, apoptosis, and cell death. Additionally, the ablation of Complexes-I, -III, -IV, and -V together did not inhibit caspase activation. Together, we demonstrate that neem limonoids target OXPHOS system to induce cancer cell death, which does not require upregulation or activation of proapoptotic Bcl-2 family proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

m-AAA Complexes Are Not Crucial for the Survival of Arabidopsis Under Optimal Growth Conditions Despite Their Importance for Mitochondrial Translation.

PubMed

Kolodziejczak, Marta; Skibior-Blaszczyk, Renata; Janska, Hanna

2018-05-01

For optimal mitochondrial activity, the mitochondrial proteome must be properly maintained or altered in response to developmental and environmental stimuli. Based on studies of yeast and humans, one of the key players in this control are m-AAA proteases, mitochondrial inner membrane-bound ATP-dependent metalloenzymes. This study focuses on the importance of m-AAA proteases in plant mitochondria, providing their first experimentally proven physiological substrate. We found that the Arabidopsis m- AAA complexes composed of AtFTSH3 and/or AtFTSH10 are involved in the proteolytic maturation of ribosomal subunit L32. Consequently, in the double Arabidopsis ftsh3/10 mutant, mitoribosome biogenesis, mitochondrial translation and functionality of OXPHOS (oxidative phosphorylation) complexes are impaired. However, in contrast to their mammalian or yeast counterparts, plant m-AAA complexes are not critical for the survival of Arabidopsis under optimal conditions; ftsh3/10 plants are only slightly smaller in size at the early developmental stage compared with plants containing m-AAA complexes. Our data suggest that a lack of significant visible morphological alterations under optimal growth conditions involves mechanisms which rely on existing functional redundancy and induced functional compensation in Arabidopsis mitochondria.

Large mitochondrial DNA deletion in an infant with addison disease.

PubMed

Duran, Gloria P; Martinez-Aguayo, A; Poggi, H; Lagos, M; Gutierrez, D; Harris, P R

2012-01-01

Mitochondrial diseases are a group of disorders caused by mutations in nuclear DNA or mitochondrial DNA, usually involving multiple organ systems. Primary adrenal insufficiency due to mitochondrial disease is extremely infrequent and has been reported in association with mitochondrial DNA deletion syndromes such as Kearns-Sayre syndrome. To report a 3-year-old boy with Addison disease, congenital glaucoma, chronic pancreatitis, and mitochondrial myopathy due to large mitochondrial DNA deletion. Molecular analysis of mitochondrial DNA samples obtained from peripheral blood, oral mucosa, and muscle tissue. A novel large mitochondrial DNA deletion of 7,372bp was identified involving almost all genes on the big arch of mtDNA. This case reaffirms the association of adrenal insufficiency and mitochondrial DNA deletions and presents new evidence that glaucoma is another manifestation of mitochondrial diseases. Due to the genetic and clinical heterogeneity of mitochondrial disorders, molecular analysis is crucial to confirm diagnosis and to allow accurate genetic counseling.

Insulin upregulates GRIM-19 and protects cardiac mitochondrial morphology in type 1 diabetic rats partly through PI3K/AKT signaling pathway.

PubMed

Li, Yong-Guang; Dong, Zhi-Feng; Chen, Kan-Kai; He, Ya-Ping; Dai, Xiao-Yan; Li, Shuai; Li, Jing-Bo; Zhu, Wei; Wei, Meng

2017-11-04

Insulin is involved in the development of diabetic heart disease and is important in the activities of mitochondrial complex I. However, the effect of insulin on cardiac mitochondrial nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 subunit of retinoic-interferon-induced mortality 19 (GRIM-19) has not been characterized. The aim of this study was to investigate the effect of insulin on the mitochondrial GRIM-19 in the hearts of rats with streptozotocin (STZ)-induced type 1 diabetes. Protein changes of GRIM-19 were evaluated by western blotting and reverse transcription-quantitative polymerase chain reaction. Furthermore, the effects of insulin on mitochondrial complex I were detected in HeLa cells and H9C2 cardiac myocytes. During the development of diabetic heart disease, the cardiac function did not change within the 8 weeks, but the mitochondrial morphology was altered. The hearts from the rats with STZ-induced diabetes exhibited reduced expression of GRIM-19. Prior to the overt cardiac dilatation, mitochondrial alterations were already present. Following subcutaneous insulin injection, it was demonstrated that GRIM-19 protein was altered, as well as the mitochondrial morphology. The phosphoinositide 3-kinase inhibitor LY294002 had an effect on insulin signaling in H9C2 cardiacmyocytes, and decreased the level of GRIM-19 by half compared with that in the insulin group. The results indicate that insulin is essential for the control of cardiac mitochondrial morphology and the GRIM-19 expression partly via PI3K/AKT signaling pathways. Copyright © 2017. Published by Elsevier Inc.

Characterization of canine mitochondrial protein expression in natural and induced forms of idiopathic dilated cardiomyopathy.

PubMed

Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

2006-06-01

To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by >or= 2-fold between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM. Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.

The complete mitochondrial genome and phylogenetic analysis of the giant panda (Ailuropoda melanoleuca).

PubMed

Peng, Rui; Zeng, Bo; Meng, Xiuxiang; Yue, Bisong; Zhang, Zhihe; Zou, Fangdong

2007-08-01

The complete mitochondrial genome sequence of the giant panda, Ailuropoda melanoleuca, was determined by the long and accurate polymerase chain reaction (LA-PCR) with conserved primers and primer walking sequence methods. The complete mitochondrial DNA is 16,805 nucleotides in length and contains two ribosomal RNA genes, 13 protein-coding genes, 22 transfer RNA genes and one control region. The total length of the 13 protein-coding genes is longer than the American black bear, brown bear and polar bear by 3 amino acids at the end of ND5 gene. The codon usage also followed the typical vertebrate pattern except for an unusual ATT start codon, which initiates the NADH dehydrogenase subunit 5 (ND5) gene. The molecular phylogenetic analysis was performed on the sequences of 12 concatenated heavy-strand encoded protein-coding genes, and suggested that the giant panda is most closely related to bears.

Alteration of the bioenergetic phenotype of mitochondria is a hallmark of breast, gastric, lung and oesophageal cancer.

PubMed Central

Isidoro, Antonio; Martínez, Marta; Fernández, Pedro L; Ortega, Alvaro D; Santamaría, Gema; Chamorro, Margarita; Reed, John C; Cuezva, José M

2004-01-01

Recent findings indicate that the expression of the beta-catalytic subunit of the mitochondrial H+-ATP synthase (beta-F1-ATPase) is depressed in liver, kidney and colon carcinomas, providing further a bioenergetic signature of cancer that is associated with patient survival. In the present study, we performed an analysis of mitochondrial and glycolytic protein markers in breast, gastric and prostate adenocarcinomas, and in squamous oesophageal and lung carcinomas. The expression of mitochondrial and glycolytic markers varied significantly in these carcinomas, when compared with paired normal tissues, with the exception of prostate cancer. Overall, the relative expression of beta-F1-ATPase was significantly reduced in breast and gastric adenocarcinomas, as well as in squamous oesophageal and lung carcinomas, strongly suggesting that alteration of the bioenergetic function of mitochondria is a hallmark of these types of cancer. PMID:14683524

A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing

PubMed Central

Green, Richard E.; Malaspinas, Anna-Sapfo; Krause, Johannes; Briggs, Adrian W.; Johnson, Philip L. F.; Uhler, Caroline; Meyer, Matthias; Good, Jeffrey M.; Maricic, Tomislav; Stenzel, Udo; Prüfer, Kay; Siebauer, Michael; Burbano, Hernán A.; Ronan, Michael; Rothberg, Jonathan M.; Egholm, Michael; Rudan, Pavao; Brajković, Dejana; Kućan, Željko; Gušić, Ivan; Wikström, Mårten; Laakkonen, Liisa; Kelso, Janet; Slatkin, Montgomery; Pääbo, Svante

2008-01-01

Summary A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000-year-old Neandertal individual using 8,341 mtDNA sequences identified among 4.8 Gb of DNA generated from ~0.3 grams of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtDNA falls outside the variation of extant human mtDNAs and allows an estimate of the divergence date between the two mtDNA lineages of 660,000±140,000 years. Of the 13 proteins encoded in the mtDNA, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtDNA was reduced compared to other primate lineages suggesting that the effective population size of Neandertals was small. PMID:18692465

Mitochondrial genomes of the jungle crow Corvus macrorhynchos (Passeriformes: Corvidae) from shed feathers and a phylogenetic analysis of genus Corvus using mitochondrial protein-coding genes.

PubMed

Krzeminska, Urszula; Wilson, Robyn; Rahman, Sadequr; Song, Beng Kah; Seneviratne, Sampath; Gan, Han Ming; Austin, Christopher M

2016-07-01

The complete mitochondrial genomes of two jungle crows (Corvus macrorhynchos) were sequenced. DNA was extracted from tissue samples obtained from shed feathers collected in the field in Sri Lanka and sequenced using the Illumina MiSeq Personal Sequencer. Jungle crow mitogenomes have a structural organization typical of the genus Corvus and are 16,927 bp and 17,066 bp in length, both comprising 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal subunit genes, and a non-coding control region. In addition, we complement already available house crow (Corvus spelendens) mitogenome resources by sequencing an individual from Singapore. A phylogenetic tree constructed from Corvidae family mitogenome sequences available on GenBank is presented. We confirm the monophyly of the genus Corvus and propose to use complete mitogenome resources for further intra- and interspecies genetic studies.

The complete mitochondrial genome of the styloperlid stonefly species Styloperla spinicercia Wu (Insecta: Plecoptera) with family-level phylogenetic analyses of the Pteronarcyoidea.

PubMed

Wang, Ying; Cao, Jinjun; Li, Weihai

2017-03-13

We present the complete mitochondrial (mt) genome sequence of the stonefly, Styloperla spinicercia Wu, 1935 (Plecoptera: Styloperlidae), the type species of the genus Styloperla and the first complete mt genome for the family Styloperlidae. The genome is circular, 16,129 base pairs long, has an A+T content of 70.7%, and contains 37 genes including the large and small ribosomal RNA (rRNA) subunits, 13 protein coding genes (PCGs), 22 tRNA genes and a large non-coding region (CR). All of the PCGs use the standard initiation codon ATN except ND1 and ND5, which start with TTG and GTG. Twelve of the PCGs stop with conventional terminal codons TAA and TAG, except ND5 which shows an incomplete terminator signal T. All tRNAs have the classic clover-leaf structures with the dihydrouridine (DHU) arm of tRNASer(AGN) forming a simple loop. Secondary structures of the two ribosomal RNAs are presented with reference to previous models. The structural elements and the variable numbers of tandem repeats are described within the control region. Phylogenetic analyses using both Bayesian (BI) and Maximum Likelihood (ML) methods support the previous hypotheses regarding family level relationships within the Pteronarcyoidea. The genetic distance calculated based on 13 PCGs and two rRNAs between Styloperla sp. and S. spinicercia is provided and interspecific divergence is discussed.

Cloning, Purification, and Characterization of a Heterodimeric β-Galactosidase from Lactobacillus kefiranofaciens ZW3.

PubMed

He, Xi; Han, Ning; Wang, Yan-Ping

2016-01-01

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Compilation of small ribosomal subunit RNA structures.

PubMed Central

Neefs, J M; Van de Peer, Y; De Rijk, P; Chapelle, S; De Wachter, R

1993-01-01

The database on small ribosomal subunit RNA structure contained 1804 nucleotide sequences on April 23, 1993. This number comprises 365 eukaryotic, 65 archaeal, 1260 bacterial, 30 plastidial, and 84 mitochondrial sequences. These are stored in the form of an alignment in order to facilitate the use of the database as input for comparative studies on higher-order structure and for reconstruction of phylogenetic trees. The elements of the postulated secondary structure for each molecule are indicated by special symbols. The database is available on-line directly from the authors by ftp and can also be obtained from the EMBL nucleotide sequence library by electronic mail, ftp, and on CD ROM disk. PMID:8332525

Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Drosophila Model of Leigh Syndrome.

PubMed

Loewen, Carin A; Ganetzky, Barry

2018-04-01

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23 , a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23 A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23 , including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease. Copyright © 2018 by the Genetics Society of America.

Mitochondrial Genome Sequence of the Legume Vicia faba

PubMed Central

Negruk, Valentine

2013-01-01

The number of plant mitochondrial genomes sequenced exceeds two dozen. However, for a detailed comparative study of different phylogenetic branches more plant mitochondrial genomes should be sequenced. This article presents sequencing data and comparative analysis of mitochondrial DNA (mtDNA) of the legume Vicia faba. The size of the V. faba circular mitochondrial master chromosome of cultivar Broad Windsor was estimated as 588,000 bp with a genome complexity of 387,745 bp and 52 conservative mitochondrial genes; 32 of them encoding proteins, 3 rRNA, and 17 tRNA genes. Six tRNA genes were highly homologous to chloroplast genome sequences. In addition to the 52 conservative genes, 114 unique open reading frames (ORFs) were found, 36 without significant homology to any known proteins and 29 with homology to the Medicago truncatula nuclear genome and to other plant mitochondrial ORFs, 49 ORFs were not homologous to M. truncatula but possessed sequences with significant homology to other plant mitochondrial or nuclear ORFs. In general, the unique ORFs revealed very low homology to known closely related legumes, but several sequence homologies were found between V. faba, Beta vulgaris, Nicotiana tabacum, Vitis vinifera, and even the monocots Oryza sativa and Zea mays. Most likely these ORFs arose independently during angiosperm evolution (Kubo and Mikami, 2007; Kubo and Newton, 2008). Computational analysis revealed in total about 45% of V. faba mtDNA sequence being homologous to the Medicago truncatula nuclear genome (more than to any sequenced plant mitochondrial genome), and 35% of this homology ranging from a few dozen to 12,806 bp are located on chromosome 1. Apparently, mitochondrial rrn5, rrn18, rps10, ATP synthase subunit alpha, cox2, and tRNA sequences are part of transcribed nuclear mosaic ORFs. PMID:23675376

Occurrence of the root-rot pathogen, Fusarium commune, in midwestern and western United States

Treesearch

J. E. Stewart; R. K. Dumroese; N. B. Klopfenstein; M. -S. Kim

2012-01-01

Fusarium commune can cause damping-off and root rot of conifer seedlings in forest nurseries. The pathogen is only reported in Oregon, Idaho, and Washington within United States. Fusarium isolates were collected from midwestern and western United States to determine occurrence of this pathogen. DNA sequences of mitochondrial small subunit gene were used to identify F....

Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis.

PubMed

Rai, Avanish; Smita, Shachi S; Singh, Anup Kumar; Shanker, Karuna; Nagegowda, Dinesh A

2013-09-01

Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.

Isolation and characterization of the pea cytochrome c oxidase Vb gene.

PubMed

Kubo, Nakao; Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Kadowaki, Koh-Ichi; Hirai, Masashi

2006-11-01

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

Genetic Identification of Orientobilharzia turkestanicum from Sheep Isolates in Iran.

PubMed

Tabaripour, Reza; Youssefi, Mohammad Reza; Tabaripour, Rabeeh

2015-01-01

Adult worms of Orientobilharzia turkestanicum live in the portal veins, or intestinal veins of cattle, sheep, goat and many other mammals causing orientobilharziasis. Orientobilharziasis causes significant economic losses to livestock industry of Iran. However, there is limited information about genotypes of O. turkestanicum in Iran. In this study, 30 isolates of O. turkestanicum obtained from sheep were characterized by sequencing mitochondrial cytochrome c oxidase subunit 1 (cox1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) gene. The mitochondrial cox1 and nad1 DNA were amplified by polymerase chain reaction (PCR) and then sequenced and compared with O. turkestanicum and that of other members of the Schistosomatidae available in Gen-Bank(™). Phylogenetic relationships between them were re-constructed using the maximum parsimony method. Phylogenetic analyses done in present study placed O. turkestanicum within the Schistosoma genus, and indicates that O. turkestanicum was phylogenetically closer to the African schistosome group than to the Asian schistosome group. Comparison of nad1 and cox1 sequences of O. turkestanicum obtained in this study with corresponding sequences available in Genbank(™) revealed some sequence variations and provided evidence for presence of microvarients in Iran.

Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

PubMed Central

Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

2013-01-01

Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

The catholic taste of broad tapeworms - multiple routes to human infection.

PubMed

Waeschenbach, Andrea; Brabec, Jan; Scholz, Tomáš; Littlewood, D Timothy J; Kuchta, Roman

2017-11-01

Broad tapeworms (Cestoda: Diphyllobothriidea) are the principal agents of widespread food-borne cestodosis. Diphyllobothriosis and diplogonoporosis, caused by members of the genera Diphyllobothrium, Diplogonoporus and Adenocephalus, are the most common fish cestodoses with an estimated 20million people infected worldwide, and has seen recent (re)emergences in Europe due to the increasing popularity of eating raw or undercooked fish. Sparganosis is a debilitating and potentially lethal disease caused by the larvae of the genus Spirometra, which occurs throughout much of the (sub)tropics and is caused by the consumption of raw snakes and frogs, and drinking water contaminated by infected copepods. Both diseases are caused by several species, but the frequency by which the transition to humans has occurred has never been studied. Using a phylogenetic framework of 30 species based on large and small nuclear ribosomal RNA subunits (ssrDNA, lsrDNA), large subunit mitochondrial ribosomal RNA (rrnL) and cytochrome c oxidase subunit I (cox1), we hypothesize that humans have been acquired asaccidental hosts four times across the tree of life of diphyllobothriideans. However, polytomies prevent an unambiguous reconstruction of the evolution of intermediate and definitive host use. The broad host spectrum and the frequency with which switching between major host groups appears to have occurred, may hold the answer as to why accidental human infection occurred multiple times across the phylogeny of diphyllobothriideans. In this study Diplogonoporus is determined to be the junior synonym of Diphyllobothrium. Furthermore, we divide the latter polyphyletic genus into (i) the resurrected genus Dibothriocephalus to include freshwater and terrestrial species including Dibothriocephalus dendriticus, Dibothriocephalus latus and Dibothriocephalus nihonkaiensis as the most common parasites of humans, and (ii) the genus Diphyllobothrium to accommodate parasites from cetaceans including the type species Diphyllobothrium stemmacephalum and Diphyllobothrium balaenopterae n. comb. known also from humans. The non-monophyletic aggregate of marine species from seals is provisionally considered as incertae sedis. Copyright © 2017 Australian Society for Parasitology. All rights reserved.

Subconductance states of mitochondrial chloride channels: implication for functionally-coupled tetramers.

PubMed

Tomasek, Milan; Misak, Anton; Grman, Marian; Tomaskova, Zuzana

2017-08-01

Recently, it has been discovered that isoforms of intracellular chloride channels (CLIC) are present in cardiac mitochondria. By reconstituting rat cardiac mitochondrial chloride channels into bilayer lipid membranes, we detected three equally separated subconductance states with conductance increment of 45 pS and < 2% occupancy. The observed rare events of channel decomposition into substates, accompanied by disrupted gating, provide an insight into channel quaternary structure. Our findings suggest that the observed channels work as four functionally coupled subunits with synchronized gating. We discuss the putative connection of channel activity from native mitochondria with the recombinant CLIC channels. However, conclusive evidence is needed to prove this connection. © 2017 Federation of European Biochemical Societies.

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet

PubMed Central

Krebs, Philippe; Fan, Weiwei; Chen, Yen-Hui; Tobita, Kimimasa; Downes, Michael R.; Wood, Malcolm R.; Sun, Lei; Xia, Yu; Ding, Ning; Spaeth, Jason M.; Moresco, Eva Marie Y.; Boyer, Thomas G.; Lo, Cecilia Wen Ya; Yen, Jeffrey; Evans, Ronald M.; Beutler, Bruce

2011-01-01

Deficiencies of subunits of the transcriptional regulatory complex Mediator generally result in embryonic lethality, precluding study of its physiological function. Here we describe a missense mutation in Med30 causing progressive cardiomyopathy in homozygous mice that, although viable during lactation, show precipitous lethality 2–3 wk after weaning. Expression profiling reveals pleiotropic changes in transcription of cardiac genes required for oxidative phosphorylation and mitochondrial integrity. Weaning mice to a ketogenic diet extends viability to 8.5 wk. Thus, we establish a mechanistic connection between Mediator and induction of a metabolic program for oxidative phosphorylation and fatty acid oxidation, in which lethal cardiomyopathy is mitigated by dietary intervention. PMID:22106289

The mitochondrial genome of the deep-sea glass sponge Lophophysema eversa (Porifera, Hexacinellida, Hyalonematidae).

PubMed

Zhang, Yanjie; Sun, Jin; Li, Xinzheng; Qiu, Jian-Wen

2016-01-01

We reported a nearly complete mitochondrial genome (mitogenome) from the glass sponge Lophophysema eversa, the second mitogenome in the order Amphidiscosida and the ninth in the class Hexactinellida. It is 20,651 base pairs in length and contains 39 genes including 13 protein-coding genes, 2 ribosomal RNA subunit genes and 24 tRNA genes. The gene content and order of L. eversa are identical to those of Tabachnickia sp., the other species with a sequenced mitogenome in Amphidiscosida, except with two additional tRNAs and three tRNA translocations. The cob gene has a +1 translational frameshift. These results will contribute to a better understanding of the phylogeny of glass sponges.

Anti-cancer analogues ME-143 and ME-344 exert toxicity by directly inhibiting mitochondrial NADH: ubiquinone oxidoreductase (Complex I)

PubMed Central

Lim, Sze Chern; Carey, Kirstyn T; McKenzie, Matthew

2015-01-01

Isoflavonoids have been shown to inhibit tumor proliferation and metastasis by activating cell death pathways. As such, they have been widely studied as potential therapies for cancer prevention. The second generation synthetic isoflavan analogues ME-143 and ME-344 also exhibit anti-cancer effects, however their specific molecular targets have not been completely defined. To identify these targets, we examined the effects of ME-143 and ME-344 on cellular metabolism and found that they are potent inhibitors of mitochondrial oxidative phosphorylation (OXPHOS) complex I (NADH: ubiquinone oxidoreductase) activity. In isolated HEK293T mitochondria, ME-143 and ME-344 reduced complex I activity to 14.3% and 28.6% of control values respectively. In addition to the inhibition of complex I, ME-344 also significantly inhibited mitochondrial complex III (ubiquinol: ferricytochrome-c oxidoreductase) activity by 10.8%. This inhibition of complex I activity (and to a lesser extent complex III activity) was associated with a reduction in mitochondrial oxygen consumption. In permeabilized HEK293T cells, ME-143 and ME-344 significantly reduced the maximum ADP-stimulated respiration rate to 62.3% and 70.0% of control levels respectively in the presence of complex I-linked substrates. Conversely, complex II-linked respiration was unaffected by either drug. We also observed that the inhibition of complex I-linked respiration caused the dissipation of the mitochondrial membrane potential (ΔΨm). Blue native (BN-PAGE) analysis revealed that prolonged loss of ΔΨm results in the destabilization of the native OXPHOS complexes. In particular, treatment of 143B osteosarcoma, HeLa and HEK293T human embryonic kidney cells with ME-344 for 4 h resulted in reduced steady-state levels of mature complex I. Degradation of the complex I subunit NDUFA9, as well as the complex IV (ferrocytochrome c: oxygen oxidoreductase) subunit COXIV, was also evident. The identification of OXPHOS complex I as a target of ME-143 and ME-344 advances our understanding of how these drugs induce cell death by disrupting mitochondrial metabolism, and will direct future work to maximize the anti-cancer capacity of these and other isoflavone-based compounds. PMID:25973307

Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging

PubMed Central

Umanskaya, Alisa; Santulli, Gaetano; Andersson, Daniel C.; Reiken, Steven R.; Marks, Andrew R.

2014-01-01

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca2+ transients, decreased intracellular Ca2+ leak and increased sarcoplasmic reticulum (SR) Ca2+ load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca2+ release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (Po). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca2+ leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders. PMID:25288763

Mitochondrial Complex I Activity Suppresses Inflammation and Enhances Bone Resorption by Tipping the Balance of Macrophage-Osteoclast Polarization

PubMed Central

Jin, Zixue; Wei, Wei; Yang, Marie; Du, Yang; Wan, Yihong

2014-01-01

SUMMARY Mitochondrial complex I (CI) deficiency is associated with multiple neurological and metabolic disorders. However, its effect on innate immunity and bone remodeling is unclear. Using deletion of the essential CI subunit Ndufs4 as a model for mitochondrial dysfunction, we report that mitochondria suppress macrophage activation and inflammation while promoting osteoclast differentiation and bone resorption via both cell-autonomous and systemic regulation. Global Ndufs4 deletion causes systemic inflammation and osteopetrosis. Hematopoietic Ndufs4 deletion causes an intrinsic lineage shift from osteoclast to macrophage. Liver Ndufs4 deletion causes a metabolic shift from fatty acid oxidation to glycolysis, accumulating fatty acids and lactate (FA/LAC) in circulation. FA/LAC further activates Ndufs4−/− macrophages via ROS induction, and diminishes osteoclast lineage commitment in Ndufs4−/− progenitors; both inflammation and osteopetrosis in Ndufs4−/− mice are attenuated by TLR4/2 deletion. Together, these findings reveal mitochondrial CI as a critical rheostat of innate immunity and skeletal homeostasis. PMID:25130399

TEFM is a potent stimulator of mitochondrial transcription elongation in vitro

PubMed Central

Posse, Viktor; Shahzad, Saba; Falkenberg, Maria; Hällberg, B. Martin; Gustafsson, Claes M.

2015-01-01

A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery. PMID:25690892

Horizontal transfer of whole mitochondria restores tumorigenic potential in mitochondrial DNA-deficient cancer cells

PubMed Central

Dong, Lan-Feng; Kovarova, Jaromira; Bajzikova, Martina; Bezawork-Geleta, Ayenachew; Svec, David; Endaya, Berwini; Sachaphibulkij, Karishma; Coelho, Ana R; Sebkova, Natasa; Ruzickova, Anna; Tan, An S; Kluckova, Katarina; Judasova, Kristyna; Zamecnikova, Katerina; Rychtarcikova, Zuzana; Gopalan, Vinod; Andera, Ladislav; Sobol, Margarita; Yan, Bing; Pattnaik, Bijay; Bhatraju, Naveen; Truksa, Jaroslav; Stopka, Pavel; Hozak, Pavel; Lam, Alfred K; Sedlacek, Radislav; Oliveira, Paulo J; Kubista, Mikael; Agrawal, Anurag; Dvorakova-Hortova, Katerina; Rohlena, Jakub; Berridge, Michael V; Neuzil, Jiri

2017-01-01

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer. DOI: http://dx.doi.org/10.7554/eLife.22187.001 PMID:28195532

Leigh disease with brainstem involvement in complex I deficiency due to assembly factor NDUFAF2 defect.

PubMed

Herzer, M; Koch, J; Prokisch, H; Rodenburg, R; Rauscher, C; Radauer, W; Forstner, R; Pilz, P; Rolinski, B; Freisinger, P; Mayr, J A; Sperl, W

2010-02-01

Mitochondrial NADH: ubiquinone oxidoreductase (complex I) deficiency accounts for most defects in mitochondrial oxidative phosphorylation. Pathogenic mutations have been described in all 7 mitochondrial and 12 of the 38 nuclear encoded subunits as well as in assembly factors by interfering with the building of the mature enzyme complex within the inner mitochondrial membrane. We now describe a male patient with a novel homozygous stop mutation in the NDUFAF2 gene. The boy presented with severe apnoea and nystagmus. MRI showed brainstem lesions without involvement of basal ganglia and thalamus, plasma lactate was normal or close to normal. He died after a fulminate course within 2 months after the first crisis. Neuropathology verified Leigh disease. We give a synopsis with other reported patients. Within the clinical spectrum of Leigh disease, patients with mutations in NDUFAF2 present with a distinct clinical pattern with predominantly brainstem involvement on MRI. The diagnosis should not be missed in spite of the normal lactate and lack of thalamus and basal ganglia changes on brain MRI.

Morphological and molecular characterisation of Aporocotyle margolisi Smith, 1967 (Digenea: Aporocotylidae) from the North Pacific hake Merluccius productus (Ayres) (Gadiformes: Merlucciidae) off Oregon, USA.

PubMed

Hernández-Orts, Jesus S; Hernández-Mena, David I; Alama-Bermejo, Gema; Kuchta, Roman; Jacobson, Kym C

2017-09-01

Aporocotylid blood flukes conspecific with Aporocotyle margolisi Smith, 1967 were collected from the bulbus arteriosus of the North Pacific hake Merluccius productus (Ayres). This study revisits the morphology of A. margolisi, including drawings, measurements and scanning electron microscopy images, and provides for the first time molecular data for the large subunit of the ribosomal RNA (28S rDNA) and the mitochondrial cytochrome c oxidase subunit 1 (cox1) genes for this species. A 28S rDNA phylogenetic study of A. margolisi, and all available Aporocotyle spp., was also performed. The distribution range of A. margolisi is extended to the Pacific coast of the USA. We provide a morphological comparison of Aporocotyle spp. from the Pacific coast in North America as well as other Aporocotyle spp. infecting hake. Comparisons with the original description revealed that the new specimens of A. margolisi were considerably larger with respect to all morphological features, except for shorter spines. Molecular results showed a close relationship between A. margolisi and A. argentinensis Smith, 1969 from the Argentine hake Merluccius hubbsi Marini. The phylogenetic relationships of Aporocotyle spp. point to a possible co-speciation of hakes species and these blood fluke parasites.

Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets.

PubMed

Bengtsson, Johan; Eriksson, K Martin; Hartmann, Martin; Wang, Zheng; Shenoy, Belle Damodara; Grelet, Gwen-Aëlle; Abarenkov, Kessy; Petri, Anna; Rosenblad, Magnus Alm; Nilsson, R Henrik

2011-10-01

The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

Oxidative Stress Induced Inflammation Initiates Functional Decline of Tear Production

PubMed Central

Uchino, Yuichi; Kawakita, Tetsuya; Miyazawa, Masaki; Ishii, Takamasa; Onouchi, Hiromi; Yasuda, Kayo; Ogawa, Yoko; Shimmura, Shigeto; Ishii, Naoaki; Tsubota, Kazuo

2012-01-01

Oxidative damage and inflammation are proposed to be involved in an age-related functional decline of exocrine glands. However, the molecular mechanism of how oxidative stress affects the secretory function of exocrine glands is unclear. We developed a novel mev-1 conditional transgenic mouse model (Tet-mev-1) using a modified tetracycline system (Tet-On/Off system). This mouse model demonstrated decreased tear production with morphological changes including leukocytic infiltration and fibrosis. We found that the mev-1 gene encodes Cyt-1, which is the cytochrome b560 large subunit of succinate-ubiquinone oxidoreductase in complex II of mitochondria (homologous to succinate dehydrogenase C subunit (SDHC) in humans). The mev-1 gene induced excessive oxidative stress associated with ocular surface epithelial damage and a decrease in protein and aqueous secretory function. This new model provides evidence that mitochondrial oxidative damage in the lacrimal gland induces lacrimal dysfunction resulting in dry eye disease. Tear volume in Tet-mev-1 mice was lower than in wild type mice and histopathological analyses showed the hallmarks of lacrimal gland inflammation by intense mononuclear leukocytic infiltration and fibrosis in the lacrimal gland of Tet-mev-1 mice. These findings strongly suggest that oxidative stress can be a causative factor for the development of dry eye disease. PMID:23071526

ENU mutagenesis identifies mice with cardiac fibrosis and hepatic steatosis caused by a mutation in the mitochondrial trifunctional protein beta-subunit.

PubMed

Kao, Hsiao-Jung; Cheng, Ching-Feng; Chen, Yen-Hui; Hung, Shuen-Iu; Huang, Cheng-Chih; Millington, David; Kikuchi, Tateki; Wu, Jer-Yuarn; Chen, Yuan-Tsong

2006-12-15

Using the metabolomics-guided screening coupled to N-ethyl-N-nitrosourea-mediated mutagenesis, we identified mice that exhibited elevated levels of long-chain acylcarnitines. Whole genome homozygosity mapping with 262 SNP markers mapped the disease gene to chromosome 5 where candidate genes Hadha and Hadhb, encoding the mitochondria trifunctional protein (MTP) alpha- and beta-subunits, respectively, are located. Direct sequencing revealed a normal alpha-subunit, but detected a nucleotide T-to-A transversion in exon 14 (c.1210T>A) of beta-subunit (Hadhb) which resulted in a missense mutation of methionine to lysine (M404K). Western blot analysis showed a significant reduction of both the alpha- and beta-subunits, consistent with reduced enzyme activity in both the long-chain 3-hydroxyacyl-CoA dehydrogenase and the long-chain 3-ketoacyl-CoA thiolase activities. These mice had a decreased weight gain and cardiac arrhythmias which manifested from a prolonged PR interval to a complete atrio-ventricular dissociation, and died suddenly between 9 and 16 months of age. Histopathological studies showed multifocal cardiac fibrosis and hepatic steatosis. This mouse model will be useful to further investigate the mechanisms underlying arrhythmogenesis relating to lipotoxic cardiomyopathy and to investigate pathophysiology and treatment strategies for human MTP deficiency.

Inhalation of Roman chamomile essential oil attenuates depressive-like behaviors in Wistar Kyoto rats.

PubMed

Kong, Yingying; Wang, Ting; Wang, Rong; Ma, Yichuan; Song, Shanshan; Liu, Juan; Hu, Weiwei; Li, Shengtian

2017-06-01

The idea of aromatherapy, using essential oils, has been considered as an alternative antidepressant treatment. In the present study, we investigated the effect of Roman chamomile essential oil inhalation for two weeks on depressive-like behaviors in Wistar-Kyoto (WKY) rats. We found that inhalation of either Roman chamomile or one of its main components α-pinene, attenuated depressive-like behavior in WKY rats in the forced swim test. Using isobaric tags for relative and absolute quantitation analysis (iTRAQ), we found that inhalation of α-pinene increased expression of proteins that are involved in oxidative phosphorylation, such as cytochrome c oxidase subunit 6C-2, cytochrome c oxidase subunit 7A2, ATPase inhibitor in the hippocampus, and cytochrome c oxidase subunit 6C-2, ATP synthase subunit e, Acyl carrier protein, and Cytochrome b-c1 complex subunit 6 in the PFC (prefrontal cortex). In addition, using the quantitative real-time polymerase chain reaction technique, we confirmed an increase of parvalbumin mRNA expression in the hippocampus, which was shown to be upregulated by 2.8-fold in iTRAQ analysis, in α-pinene treated WKY rats. These findings collectively suggest the involvement of mitochondrial functions and parvalbumin-related signaling in the antidepressant effect of α-pinene inhalation.

Cardioprotective Effect of Danshensu against Ischemic/Reperfusion Injury via c-Subunit of ATP Synthase Inhibition

PubMed Central

Zhao, JingYi; Fan, Zixuan; Bao, Jiadi; Sun, Dawei; Sun, Chun

2017-01-01

Mitochondrial permeability transition pore (MPTP) opening is the main culprit of ischemic/reperfusion (IR) injury. It is reported that c-subunit of ATP synthase is the core component of MPTP. Danshensu (DSS), a monomer isolated from the traditional Chinese herb Danshen, has showed cardioprotective effect against IR injury through unknown mechanism. In this study, rat hearts were suspended in Langendorff instrument and perfused with Krebs-Henseleit (KH) buffer containing DSS for 60 minutes, followed by 30 minutes of global ischemia. Parameters including heart rate, left ventricular developed pressure, and the rate of left ventricle diastolic pressure change were recorded to assess their cardiac function. All these indexes were improved in DSS group. The rate of cardiomyocytes apoptosis and MPTP opening were both inhibited in DSS group. In addition, DSS administration leads to downregulation of c-subunit of ATP synthase in both mRNA and protein levels. Consistently, when c-subunit of ATP synthase was overexpressed in H9C2 cells through pcDNA3/5G1 plasmid transfection, MPTP opening was enhanced when the cardioprotective effect of DSS also tapers. In conclusion, DSS could alleviate cardiac IR injury via inhibiting c-subunit of ATP synthase expression. PMID:29250127

Identification of a Novel HADHB Gene Mutation in an Iranian Patient with Mitochondrial Trifunctional Protein Deficiency.

PubMed

Shahrokhi, Mahdiyeh; Shafiei, Mohammad; Galehdari, Hamid; Shariati, Gholamreza

2017-01-01

Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of eight parts (subunits): four α-subunits containing LCEH (long-chain 2,3-enoyl-CoA  hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and four β-subunits that possess LCKT (long-chain  3-ketoacyl-CoA thiolase) activity which catalyzes three out of four steps in β-oxidation spiral of long-chain fatty acid. Its deficiency is an autosomal recessive disorder that causes a clinical spectrum of diseases. A blood spot was collected from the patient's original newborn screening card with parental informed consent. A newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS were performed. After isolation of DNA and Amplification of all exons of the HADHA and HADHB, directly Sequence analyses of all exons and the flanking introns both of genes were performed. Here, we report a novel mutation in a patient with MTP deficiency diagnosed with newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS and then confirmed by enzyme analysis in cultured fibroblasts and direct sequencing of the HADHA and HADHB genes. Molecular analysis of causative genes showed a missense mutation (p.Q385P) c.1154A > C in exon 14 of HADHB gene. Since this mutation was not found in 50 normal control cases; so it was concluded that c.1154A > C mutation was a causative mutation. Phenotype analysis of this mutation predicted pathogenesis which reduces the stability of the MTP protein complex.

Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients.

PubMed

Poyau, A; Buchet, K; Bouzidi, M F; Zabot, M T; Echenne, B; Yao, J; Shoubridge, E A; Godinot, C

2000-02-01

We have studied the fibroblasts of three patients suffering from Leigh syndrome associated with cytochrome c oxidase deficiency (LS-COX-). Their mitochondrial DNA was functional and all nuclear COX subunits had a normal sequence. The expression of transcripts encoding mitochondrial and nuclear COX subunits was normal or slightly increased. Similarly, the OXA1 transcript coding for a protein involved in COX assembly was increased. However, several COX-protein subunits were severely depressed, indicating deficient COX assembly. Surf1, a factor involved in COX biogenesis, was recently reported as mutated in LS-COX- patients, all mutations predicting a truncated protein. Sequence analysis of SURF1 gene in our three patients revealed seven heterozygous mutations, six of which were new : an insertion, a nonsense mutation, a splicing mutation of intron 7 in addition to three missense mutations. The mutation G385 A (Gly124-->Glu) changes a Gly that is strictly conserved in Surfl homologs of 12 species. The substitution G618 C (Asp202-->His), changing an Asp that is conserved only in mammals, appears to be a polymorphism. The mutation T751 C changes Ile246 to Thr, a position at which a hydrophobic amino acid is conserved in all eukaryotic and some bacterial species. Replacing Ile246 by Thr disrupts a predicted beta sheet structure present in all higher eukaryotes. COX activity could be restored in fibroblasts of the three patients by complementation with a retroviral vector containing normal SURF1 cDNA. These mutations identify domains essential to Surf1 protein structure and/or function.

Dual African Origins of Global Aedes aegypti s.l. Populations Revealed by Mitochondrial DNA

PubMed Central

Moore, Michelle; Sylla, Massamba; Goss, Laura; Burugu, Marion Warigia; Sang, Rosemary; Kamau, Luna W.; Kenya, Eucharia Unoma; Bosio, Chris; Munoz, Maria de Lourdes; Sharakova, Maria; Black, William Cormack

2013-01-01

Background Aedes aegypti is the primary global vector to humans of yellow fever and dengue flaviviruses. Over the past 50 years, many population genetic studies have documented large genetic differences among global populations of this species. These studies initially used morphological polymorphisms, followed later by allozymes, and most recently various molecular genetic markers including microsatellites and mitochondrial markers. In particular, since 2000, fourteen publications and four unpublished datasets have used sequence data from the NADH dehydrogenase subunit 4 mitochondrial gene to compare Ae. aegypti collections and collectively 95 unique mtDNA haplotypes have been found. Phylogenetic analyses in these many studies consistently resolved two clades but no comprehensive study of mtDNA haplotypes have been made in Africa, the continent in which the species originated. Methods and Findings ND4 haplotypes were sequenced in 426 Ae. aegypti s.l. from Senegal, West Africa and Kenya, East Africa. In Senegal 15 and in Kenya 7 new haplotypes were discovered. When added to the 95 published haplotypes and including 6 African Aedes species as outgroups, phylogenetic analyses showed that all but one Senegal haplotype occurred in a basal clade while most East African haplotypes occurred in a second clade arising from the basal clade. Globally distributed haplotypes occurred in both clades demonstrating that populations outside Africa consist of mixtures of mosquitoes from both clades. Conclusions Populations of Ae. aegypti outside Africa consist of mosquitoes arising from one of two ancestral clades. One clade is basal and primarily associated with West Africa while the second arises from the first and contains primarily mosquitoes from East Africa PMID:23638196

Genetic diversity and population genetics of mosquitoes (Diptera: Culicidae: Culex spp.) from the Sonoran Desert of North America.

PubMed

Pfeiler, Edward; Flores-López, Carlos A; Mada-Vélez, Jesús Gerardo; Escalante-Verdugo, Juan; Markow, Therese A

2013-01-01

The population genetics and phylogenetic relationships of Culex mosquitoes inhabiting the Sonoran Desert region of North America were studied using mitochondrial DNA and microsatellite molecular markers. Phylogenetic analyses of mitochondrial cytochrome c oxidase subunit I (COI) from mosquitoes collected over a wide geographic area, including the Baja California peninsula, and mainland localities in southern Arizona, USA and Sonora, Mexico, showed several well-supported partitions corresponding to Cx. quinquefasciatus, Cx. tarsalis, and two unidentified species, Culex sp. 1 and sp. 2. Culex quinquefasciatus was found at all localities and was the most abundant species collected. Culex tarsalis was collected only at Tucson, Arizona and Guaymas, Sonora. The two unidentified species of Culex were most abundant at Navojoa in southern Sonora. Haplotype and nucleotide diversities in the COI gene segment were substantially lower in Cx. quinquefasciatus compared with the other three species. Analysis of molecular variance revealed little structure among seven populations of Cx. quinquefasciatus, whereas significant structure was found between the two populations of Cx. tarsalis. Evidence for an historical population expansion beginning in the Pleistocene was found for Cx. tarsalis. Possible explanations for the large differences in genetic diversity between Cx. quinquefasciatus and the other species of Culex are presented.

Genetic Diversity and Population Genetics of Mosquitoes (Diptera: Culicidae: Culex spp.) from the Sonoran Desert of North America

PubMed Central

Pfeiler, Edward; Flores-López, Carlos A.; Mada-Vélez, Jesús Gerardo; Escalante-Verdugo, Juan; Markow, Therese A.

2013-01-01

The population genetics and phylogenetic relationships of Culex mosquitoes inhabiting the Sonoran Desert region of North America were studied using mitochondrial DNA and microsatellite molecular markers. Phylogenetic analyses of mitochondrial cytochrome c oxidase subunit I (COI) from mosquitoes collected over a wide geographic area, including the Baja California peninsula, and mainland localities in southern Arizona, USA and Sonora, Mexico, showed several well-supported partitions corresponding to Cx. quinquefasciatus, Cx. tarsalis, and two unidentified species, Culex sp. 1 and sp. 2. Culex quinquefasciatus was found at all localities and was the most abundant species collected. Culex tarsalis was collected only at Tucson, Arizona and Guaymas, Sonora. The two unidentified species of Culex were most abundant at Navojoa in southern Sonora. Haplotype and nucleotide diversities in the COI gene segment were substantially lower in Cx. quinquefasciatus compared with the other three species. Analysis of molecular variance revealed little structure among seven populations of Cx. quinquefasciatus, whereas significant structure was found between the two populations of Cx. tarsalis. Evidence for an historical population expansion beginning in the Pleistocene was found for Cx. tarsalis. Possible explanations for the large differences in genetic diversity between Cx. quinquefasciatus and the other species of Culex are presented. PMID:24302868

Effects of habitat fragmentation on the genetic diversity and differentiation of Dendrolimus punctatus (Lepidoptera: Lasiocampidae) in Thousand Island Lake, China, based on mitochondrial COI gene sequences.

PubMed

Lv, K; Wang, J-R; Li, T-Q; Zhou, J; Gu, J-Q; Zhou, G-X; Xu, Z-H

2018-05-10

Thousand Island Lake (TIL) is a typical fragmented landscape and an ideal model to study ecological effects of fragmentation. Partial fragments of the mitochondrial cytochrome oxidase subunit I gene of 23 island populations of Dendrolimus punctatus in TIL were sequenced, 141 haplotypes being identified. The number of haplotypes increased significantly with the increase in island area and shape index, whereas no significant correlation was detected between three island attributes (area, shape and isolation) and haplotype diversity. However, the correlation with number of haplotypes was no longer significant when the 'outlier' island JSD (the largest island) was not included. Additionally, we found no significant relationship between geographic distance and genetic distance. Geographic isolation did not obstruct the gene flow among D. punctatus populations, which might be because of the high dispersal capacity of this pine moth. Fragmentation resulted in the conversion of large and continuous habitats into isolated, small and insular patches, which was the primary effect on the genetic diversity of D. punctatus in TIL. The conclusion to emphasize from our research is that habitat fragmentation reduced the biological genetic diversity to some extent, further demonstrating the importance of habitat continuity in biodiversity protection.

Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F₁F₀-ATP synthase

PubMed Central

Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent

2013-01-01

We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53+/+ cells simultaneously show increased O₂ consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F₁F₀-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F₁F₀-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria. PMID:23966169

Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F₁F0-ATP synthase.

PubMed

Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent

2013-09-01

We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53(+/+) cells simultaneously show increased O₂ consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F₁F₀-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F₁F₀-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria.

Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats.

PubMed

Akude, Eli; Zherebitskaya, Elena; Chowdhury, Subir K Roy; Smith, Darrell R; Dobrowsky, Rick T; Fernyhough, Paul

2011-01-01

Impairments in mitochondrial function have been proposed to play a role in the etiology of diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in axons of sensory neurons in type 1 diabetes is due to abnormal activity of the respiratory chain and an altered mitochondrial proteome. Proteomic analysis using stable isotope labeling with amino acids in cell culture (SILAC) determined expression of proteins in mitochondria from dorsal root ganglia (DRG) of control, 22-week-old streptozotocin (STZ)-diabetic rats, and diabetic rats treated with insulin. Rates of oxygen consumption and complex activities in mitochondria from DRG were measured. Fluorescence imaging of axons of cultured sensory neurons determined the effect of diabetes on mitochondrial polarization status, oxidative stress, and mitochondrial matrix-specific reactive oxygen species (ROS). Proteins associated with mitochondrial dysfunction, oxidative phosphorylation, ubiquinone biosynthesis, and the citric acid cycle were downregulated in diabetic samples. For example, cytochrome c oxidase subunit IV (COX IV; a complex IV protein) and NADH dehydrogenase Fe-S protein 3 (NDUFS3; a complex I protein) were reduced by 29 and 36% (P < 0.05), respectively, in diabetes and confirmed previous Western blot studies. Respiration and mitochondrial complex activity was significantly decreased by 15 to 32% compared with control. The axons of diabetic neurons exhibited oxidative stress and depolarized mitochondria, an aberrant adaption to oligomycin-induced mitochondrial membrane hyperpolarization, but reduced levels of intramitochondrial superoxide compared with control. Abnormal mitochondrial function correlated with a downregulation of mitochondrial proteins, with components of the respiratory chain targeted in lumbar DRG in diabetes. The reduced activity of the respiratory chain was associated with diminished superoxide generation within the mitochondrial matrix and did not contribute to oxidative stress in axons of diabetic neurons. Alternative pathways involving polyol pathway activity appear to contribute to raised ROS in axons of diabetic neurons under high glucose concentration.

Deletion of PRKAA triggers mitochondrial fission by inhibiting the autophagy-dependent degradation of DNM1L.

PubMed

Wang, Qilong; Wu, Shengnan; Zhu, Huaiping; Ding, Ye; Dai, Xiaoyan; Ouyang, Changhan; Han, Young-Min; Xie, Zhonglin; Zou, Ming-Hui

2017-02-01

PRKAA (protein kinase, AMP-activated, α catalytic subunit) regulates mitochondrial biogenesis, function, and turnover. However, the molecular mechanisms by which PRKAA regulates mitochondrial dynamics remain poorly characterized. Here, we report that PRKAA regulated mitochondrial fission via the autophagy-dependent degradation of DNM1L (dynamin 1-like). Deletion of Prkaa1/AMPKα1 or Prkaa2/AMPKα2 resulted in defective autophagy, DNM1L accumulation, and aberrant mitochondrial fragmentation in the mouse aortic endothelium. Furthermore, autophagy inhibition by chloroquine treatment or ATG7 small interfering RNA (siRNA) transfection, upregulated DNM1L expression and triggered DNM1L-mediated mitochondrial fragmentation. In contrast, autophagy activation by overexpression of ATG7 or chronic administration of rapamycin, the MTOR inhibitor, promoted DNM1L degradation and attenuated mitochondrial fragmentation in Prkaa2-deficient (prkaa2 -/- ) mice, suggesting that defective autophagy contributes to enhanced DNM1L expression and mitochondrial fragmentation. Additionally, the autophagic receptor protein SQSTM1/p62, which bound to DNM1L and led to its translocation into the autophagosome, was involved in DNM1L degradation by the autophagy-lysosome pathway. Gene silencing of SQSTM1 markedly reduced the association between SQSTM1 and DNM1L, impaired the degradation of DNM1L, and enhanced mitochondrial fragmentation in PRKAA-deficient endothelial cells. Finally, the genetic (DNM1L siRNA) or pharmacological (mdivi-1) inhibition of DNMA1L ablated mitochondrial fragmentation in the mouse aortic endothelium and prevented the acetylcholine-induced relaxation of isolated mouse aortas. This suggests that aberrant DNM1L is responsible for enhanced mitochondrial fragmentation and endothelial dysfunction in prkaa knockout mice. Overall, our results show that PRKAA deletion promoted mitochondrial fragmentation in vascular endothelial cells by inhibiting the autophagy-dependent degradation of DNM1L.

Mind the gap! The mitochondrial control region and its power as a phylogenetic marker in echinoids.

PubMed

Bronstein, Omri; Kroh, Andreas; Haring, Elisabeth

2018-05-30

In Metazoa, mitochondrial markers are the most commonly used targets for inferring species-level molecular phylogenies due to their extremely low rate of recombination, maternal inheritance, ease of use and fast substitution rate in comparison to nuclear DNA. The mitochondrial control region (CR) is the main non-coding area of the mitochondrial genome and contains the mitochondrial origin of replication and transcription. While sequences of the cytochrome oxidase subunit 1 (COI) and 16S rRNA genes are the prime mitochondrial markers in phylogenetic studies, the highly variable CR is typically ignored and not targeted in such analyses. However, the higher substitution rate of the CR can be harnessed to infer the phylogeny of closely related species, and the use of a non-coding region alleviates biases resulting from both directional and purifying selection. Additionally, complete mitochondrial genome assemblies utilizing next generation sequencing (NGS) data often show exceptionally low coverage at specific regions, including the CR. This can only be resolved by targeted sequencing of this region. Here we provide novel sequence data for the echinoid mitochondrial control region in over 40 species across the echinoid phylogenetic tree. We demonstrate the advantages of directly targeting the CR and adjacent tRNAs to facilitate complementing low coverage NGS data from complete mitochondrial genome assemblies. Finally, we test the performance of this region as a phylogenetic marker both in the lab and in phylogenetic analyses, and demonstrate its superior performance over the other available mitochondrial markers in echinoids. Our target region of the mitochondrial CR (1) facilitates the first thorough investigation of this region across a wide range of echinoid taxa, (2) provides a tool for complementing missing data in NGS experiments, and (3) identifies the CR as a powerful, novel marker for phylogenetic inference in echinoids due to its high variability, lack of selection, and high compatibility across the entire class, outperforming conventional mitochondrial markers.

Melatonin-induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence.

PubMed

He, Bin; Yin, Chao; Gong, Yabin; Liu, Jie; Guo, Huiduo; Zhao, Ruqian

2018-01-01

Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence. © 2017 Wiley Periodicals, Inc.

Protective effects of peroxisome proliferator-activated receptor agonists on human podocytes: proposed mechanisms of action

PubMed Central

Miglio, Gianluca; Rosa, Arianna Carolina; Rattazzi, Lorenza; Grange, Cristina; Camussi, Giovanni; Fantozzi, Roberto

2012-01-01

BACKGROUND AND PURPOSE Peroxisome proliferator-activated receptor (PPAR) agonists exert anti-albuminuric effects. However, the nephroprotective effects of these drugs remain to be fully understood. We have investigated whether gemfibrozil, GW0742 and pioglitazone protect human podocytes against nutrient deprivation (ND)-induced cell death and the role of mitochondrial biogenesis as a cytoprotective process. EXPERIMENTAL APPROACH Immortalized human podocytes were pre-treated with the PPAR agonists and exposed to ND (5 h) under normoxia, hypoxia or in the presence of pyruvate. Cell death was measured at the end of the ND and of the recovery phase (24 h). Mitochondrial mass, cytochrome c oxidase (COX) subunits 1 and 4 were measured as markers of mitochondrial cell content, while membrane potential as an index of mitochondrial function. PGC-1α, NRF1 and Tfam expression was studied, as crucial regulators of mitochondrial biogenesis. KEY RESULTS Cell pre-treatment with gemfibrozil, GW0742, or pioglitazone significantly decreased the ND-induced cell loss, necrosis and apoptosis. These effects were attenuated by hypoxia and potentiated by pyruvate. Pre-treatment with these drugs significantly increased mitochondrial cell content, while it did not affect mitochondrial function. In all these experiments pioglitazone exerted significantly larger effects than gemfibrozil or GW0742. CONCLUSIONS AND IMPLICATIONS Gemfibrozil, GW0742 and pioglitazone may exert direct protective effects on human podocytes. Mitochondrial biogenesis is a cell response to the PPAR agonists related to their cytoprotective activity. These results provide a mechanistic support to the clinical evidence indicating PPAR agonists as disease-modifying agents for glomerular diseases. PMID:22594945

Codon usage bias and phylogenetic analysis of mitochondrial ND1 gene in pisces, aves, and mammals.

PubMed

Uddin, Arif; Choudhury, Monisha Nath; Chakraborty, Supriyo

2018-01-01

The mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) gene is a subunit of the respiratory chain complex I and involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). To understand the pattern of compositional properties, codon usage and expression level of mitochondrial ND1 genes in pisces, aves, and mammals, we used bioinformatic approaches as no work was reported earlier. In this study, a perl script was used for calculating nucleotide contents and different codon usage bias parameters. The codon usage bias of MT-ND1 was low but the expression level was high as revealed from high ENC and CAI value. Correspondence analysis (COA) suggests that the pattern of codon usage for MT-ND1 gene is not same across species and that compositional constraint played an important role in codon usage pattern of this gene among pisces, aves, and mammals. From the regression equation of GC12 on GC3, it can be inferred that the natural selection might have played a dominant role while mutation pressure played a minor role in influencing the codon usage patterns. Further, ND1 gene has a discrepancy with cytochrome B (CYB) gene in preference of codons as evident from COA. The codon usage bias was low. It is influenced by nucleotide composition, natural selection, mutation pressure, length (number) of amino acids, and relative dinucleotide composition. This study helps in understanding the molecular biology, genetics, evolution of MT-ND1 gene, and also for designing a synthetic gene.

Proteomics Analysis of Human Skeletal Muscle Reveals Novel Abnormalities in Obesity and Type 2 Diabetes

PubMed Central

Hwang, Hyonson; Bowen, Benjamin P.; Lefort, Natalie; Flynn, Charles R.; De Filippis, Elena A.; Roberts, Christine; Smoke, Christopher C.; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J.

2010-01-01

OBJECTIVE Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here. RESEARCH DESIGN AND METHODS Muscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry–based quantification using normalized spectral abundance factors. RESULTS Of 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased). CONCLUSIONS The results confirm the reduction in mitochondrial proteins in insulin-resistant muscle and suggest that changes in muscle structure, protein degradation, and folding also characterize insulin resistance. PMID:19833877

Lipoic acid biosynthesis defects.

PubMed

Mayr, Johannes A; Feichtinger, René G; Tort, Frederic; Ribes, Antonia; Sperl, Wolfgang

2014-07-01

Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metabolism, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metabolism, branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metabolism with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulphur-cluster-dependent enzyme, a number of recently identified defects in mitochondrial iron-sulphur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clinical spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.

Different effects of the nonsteroidal anti-inflammatory drugs meclofenamate sodium and naproxen sodium on proteasome activity in cardiac cells.

PubMed

Ghosh, Rajeshwary; Hwang, Soyun M; Cui, Ziyou; Gilda, Jennifer E; Gomes, Aldrin V

2016-05-01

The use of nonsteroidal anti-inflammatory drugs (NSAIDs) like meclofenamate sodium (MS), used to reduce pain, has been associated with an increased risk of cardiovascular disease (CVD). Naproxen (NAP), another NSAID, is not associated with increased risk of CVD. The molecular mechanism(s) by which NSAIDs induce CVD is unknown. We investigated the effects of MS and NAP on protein homeostasis and cardiotoxicity in rat cardiac H9c2 cells and murine neonatal cardiomyocytes. MS, but not NAP, significantly inhibited proteasome activity and reduced cardiac cell viability at pharmacological levels found in humans. Although proteasome subunit gene and protein expression were unaffected by NSAIDs, MS treated cell lysates showed higher 20S proteasome content, while purified proteasomes from MS treated cells had lower proteasome activity and higher levels of oxidized subunits than proteasomes from control cells. Addition of exogenous proteasome to MS treated cells improved cell viability. Both MS and NAP increased ROS production, but the rate of ROS production was greater in MS than in NAP treated cells. The ROS production is likely from mitochondria, as MS inhibited mitochondrial Complexes I and III, major sources of ROS, while NAP inhibited Complex I. MS also impaired mitochondrial membrane potential while NAP did not. Antioxidants were able to prevent the reduced cell viability caused by MS treatment. These results suggest that NSAIDs induce cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction and may explain why some NSAIDs should not be given to patients for long periods. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Chemical Form of Mitochondrial Iron in Friedreich's Ataxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popescu, B.F.Gh.; Pickering, I.J.; George, G.N.

2007-07-12

Friedreich's ataxia (FRDA) results from cellular damage caused by a deficiency in the mitochondrial matrix protein frataxin. To address the effect of frataxin deficiency on mitochondrial iron chemistry, the heavy mitochondrial fraction (HMF) was isolated from primary fibroblasts from FRDA affected and unaffected individuals. X-ray absorption spectroscopy was used to characterize the chemical form of iron. Near K-edge spectra were fitted with a series of model iron compounds to determine the proportion of each iron species. Most of the iron in both affected and unaffected fibroblasts was ferrihydrite. The iron K-edge from unaffected HMFs were best fitted with poorly organizedmore » ferrihydrite modeled by frataxin whereas HMFs from affected cells were best fitted with highly organized ferrihydrite modeled by ferritin. Both had several minor iron species but these did not differ consistently with disease. Since the iron K-edge spectra of ferritin and frataxin are very similar, we present additional evidence for the presence of ferritin-bound iron in HMF. The predominant ferritin subunit in HMFs from affected cells resembled mitochondrial ferritin (MtFt) in size and antigenicity. Western blotting of native gels showed that HMF from affected cells had 3-fold more holoferritin containing stainable iron. We conclude that most of the iron in fibroblast HMF from both affected and unaffected cells is ferrihydrite but only FRDA affected cells mineralize significant iron in mitochondrial ferritin.« less

TRPM2 Channels Protect against Cardiac Ischemia-Reperfusion Injury

PubMed Central

Miller, Barbara A.; Hoffman, Nicholas E.; Merali, Salim; Zhang, Xue-Qian; Wang, JuFang; Rajan, Sudarsan; Shanmughapriya, Santhanam; Gao, Erhe; Barrero, Carlos A.; Mallilankaraman, Karthik; Song, Jianliang; Gu, Tongda; Hirschler-Laszkiewicz, Iwona; Koch, Walter J.; Feldman, Arthur M.; Madesh, Muniswamy; Cheung, Joseph Y.

2014-01-01

Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca2+ uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca2+ uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca2+ uptake was likely due to both lower ψm and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels. PMID:24492610

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

PubMed

Folmes, Clifford D L; Martinez-Fernandez, Almudena; Perales-Clemente, Ester; Li, Xing; McDonald, Amber; Oglesbee, Devin; Hrstka, Sybil C; Perez-Terzic, Carmen; Terzic, Andre; Nelson, Timothy J

2013-07-01

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. Copyright © 2013 AlphaMed Press.

Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus) and humans.

PubMed

Zsurka, Gábor; Kudina, Tatiana; Peeva, Viktoriya; Hallmann, Kerstin; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2010-09-02

We have analyzed the complete mitochondrial genomes of 22 Pan paniscus (bonobo, pygmy chimpanzee) individuals to assess the detailed mitochondrial DNA (mtDNA) phylogeny of this close relative of Homo sapiens. We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i) Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii) A comparison of the ratios of non-synonymous to synonymous changes (dN/dS) among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos. Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans.

Distinct patterns of mitochondrial genome diversity in bonobos (Pan paniscus) and humans

PubMed Central

2010-01-01

Background We have analyzed the complete mitochondrial genomes of 22 Pan paniscus (bonobo, pygmy chimpanzee) individuals to assess the detailed mitochondrial DNA (mtDNA) phylogeny of this close relative of Homo sapiens. Results We identified three major clades among bonobos that separated approximately 540,000 years ago, as suggested by Bayesian analysis. Incidentally, we discovered that the current reference sequence for bonobo likely is a hybrid of the mitochondrial genomes of two distant individuals. When comparing spectra of polymorphic mtDNA sites in bonobos and humans, we observed two major differences: (i) Of all 31 bonobo mtDNA homoplasies, i.e. nucleotide changes that occurred independently on separate branches of the phylogenetic tree, 13 were not homoplasic in humans. This indicates that at least a part of the unstable sites of the mitochondrial genome is species-specific and difficult to be explained on the basis of a mutational hotspot concept. (ii) A comparison of the ratios of non-synonymous to synonymous changes (dN/dS) among polymorphic positions in bonobos and in 4902 Homo sapiens mitochondrial genomes revealed a remarkable difference in the strength of purifying selection in the mitochondrial genes of the F0F1-ATPase complex. While in bonobos this complex showed a similar low value as complexes I and IV, human haplogroups displayed 2.2 to 7.6 times increased dN/dS ratios when compared to bonobos. Conclusions Some variants of mitochondrially encoded subunits of the ATPase complex in humans very likely decrease the efficiency of energy conversion leading to production of extra heat. Thus, we hypothesize that the species-specific release of evolutionary constraints for the mitochondrial genes of the proton-translocating ATPase is a consequence of altered heat homeostasis in modern humans. PMID:20813043

New Model of Action for Mood Stabilizers: Phosphoproteome from Rat Pre-Frontal Cortex Synaptoneurosomal Preparations

PubMed Central

Corena-McLeod, Maria; Walss-Bass, Consuelo; Oliveros, Alfredo; Gordillo Villegas, Andres; Ceballos, Carolina; Charlesworth, Cristine M.; Madden, Benjamin; Linser, Paul J.; Van Ekeris, Leslie; Smith, Kristin; Richelson, Elliott

2013-01-01

Background Mitochondrial short and long-range movements are necessary to generate the energy needed for synaptic signaling and plasticity. Therefore, an effective mechanism to transport and anchor mitochondria to pre- and post-synaptic terminals is as important as functional mitochondria in neuronal firing. Mitochondrial movement range is regulated by phosphorylation of cytoskeletal and motor proteins in addition to changes in mitochondrial membrane potential. Movement direction is regulated by serotonin and dopamine levels. However, data on mitochondrial movement defects and their involvement in defective signaling and neuroplasticity in relationship with mood disorders is scarce. We have previously reported the effects of lithium, valproate and a new antipsychotic, paliperidone on protein expression levels at the synaptic level. Hypothesis Mitochondrial function defects have recently been implicated in schizophrenia and bipolar disorder. We postulate that mood stabilizer treatment has a profound effect on mitochondrial function, synaptic plasticity, mitochondrial migration and direction of movement. Methods Synaptoneurosomal preparations from rat pre-frontal cortex were obtained after 28 daily intraperitoneal injections of lithium, valproate and paliperidone. Phosphorylated proteins were identified using 2D-DIGE and nano LC-ESI tandem mass spectrometry. Results Lithium, valproate and paliperidone had a substantial and common effect on the phosphorylation state of specific actin, tubulin and myosin isoforms as well as other proteins associated with neurofilaments. Furthermore, different subunits from complex III and V of the electron transfer chain were heavily phosphorylated by treatment with these drugs indicating selective phosphorylation. Conclusions Mood stabilizers have an effect on mitochondrial function, mitochondrial movement and the direction of this movement. The implications of these findings will contribute to novel insights regarding clinical treatment and the mode of action of these drugs. PMID:23690912

A novel mutation MT-COIII m.9267G>C and MT-COI m.5913G>A mutation in mitochondrial genes in a Tunisian family with maternally inherited diabetes and deafness (MIDD) associated with severe nephropathy.

PubMed

Tabebi, Mouna; Mkaouar-Rebai, Emna; Mnif, Mouna; Kallabi, Fakhri; Ben Mahmoud, Afif; Ben Saad, Wafa; Charfi, Nadia; Keskes-Ammar, Leila; Kamoun, Hassen; Abid, Mohamed; Fakhfakh, Faiza

2015-04-10

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia, maternal transmission and its association with a bilateral hearing impairment. Several studies reported mutations in mitochondrial genes as potentially pathogenic for diabetes, since mitochondrial oxidative phosphorylation plays an important role in glucose-stimulated insulin secretion from beta cells. In the present report, we studied a Tunisian family with mitochondrial diabetes (MD) and deafness associated with nephropathy. The mutational analysis screening revealed the presence of a novel heteroplasmic mutation m.9276G>C in the mitochondrial COIII gene, detected in mtDNA extracted from leukocytes of a mother and her two daughters indicating that this mutation is maternally transmitted and suggest its implication in the observed phenotype. Bioinformatic tools showed that m.9267G>C mutation (p.A21P) is « deleterious » and it can modify the function and the stability of the MT-COIII protein by affecting the assembly of mitochondrial COX subunits and the translocation of protons then reducing the activity of the respective OXPHOS complexes of ATP synthesis. The nonsynonymous mutation (p.A21P) has not been reported before, it is the first mutation described in the COXIII gene which is related to insulin dependent mitochondrial diabetes and deafness and could be specific to the Tunisian population. The m.9267G>C mutation was present with a nonsynonymous inherited mitochondrial homoplasmic variation MT-COI m.5913 G>A (D4N) responsible of high blood pressure, a clinical feature detected in all explored patients. Copyright © 2015. Published by Elsevier Inc.

Posttranslational modification of mitochondrial transcription factor A in impaired mitochondria biogenesis: implications in diabetic retinopathy and metabolic memory phenomenon.

PubMed

Santos, Julia M; Mishra, Manish; Kowluru, Renu A

2014-04-01

Mitochondrial transcription factor A (TFAM) is one of the key regulators of the transcription of mtDNA. In diabetes, despite increase in gene transcripts of TFAM, its protein levels in the mitochondria are decreased and mitochondria copy numbers become subnormal. The aim of this study is to investigate the mechanism(s) responsible for decreased mitochondrial TFAM in diabetes. Using retinal endothelial cells, we have investigated the effect of overexpression of cytosolic chaperone, Hsp70, and TFAM on glucose-induced decrease in mitochondrial TFAM levels, and the transcription of mtDNA-encoded genes, NADH dehydrogenase subunit 6 (ND6) and cytochrome b (Cytb). To investigate the role of posttranslational modifications in subnormal mitochondrial TFAM, ubiquitination of TFAM was assessed, and the results were confirmed in the retina from streptozotocin-induced diabetic rats. While overexpression of Hsp70 failed to prevent glucose-induced decrease in mitochondrial TFAM and transcripts of ND6 and Cytb, overexpression of TFAM ameliorated decrease in its mitochondrial protein levels and transcriptional activity. TFAM was ubiquitinated by high glucose, and PYR-41, an inhibitor of ubiquitination, prevented TFAM ubiquitination and restored the transcriptional activity. Similarly, TFAM was ubiquitinated in the retina from diabetic rats, and it continued to be modified after reinstitution of normal glycemia. Our results clearly imply that the ubiquitination of TFAM impedes its transport to the mitochondria resulting in subnormal mtDNA transcription and mitochondria dysfunction, and inhibition of ubiquitination restores mitochondrial homeostasis. Reversal of hyperglycemia does not provide any benefit to TFAM ubiquitination. Thus, strategies targeting posttranslational modification could provide an avenue to preserve mitochondrial homeostasis, and inhibit the development/progression of diabetic retinopathy. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents.

PubMed Central

Steenkamp, D J

1988-01-01

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2'-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered. Images Fig. 4. Fig. 5. Fig. 6. PMID:3145738

Mitochondrial respiratory chain complexes as sources and targets of thiol-based redox-regulation.

PubMed

Dröse, Stefan; Brandt, Ulrich; Wittig, Ilka

2014-08-01

The respiratory chain of the inner mitochondrial membrane is a unique assembly of protein complexes that transfers the electrons of reducing equivalents extracted from foodstuff to molecular oxygen to generate a proton-motive force as the primary energy source for cellular ATP-synthesis. Recent evidence indicates that redox reactions are also involved in regulating mitochondrial function via redox-modification of specific cysteine-thiol groups in subunits of respiratory chain complexes. Vice versa the generation of reactive oxygen species (ROS) by respiratory chain complexes may have an impact on the mitochondrial redox balance through reversible and irreversible thiol-modification of specific target proteins involved in redox signaling, but also pathophysiological processes. Recent evidence indicates that thiol-based redox regulation of the respiratory chain activity and especially S-nitrosylation of complex I could be a strategy to prevent elevated ROS production, oxidative damage and tissue necrosis during ischemia-reperfusion injury. This review focuses on the thiol-based redox processes involving the respiratory chain as a source as well as a target, including a general overview on mitochondria as highly compartmentalized redox organelles and on methods to investigate the redox state of mitochondrial proteins. This article is part of a Special Issue entitled: Thiol-Based Redox Processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Involvement of a putative substrate binding site in the biogenesis and assembly of phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae.

PubMed

Di Bartolomeo, Francesca; Doan, Kim Nguyen; Athenstaedt, Karin; Becker, Thomas; Daum, Günther

2017-07-01

In the yeast Saccharomyces cerevisiae, the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) produces the largest amount of cellular phosphatidylethanolamine (PE). Psd1p is synthesized as a larger precursor on cytosolic ribosomes and then imported into mitochondria in a three-step processing event leading to the formation of an α-subunit and a β-subunit. The α-subunit harbors a highly conserved motif, which was proposed to be involved in phosphatidylserine (PS) binding. Here, we present a molecular analysis of this consensus motif for the function of Psd1p by using Psd1p variants bearing either deletions or point mutations in this region. Our data show that mutations in this motif affect processing and stability of Psd1p, and consequently the enzyme's activity. Thus, we conclude that this consensus motif is essential for structural integrity and processing of Psd1p. Copyright © 2017 Elsevier B.V. All rights reserved.

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase.

PubMed Central

Steenkamp, D J

1987-01-01

The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA. Images Fig. 2. Fig. 3. Fig. 5. PMID:3115254

Cancer-associated Isocitrate Dehydrogenase Mutations Inactivate NADPH-dependent Reductive Carboxylation*

PubMed Central

Leonardi, Roberta; Subramanian, Chitra; Jackowski, Suzanne; Rock, Charles O.

2012-01-01

Isocitrate dehydrogenase (IDH) is a reversible enzyme that catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate (ICT) to α-ketoglutarate (αKG) and the NADPH/CO2-dependent reductive carboxylation of αKG to ICT. Reductive carboxylation by IDH1 was potently inhibited by NADP+ and, to a lesser extent, by ICT. IDH1 and IDH2 with cancer-associated mutations at the active site arginines were unable to carry out the reductive carboxylation of αKG. These mutants were also defective in ICT decarboxylation and converted αKG to 2-hydroxyglutarate using NADPH. These mutant proteins were thus defective in both of the normal reactions of IDH. Biochemical analysis of heterodimers between wild-type and mutant IDH1 subunits showed that the mutant subunit did not inactivate reductive carboxylation by the wild-type subunit. Cells expressing the mutant IDH are thus deficient in their capacity for reductive carboxylation and may be compromised in their ability to produce acetyl-CoA under hypoxia or when mitochondrial function is otherwise impaired. PMID:22442146

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae.

PubMed

Weber, Heike E; Gottardi, Manuela; Brückner, Christine; Oreb, Mislav; Boles, Eckhard; Tripp, Joanna

2017-05-15

Biotechnological production of cis , cis -muconic acid from renewable feedstocks is an environmentally sustainable alternative to conventional, petroleum-based methods. Even though a heterologous production pathway for cis , cis -muconic acid has already been established in the host organism Saccharomyces cerevisiae , the generation of industrially relevant amounts of cis , cis -muconic acid is hampered by the low activity of the bacterial protocatechuic acid (PCA) decarboxylase AroY isomeric subunit C iso (AroY-C iso ), leading to secretion of large amounts of the intermediate PCA into the medium. In the present study, we show that the activity of AroY-C iso in S. cerevisiae strongly depends on the strain background. We could demonstrate that the strain dependency is caused by the presence or absence of an intact genomic copy of PAD1 , which encodes a mitochondrial enzyme responsible for the biosynthesis of a prenylated form of the cofactor flavin mononucleotide (prFMN). The inactivity of AroY-C iso in strain CEN.PK2-1 could be overcome by plasmid-borne expression of Pad1 or its bacterial homologue AroY subunit B (AroY-B). Our data reveal that the two enzymes perform the same function in decarboxylation of PCA by AroY-C iso , although coexpression of Pad1 led to higher decarboxylase activity. Conversely, AroY-B can replace Pad1 in its function in decarboxylation of phenylacrylic acids by ferulic acid decarboxylase Fdc1. Targeting of the majority of AroY-B to mitochondria by fusion to a heterologous mitochondrial targeting signal did not improve decarboxylase activity of AroY-C iso , suggesting that mitochondrial localization has no major impact on cofactor biosynthesis. IMPORTANCE In Saccharomyces cerevisiae , the decarboxylation of protocatechuic acid (PCA) to catechol is the bottleneck reaction in the heterologous biosynthetic pathway for production of cis , cis -muconic acid, a valuable precursor for the production of bulk chemicals. In our work, we demonstrate the importance of the strain background for the activity of a bacterial PCA decarboxylase in S. cerevisiae Inactivity of the decarboxylase is due to a nonsense mutation in a gene encoding a mitochondrial enzyme involved in the biosynthesis of a cofactor required for decarboxylase function. Our study reveals functional interchangeability of Pad1 and a bacterial homologue, irrespective of their intracellular localization. Our results open up new possibilities to improve muconic acid production by engineering cofactor supply. Furthermore, the results have important implications for the choice of the production strain. Copyright © 2017 American Society for Microbiology.

Two novel kinases phosphorylate tau and the KSP site of heavy neurofilament subunits in high stoichiometric ratios.

PubMed

Roder, H M; Ingram, V M

1991-11-01

We have identified, purified, and characterized two neurofilament/tau kinases from bovine brain, PK36 and PK40, with apparent Mr of 36,000 and 40,000 and with novel biochemical properties. A specially designed immunoassay for phosphorylated epitopes in neurofilament (NF) proteins was used in the early stages of the purification. Neither kinase is closely associated with the cytoskeleton. Both kinases phosphorylate bovine intermediate (NF-M) and heavy (NF-H) NF subunits and also bovine tau at the expected KSP sequences, though other sites cannot be ruled out. In human paired helical filaments, tau, phosphorylated at these same KSP sites, is a major characterized constituent. Neither kinase is activated by the usual second messengers. Tau and the above NF subunits are phosphorylated in high stoichiometric ratios. In the intermediate NF subunit, all the expected sites appear to be phosphorylated, but in the heavy NF subunit only 7 out of the greater than 40 expected sites can be phosphorylated by our kinases. We demonstrate that both kinases can induce considerable shifts of apparent Mr with SDS-PAGE for tau and, for the first time in vitro, also for the intermediate NF subunit. Interestingly, PK36 and particularly PK40 are strongly inhibited by an excess of free ATP. We propose that during normal aging, and in Alzheimer's disease, age-related mitochondrial dysfunction would reduce ATP levels, which in turn might release the neurofilament/tau kinase from inhibition with consequent paired helical filament formation.

Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

PubMed

Avni, A; Avital, S; Gromet-Elhanan, Z

1991-04-25

Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.

Charting organellar importomes by quantitative mass spectrometry

PubMed Central

Peikert, Christian D.; Mani, Jan; Morgenstern, Marcel; Käser, Sandro; Knapp, Bettina; Wenger, Christoph; Harsman, Anke; Oeljeklaus, Silke; Schneider, André; Warscheid, Bettina

2017-01-01

Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles. PMID:28485388

Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts.

PubMed

Kuniyasu, Akihiko; Kaneko, Kazuyoshi; Kawahara, Kohichi; Nakayama, Hitoshi

2003-09-25

Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.