Peng, Yingjie; Zhong, Chen; Huang, Wei; Ding, Jianping
2008-09-01
Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.
Peng, Yingjie; Zhong, Chen; Huang, Wei; Ding, Jianping
2008-01-01
Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca2+, which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118–124 and residues 284–287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca2+ in the pseudo-Michaelis complex or with NADPH, AKG, and Ca2+ in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes. PMID:18552125
Mitochondrial genome evolution in the Saccharomyces sensu stricto complex.
Ruan, Jiangxing; Cheng, Jian; Zhang, Tongcun; Jiang, Huifeng
2017-01-01
Exploring the evolutionary patterns of mitochondrial genomes is important for our understanding of the Saccharomyces sensu stricto (SSS) group, which is a model system for genomic evolution and ecological analysis. In this study, we first obtained the complete mitochondrial sequences of two important species, Saccharomyces mikatae and Saccharomyces kudriavzevii. We then compared the mitochondrial genomes in the SSS group with those of close relatives, and found that the non-coding regions evolved rapidly, including dramatic expansion of intergenic regions, fast evolution of introns and almost 20-fold higher rearrangement rates than those of the nuclear genomes. However, the coding regions, and especially the protein-coding genes, are more conserved than those in the nuclear genomes of the SSS group. The different evolutionary patterns of coding and non-coding regions in the mitochondrial and nuclear genomes may be related to the origin of the aerobic fermentation lifestyle in this group. Our analysis thus provides novel insights into the evolution of mitochondrial genomes.
Szabóová, Dana; Bielik, Peter; Poláková, Silvia; Šoltys, Katarína; Jatzová, Katarína; Szemes, Tomáš
2017-01-01
Abstract The yeast Saccharomyces are widely used to test ecological and evolutionary hypotheses. A large number of nuclear genomic DNA sequences are available, but mitochondrial genomic data are insufficient. We completed mitochondrial DNA (mtDNA) sequencing from Illumina MiSeq reads for all Saccharomyces species. All are circularly mapped molecules decreasing in size with phylogenetic distance from Saccharomyces cerevisiae but with similar gene content including regulatory and selfish elements like origins of replication, introns, free-standing open reading frames or GC clusters. Their most profound feature is species-specific alteration in gene order. The genetic code slightly differs from well-established yeast mitochondrial code as GUG is used rarely as the translation start and CGA and CGC code for arginine. The multilocus phylogeny, inferred from mtDNA, does not correlate with the trees derived from nuclear genes. mtDNA data demonstrate that Saccharomyces cariocanus should be assigned as a separate species and Saccharomyces bayanus CBS 380T should not be considered as a distinct species due to mtDNA nearly identical to Saccharomyces uvarum mtDNA. Apparently, comparison of mtDNAs should not be neglected in genomic studies as it is an important tool to understand the origin and evolutionary history of some yeast species. PMID:28992063
Peris, David; Arias, Armando; Orlić, Sandi; Belloch, Carmela; Pérez-Través, Laura; Querol, Amparo; Barrio, Eladio
2017-03-01
Horizontal gene transfer (HGT) in eukaryotic plastids and mitochondrial genomes is common, and plays an important role in organism evolution. In yeasts, recent mitochondrial HGT has been suggested between S. cerevisiae and S. paradoxus. However, few strains have been explored given the lack of accurate mitochondrial genome annotations. Mitochondrial genome sequences are important to understand how frequent these introgressions occur, and their role in cytonuclear incompatibilities and fitness. Indeed, most of the Bateson-Dobzhansky-Muller genetic incompatibilities described in yeasts are driven by cytonuclear incompatibilities. We herein explored the mitochondrial inheritance of several worldwide distributed wild Saccharomyces species and their hybrids isolated from different sources and geographic origins. We demonstrated the existence of several recombination points in mitochondrial region COX2-ORF1, likely mediated by either the activity of the protein encoded by the ORF1 (F-SceIII) gene, a free-standing homing endonuclease, or mostly facilitated by A+T tandem repeats and regions of integration of GC clusters. These introgressions were shown to occur among strains of the same species and among strains of different species, which suggests a complex model of Saccharomyces evolution that involves several ancestral hybridization events in wild environments. Copyright © 2017 Elsevier Inc. All rights reserved.
2018-01-01
ABSTRACT The mitochondrial genomes of Saccharomyces cerevisiae strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of S. cerevisiae strain W303 gave the S. cerevisiae CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology. PMID:29700138
Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae
Chatre, Laurent; Ricchetti, Miria
2011-01-01
The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in. PMID:21408151
Nuclear mitochondrial DNA activates replication in Saccharomyces cerevisiae.
Chatre, Laurent; Ricchetti, Miria
2011-03-08
The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in.
Bonnefoy, Nathalie; Fox, Thomas D
2007-01-01
Saccharomyces cerevisiae is currently the only species in which genetic transformation of mitochondria can be used to generate a wide variety of defined alterations in mitochondrial deoxyribonucleic acid (mtDNA). DNA sequences can be delivered into yeast mitochondria by microprojectile bombardment (biolistic transformation) and subsequently incorporated into mtDNA by the highly active homologous recombination machinery present in the organelle. Although transformation frequencies are relatively low, the availability of strong mitochondrial selectable markers for the yeast system, both natural and synthetic, makes the isolation of transformants routine. The strategies and procedures reviewed here allow the researcher to insert defined mutations into endogenous mitochondrial genes and to insert new genes into mtDNA. These methods provide powerful in vivo tools for the study of mitochondrial biology.
Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae.
Vambutas, A; Ackerman, S H; Tzagoloff, A
1991-11-01
C155 and E252 are respiratory-defective mutants of Saccharomyces cerevisiae, previously assigned to complementation groups G37 and G142, respectively. The following evidence suggested that both mutants were likely to have lesions in components of the mitochondrial translational machinery: C155 and E252 display a pleiotropic deficiency in cytochromes a, a3 and b; both strains are severly limited in their ability to incorporate radioactive methionine into the mitochondrial translation products and, in addition, display a tendency to loose wild-type mitochondrial DNA. This set of characteristics is commonly found in strains affected in mitochondrial protein synthesis. To identify the biochemical lesions, each mutant was transformed with a wild-type yeast genomic library and clones complemented for the respiratory defect were selected for growth on a non-fermentable substrate. Analysis of the cloned genes revealed that C155 has a mutation in a protein which has high sequence similarity to bacterial elongation factor G and that E252 has a mutation in a protein homologous to bacterial initiation factor 2. Disruption of the chromosomal copy of each gene in a wild-type haploid yeast induced a phenotype analogous to that of the original mutants, but does not affect cell viability. These results indicate that both gene products function exclusively in mitochondrial protein synthesis. Subcloning of the IFM1 gene, coding for the mitochondrial initiation factor, indicates that the amino-terminal 423 residues of the protein are sufficient to promote peptide-chain initiation in vivo.
Vest, Katherine E.; Leary, Scot C.; Winge, Dennis R.; Cobine, Paul A.
2013-01-01
Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria. PMID:23846699
Vest, Katherine E; Leary, Scot C; Winge, Dennis R; Cobine, Paul A
2013-08-16
Saccharomyces cerevisiae must import copper into the mitochondrial matrix for eventual assembly of cytochrome c oxidase. This copper is bound to an anionic fluorescent molecule known as the copper ligand (CuL). Here, we identify for the first time a mitochondrial carrier family protein capable of importing copper into the matrix. In vitro transport of the CuL into the mitochondrial matrix was saturable and temperature-dependent. Strains with a deletion of PIC2 grew poorly on copper-deficient non-fermentable medium supplemented with silver and under respiratory conditions when challenged with a matrix-targeted copper competitor. Mitochondria from pic2Δ cells had lower total mitochondrial copper and exhibited a decreased capacity for copper uptake. Heterologous expression of Pic2 in Lactococcus lactis significantly enhanced CuL transport into these cells. Therefore, we propose a novel role for Pic2 in copper import into mitochondria.
Langkjær, R. B.; Casaregola, S.; Ussery, D. W.; Gaillardin, C.; Piškur, J.
2003-01-01
The complete sequences of mitochondrial DNA (mtDNA) from the two budding yeasts Saccharomyces castellii and Saccharomyces servazzii, consisting of 25 753 and 30 782 bp, respectively, were analysed and compared to Saccharomyces cerevisiae mtDNA. While some of the traits are very similar among Saccharomyces yeasts, others have highly diverged. The two mtDNAs are much more compact than that of S.cerevisiae and contain fewer introns and intergenic sequences, although they have almost the same coding potential. A few genes contain group I introns, but group II introns, otherwise found in S.cerevisiae mtDNA, are not present. Surprisingly, four genes (ATP6, COX2, COX3 and COB) in the mtDNA of S.servazzii contain, in total, five +1 frameshifts. mtDNAs of S.castellii, S.servazzii and S.cerevisiae contain all genes on the same strand, except for one tRNA gene. On the other hand, the gene order is very different. Several gene rearrangements have taken place upon separation of the Saccharomyces lineages, and even a part of the transcription units have not been preserved. It seems that the mechanism(s) involved in the generation of the rearrangements has had to ensure that all genes stayed encoded by the same DNA strand. PMID:12799436
Kursu, V. A. Samuli; Pietikäinen, Laura P.; Fontanesi, Flavia; Aaltonen, Mari J.; Suomi, Fumi; Nair, Remya Raghavan; Schonauer, Melissa S.; Dieckmann, Carol L.; Barrientos, Antoni; Hiltunen, J. Kalervo; Kastaniotis, Alexander J.
2014-01-01
Summary Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild heme deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a coordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell. PMID:24102902
Kursu, V A Samuli; Pietikäinen, Laura P; Fontanesi, Flavia; Aaltonen, Mari J; Suomi, Fumi; Raghavan Nair, Remya; Schonauer, Melissa S; Dieckmann, Carol L; Barrientos, Antoni; Hiltunen, J Kalervo; Kastaniotis, Alexander J
2013-11-01
Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell. © 2013 John Wiley & Sons Ltd.
Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna
2015-09-01
Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase*║
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
Akdoğan, Emel; Tardu, Mehmet; Garipler, Görkem; Baytek, Gülkız; Kavakli, İ. Halil; Dunn, Cory D.
2016-01-01
Damage to the mitochondrial genome (mtDNA) can lead to diseases for which there are no clearly effective treatments. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. We found that restricting glucose or otherwise reducing the activity of the protein kinase A (PKA) pathway can lead to improved proliferation of Saccharomyces cerevisiae cells lacking mtDNA and that the transcriptional response to mtDNA loss is reduced in cells with diminished PKA activity. We have excluded many pathways and proteins from being individually responsible for the benefits provided to cells lacking mtDNA by PKA inhibition, and we found that robust import of mitochondrial polytopic membrane proteins may be required in order for cells without mtDNA to receive the full benefits of PKA reduction. Finally, we have discovered that the transcription of genes involved in arginine biosynthesis and aromatic amino acid catabolism is altered after mtDNA damage. Our results highlight the potential importance of nutrient detection and availability on the outcome of mitochondrial dysfunction. PMID:26751567
Analysis of repeat-mediated deletions in the mitochondrial genome of Saccharomyces cerevisiae.
Phadnis, Naina; Sia, Rey A; Sia, Elaine A
2005-12-01
Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions.
Analysis of Repeat-Mediated Deletions in the Mitochondrial Genome of Saccharomyces cerevisiae
Phadnis, Naina; Sia, Rey A.; Sia, Elaine A.
2005-01-01
Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions. PMID:16157666
Martin, N C; Underbrink-Lyon, K
1981-01-01
We have used a cloned yeast mitochondrial tRNAUCNSer gene as a probe to detect RNA species that are transcripts from this gene in wild-type Saccharomyces cerevisiae and in petite deletion mutants. In RNA from wild-type cells, the tRNA is the most prominent transcript of the gene. In RNA from deletion mutants that retain this gene but have lost other regions of mtDNA, high molecular weight transcripts containing the tRNAUCNSer sequences accumulate but tRNAUCNSer is not made. tRNAUCNSer synthesis can be restored in these mutants when they are mated to other deletion mutants that retain a different portion of the mitochondrial genome. Protein synthesis is not necessary for the restoration, and the restoration is not due to a nuclear effect or to an effect of mating alone, because strains without mtDNA are not able to restore tRNA synthesis. These results definitively demonstrate the existence of a yeast mitochondrial locus that is necessary for tRNA synthesis and, because the restoration of tRNAUCNSer synthesis appears to result from intergenic complementation, not recombination, indicate that this locus acts in trans. Images PMID:6795621
Environmental Factors Can Influence Mitochondrial Inheritance in the Saccharomyces Yeast Hybrids.
Hsu, Yu-Yi; Chou, Jui-Yu
2017-01-01
Mitochondria play a critical role in the generation of metabolic energy and are crucial for eukaryotic cell survival and proliferation. In most sexual eukaryotes, mitochondrial DNA (mtDNA) is inherited from only one parent in non-Mendelian inheritance in contrast to the inheritance of nuclear DNA. The model organism Saccharomyces cerevisiae is commonly used to study mitochondrial biology. It has two mating types: MATa and MATα. Previous studies have suggested that the mtDNA inheritance patterns in hybrid diploid cells depend on the genetic background of parental strains. However, the underlying mechanisms remain unclear. To elucidate the mechanisms, we examined the effects of environmental factors on the mtDNA inheritance patterns in hybrids obtained by crossing S. cerevisiae with its close relative S. paradoxus. The results demonstrated that environmental factors can influence mtDNA transmission in hybrid diploids, and that the inheritance patterns are strain dependent. The fitness competition assay results showed that the fitness differences can explain the mtDNA inheritance patterns under specific conditions. However, in this study, we found that fitness differences cannot fully be explained by mitochondrial activity in hybrids under stress conditions.
Environmental Factors Can Influence Mitochondrial Inheritance in the Saccharomyces Yeast Hybrids
Hsu, Yu-Yi; Chou, Jui-Yu
2017-01-01
Mitochondria play a critical role in the generation of metabolic energy and are crucial for eukaryotic cell survival and proliferation. In most sexual eukaryotes, mitochondrial DNA (mtDNA) is inherited from only one parent in non-Mendelian inheritance in contrast to the inheritance of nuclear DNA. The model organism Saccharomyces cerevisiae is commonly used to study mitochondrial biology. It has two mating types: MATa and MATα. Previous studies have suggested that the mtDNA inheritance patterns in hybrid diploid cells depend on the genetic background of parental strains. However, the underlying mechanisms remain unclear. To elucidate the mechanisms, we examined the effects of environmental factors on the mtDNA inheritance patterns in hybrids obtained by crossing S. cerevisiae with its close relative S. paradoxus. The results demonstrated that environmental factors can influence mtDNA transmission in hybrid diploids, and that the inheritance patterns are strain dependent. The fitness competition assay results showed that the fitness differences can explain the mtDNA inheritance patterns under specific conditions. However, in this study, we found that fitness differences cannot fully be explained by mitochondrial activity in hybrids under stress conditions. PMID:28081193
Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.
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.
Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae
Prasai, Kanchanjunga; Robinson, Lucy C.; Scott, Rona S.; Tatchell, Kelly
2017-01-01
Abstract The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ− cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. PMID:28549155
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.
RPO41-independent maintenance of [rho-] mitochondrial DNA in Saccharomyces cerevisiae.
Fangman, W L; Henly, J W; Brewer, B J
1990-01-01
A subset of promoters in the mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae has been proposed to participate in replication initiation, giving rise to a primer through site-specific cleavage of an RNA transcript. To test whether transcription is essential for mtDNA maintenance, we examined two simple mtDNA deletion ([rho-]) genomes in yeast cells. One genome (HS3324) contains a consensus promoter (ATATAAGTA) for the mitochondrial RNA polymerase encoded by the nuclear gene RPO41, and the other genome (4a) does not. As anticipated, in RPO41 cells transcripts from the HS3324 genome were more abundant than were transcripts from the 4a genome. When the RPO41 gene was disrupted, both [rho-] genomes were efficiently maintained. The level of transcripts from HS3324 mtDNA was decreased greater than 400-fold in cells carrying the RPO41 disrupted gene; however, the low-level transcripts from 4a mtDNA were undiminished. These results indicate that replication of [rho-] genomes can be initiated in the absence of wild-type levels of the RPO41-encoded RNA polymerase.
Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae.
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
Evidence for double-strand break mediated mitochondrial DNA replication in Saccharomyces cerevisiae.
Prasai, Kanchanjunga; Robinson, Lucy C; Scott, Rona S; Tatchell, Kelly; Harrison, Lynn
2017-07-27
The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ- cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter cells. Interestingly, we find that mitochondrial-targeted bKu does not decrease mtDNA content in human MCF7 cells. This finding is in agreement with the fact that human mtDNA replication, typically, is not initiated by a DSB. Therefore, this study provides evidence that DSB-mediated replication is the predominant form of mtDNA replication in ρ+ yeast cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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.
Palmieri, Ferdinando; Agrimi, Gennaro; Blanco, Emanuela; Castegna, Alessandra; Di Noia, Maria A; Iacobazzi, Vito; Lasorsa, Francesco M; Marobbio, Carlo M T; Palmieri, Luigi; Scarcia, Pasquale; Todisco, Simona; Vozza, Angelo; Walker, John
2006-01-01
The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane alpha-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established.
Santangelo, G M; Tornow, J
1997-12-01
As part of an effort to identify random carbon-source-regulated promoters in the Saccharomyces cerevisiae genome, we discovered that a mitochondrial DNA fragment is capable of directing glucose-repressible expression of a reporter gene. This fragment (CR24) originated from the mitochondrial genome adjacent to a transcription initiation site. Mutational analyses identified a GC cluster within the fragment that is required for transcriptional induction. Repression of nuclear CR24-driven transcription required Reg1p, indicating that this mitochondrially derived promoter is a member of a large group of glucose-repressible nuclear promoters that are similarly regulated by Reg1p. In vivo and in vitro binding assays indicated the presence of factors, located within the nucleus and the mitochondria, that bind to the GC cluster. One or more of these factors may provide a regulatory link between the nucleus and mitochondria.
Mutagenic effect of freezing on mitochondrial DNA of Saccharomyces cerevisiae.
Stoycheva, T; Venkov, P; Tsvetkov, Ts
2007-06-01
Although suggested in some studies, the mutagenic effect of freezing has not been proved by induction and isolation of mutants. Using a well-defined genetic model, we supply in this communication evidence for the mutagenic effect of freezing on mitochondrial DNA (mtDNA) of the yeast Saccharomyces cerevisiae. The cooling for 2 h at +4 degrees C, followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C resulted in induction of respiratory mutations. The immediate freezing in liquid nitrogen was without mutagenic effect. The study of the stepwise procedure showed that the induction of respiratory mutants takes place during the freezing at -10 and -20 degrees C of cells pre-cooled at +4 degrees C. The genetic crosses of freeze-induced mutants evidenced their mitochondrial rho- origin. The freeze-induced rho- mutants are most likely free of simultaneous nuclear mutations. The extracellular presence of cryoprotectants did not prevent the mutagenic effect of freezing while accumulation of cryoprotectors inside cells completely escaped mtDNA from cryodamage. Although the results obtained favor the notion that the mutagenic effect of freezing on yeast mtDNA is due to formation and growth of intracellular ice crystals, other reasons, such as impairment of mtDNA replication or elevated levels of ROS production are discussed as possible explanations of the mutagenic effect of freezing. It is concluded that: (i) freezing can be used as a method for isolation of mitochondrial mutants in S. cerevisiae and (ii) given the substantial development in cryopreservation of cells and tissues, special precautions should be made to avoid mtDNA damage during the cryopreservation procedures.
Wen, Kuo-Kuang; McKane, Melissa; Stokasimov, Ema; Rubenstein, Peter A.
2011-01-01
In the Saccharomyces cerevisiae actin-profilin interface, Ala167 of the actin barbed end W-loop and His372 near the C terminus form a clamp around a profilin segment containing residue Arg81 and Tyr79. Modeling suggests that altering steric packing in this interface regulates actin activity. An actin A167E mutation could increase interface crowding and alter actin regulation, and A167E does cause growth defects and mitochondrial dysfunction. We assessed whether a profilin Y79S mutation with its decreased mass could compensate for actin A167E crowding and rescue the mutant phenotype. Y79S profilin alone caused no growth defect in WT actin cells under standard conditions in rich medium and rescued the mitochondrial phenotype resulting from both the A167E and H372R actin mutations in vivo consistent with our model. Rescue did not result from effects of profilin on actin nucleotide exchange or direct effects of profilin on actin polymerization. Polymerization of A167E actin was less stimulated by formin Bni1 FH1-FH2 fragment than was WT actin. Addition of WT profilin to mixtures of A167E actin and formin fragment significantly altered polymerization kinetics from hyperbolic to a decidedly more sigmoidal behavior. Substitution of Y79S profilin in this system produced A167E behavior nearly identical to that of WT actin. A167E actin caused more dynamic actin cable behavior in vivo than observed with WT actin. Introduction of Y79S restored cable movement to a more normal phenotype. Our studies implicate the importance of the actin-profilin interface for formin-dependent actin and point to the involvement of formin and profilin in the maintenance of mitochondrial integrity and function. PMID:21956104
Nagarajan, Prabha; Prevost, Christopher T; Stein, Alexis; Kasimer, Rachel; Kalifa, Lidza; Sia, Elaine A
2017-06-01
The structure-specific nuclease, Rad27p/FEN1, plays a crucial role in DNA repair and replication mechanisms in the nucleus. Genetic assays using the rad27-∆ mutant have shown altered rates of DNA recombination, microsatellite instability, and point mutation in mitochondria. In this study, we examined the role of Rad27p in mitochondrial mutagenesis and double-strand break (DSB) repair in Saccharomyces cerevisiae Our findings show that Rad27p is essential for efficient mitochondrial DSB repair by a pathway that generates deletions at a region flanked by direct repeat sequences. Mutant analysis suggests that both exonuclease and endonuclease activities of Rad27p are required for its role in mitochondrial DSB repair. In addition, we found that the nuclease activities of Rad27p are required for the prevention of mitochondrial DNA (mtDNA) point mutations, and in the generation of spontaneous mtDNA rearrangements. Overall, our findings underscore the importance of Rad27p in the maintenance of mtDNA, and demonstrate that it participates in multiple DNA repair pathways in mitochondria, unlinked to nuclear phenotypes. Copyright © 2017 by the Genetics Society of America.
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.
Saccharomyces cerevisiae and non-Saccharomyces yeasts in grape varieties of the São Francisco Valley
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
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.
Seguin, Alexandra; Santos, Renata; Pain, Debkumar; Dancis, Andrew; Camadro, Jean-Michel; Lesuisse, Emmanuel
2011-02-25
Saccharomyces cerevisiae cells lacking the yeast frataxin homologue (Δyfh1) accumulate iron in the mitochondria in the form of nanoparticles of ferric phosphate. The phosphate content of Δyfh1 mitochondria was higher than that of wild-type mitochondria, but the proportion of mitochondrial phosphate that was soluble was much lower in Δyfh1 cells. The rates of phosphate and iron uptake in vitro by isolated mitochondria were higher for Δyfh1 than wild-type mitochondria, and a significant proportion of the phosphate and iron rapidly became insoluble in the mitochondrial matrix, suggesting co-precipitation of these species after oxidation of iron by oxygen. Increasing the amount of phosphate in the medium decreased the amount of iron accumulated by Δyfh1 cells and improved their growth in an iron-dependent manner, and this effect was mostly transcriptional. Overexpressing the major mitochondrial phosphate carrier, MIR1, slightly increased the concentration of soluble mitochondrial phosphate and significantly improved various mitochondrial functions (cytochromes, [Fe-S] clusters, and respiration) in Δyfh1 cells. We conclude that in Δyfh1 cells, soluble phosphate is limiting, due to its co-precipitation with iron.
Murai, Masatoshi; Okuda, Ayaka; Yamamoto, Takenori; Shinohara, Yasuo; Miyoshi, Hideto
2017-01-31
The role of the voltage-dependent anion channel (VDAC) as a metabolic gate of the mitochondrial outer membrane has been firmly established; however, its involvement in the regulation of mitochondrial permeability transition (PT) remains extremely controversial. Although some low-molecular-weight chemicals have been proposed to modulate the regulatory role of VDAC in the induction of PT, direct binding between these chemicals and VDAC has not yet been demonstrated. In the present study, we investigated whether the ubiquinone molecule directly binds to VDAC in Saccharomyces cerevisiae mitochondria through a photoaffinity labeling technique using two photoreactive ubiquinones (PUQ-1 and PUQ-2). The results of the labeling experiments demonstrated that PUQ-1 and PUQ-2 specifically bind to VDAC1 and that the labeled position is located in the C-terminal region Phe221-Lys234, connecting the 15th and 16th β-strand sheets. Mutations introduced in this region (R224A, Y225A, D228A, and Y225A/D228A) hardly affected the binding affinity of PUQ-1. PUQ-1 and PUQ-2 both significantly suppressed the Ca 2+ -induced mitochondrial PT (monitored by mitochondrial swelling) at the one digit μM level. Thus, the results of the present study provided, for the first time to our knowledge, direct evidence indicating that the ubiquinone molecule specifically binds to VDAC1 through its quinone-head ring.
Silva, C R; Caldeira-de-Araújo, A; Leitão, A C; Pádula, M
2014-11-27
Cassia angustifolia Vahl. (senna) is commonly used in self-medication and is frequently used to treat intestine constipation. A previous study involving bacteria and plasmid DNA suggested the possible toxicity of the aqueous extract of senna (SAE). The aim of this study was to extend the knowledge concerning SAE genotoxicity mechanisms because of its widespread use and its risks to human health. We investigated the impact of SAE on nuclear DNA and on the stability of mitochondrial DNA in Saccharomyces cerevisiae (wt, ogg1, msh6, and ogg1msh6) strains, monitoring the formation of petite mutants. Our results demonstrated that SAE specifically increased Can(R) mutagenesis only in the msh6 mutant, supporting the view that SAE can induce misincorporation errors in DNA. We observed a significant increase in the frequency of petite colonies in all studied strains. Our data indicate that SAE has genotoxic activity towards both mitochondrial and nuclear DNA.
Lv, Xiaomei; Wang, Fan; Zhou, Pingping; Ye, Lidan; Xie, Wenping; Xu, Haoming; Yu, Hongwei
2016-09-21
Microbial production of isoprene from renewable feedstock is a promising alternative to traditional petroleum-based processes. Currently, efforts to improve isoprenoid production in Saccharomyces cerevisiae mainly focus on cytoplasmic engineering, whereas comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we propose dual metabolic engineering of cytoplasmic and mitochondrial acetyl-CoA utilization to boost isoprene synthesis in S. cerevisiae. This strategy increases isoprene production by 2.1-fold and 1.6-fold relative to the recombinant strains with solely mitochondrial or cytoplasmic engineering, respectively. By combining a modified reiterative recombination system for rapid pathway assembly, a two-phase culture process for dynamic metabolic regulation, and aerobic fed-batch fermentation for sufficient supply of acetyl-coA and carbon, we achieve 2527, mg l(-1) of isoprene, which is the highest ever reported in engineered eukaryotes. We propose this strategy as an efficient approach to enhancing isoprene production in yeast, which might open new possibilities for bioproduction of other value-added chemicals.
Bradshaw, Elliot; Yoshida, Minoru; Ling, Feng
2017-01-01
Small mitochondrial genomes can behave as selfish elements by displacing wild-type genomes regardless of their detriment to the host organism. In the budding yeast Saccharomyces cerevisiae, small hypersuppressive mtDNA transiently coexist with wild-type in a state of heteroplasmy, wherein the replicative advantage of the small mtDNA outcompetes wild-type and produces offspring without respiratory capacity in >95% of colonies. The cytosolic enzyme ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and its inhibition has been correlated with increased petite colony formation, reflecting loss of respiratory function. Here, we used heteroplasmic diploids containing wild-type (rho+) and suppressive (rho−) or hypersuppressive (HS rho−) mitochondrial genomes to explore the effects of RNR activity on mtDNA heteroplasmy in offspring. We found that the proportion of rho+ offspring was significantly increased by RNR overexpression or deletion of its inhibitor, SML1, while reducing RNR activity via SML1 overexpression produced the opposite effects. In addition, using Ex Taq and KOD Dash polymerases, we observed a replicative advantage for small over large template DNA in vitro, but only at low dNTP concentrations. These results suggest that dNTP insufficiency contributes to the replicative advantage of small mtDNA over wild-type and cytosolic dNTP synthesis by RNR is an important regulator of heteroplasmy involving small mtDNA molecules in yeast. PMID:28717049
Fizikova, A Iu; Padkina, M V; Sambuk, E V
2009-06-01
The cyclin-dependent protein kinase Pho85 is involved in the regulation of phosphate metabolism in yeast Saccharomyces cerevisiae. Mutations in the PH085 gene lead to constitutive synthesis of Pho5 acidic phosphatase, a delay in cell growth on media containing nonfermentable carbon sources, and other pleiotropic effects. In this work, it was shown that the accumulation of respiratory incompetent cells occurs with high frequency in strains carrying pho85 mutations as early as during the first cell divisions, and the number of these cells at the early logarithmic growth phase of the culture promptly reaches virtually 100%. Cytological analysis revealed a high accumulation rate of [rho(0)] cells the background of gene pho85 that may be related to disturbances in the distribution of mitochondrial nucleoids rather than to changes in morphology of mitochondria and a delay in their transport into the bud. Genetic analysis revealed that the appearing secondary mutations pho4, pho81, pho84, and pho87 stabilize nucleoids and hamper the loss of mitochondrial DNA caused by pho85. These results provide evidence for the influence of intracellular phosphate concentration on the inheritance of mitochondrial nucleoids, but it is fully probable that the occurrence of mutation pho4 in the background of gene pho85 may change the expression level of other genes required for the stabilization of mitochondrial functions.
The role of Pif1p, a DNA helicase in Saccharomyces cerevisiae, in maintaining mitochondrial DNA.
Cheng, Xin; Dunaway, Stephen; Ivessa, Andreas S
2007-05-01
Mitochondrial DNA (mtDNA) is highly susceptible to oxidative and chemically induced damage, and these insults lead to a number of diseases. In Saccharomyces cerevisiae, the DNA helicase Pif1p is localized to the nucleus and mitochondria. We show that pif1 mutant cells are sensitive to ethidium bromide-induced damage and this mtDNA is prone to fragmentation. We also show that Pif1p associates with mtDNA. In pif1 mutant cells, mtDNA breaks at specific sites that exhibit Pif1-dependent recombination. We conclude that Pif1p participates in the protection from double-stranded (ds) DNA breaks or alternatively in the repair process of dsDNA breaks in mtDNA.
Ramos-Gomez, Minerva; Olivares-Marin, Ivanna Karina; Canizal-García, Melina; González-Hernández, Juan Carlos; Nava, Gerardo M; Madrigal-Perez, Luis Alberto
2017-06-01
A broad range of health benefits have been attributed to resveratrol (RSV) supplementation in mammalian systems, including the increases in longevity. Nonetheless, despite the growing number of studies performed with RSV, the molecular mechanism by which it acts still remains unknown. Recently, it has been proposed that inhibition of the oxidative phosphorylation activity is the principal mechanism of RSV action. This mechanism suggests that RSV might induce mitochondrial dysfunction resulting in oxidative damage to cells with a concomitant decrease of cell viability and cellular life span. To prove this hypothesis, the chronological life span (CLS) of Saccharomyces cerevisiae was studied as it is accepted as an important model of oxidative damage and aging. In addition, oxygen consumption, mitochondrial membrane potential, and hydrogen peroxide (H 2 O 2 ) release were measured in order to determine the extent of mitochondrial dysfunction. The results demonstrated that the supplementation of S. cerevisiae cultures with 100 μM RSV decreased CLS in a glucose-dependent manner. At high-level glucose, RSV supplementation increased oxygen consumption during the exponential phase yeast cultures, but inhibited it in chronologically aged yeast cultures. However, at low-level glucose, oxygen consumption was inhibited in yeast cultures in the exponential phase as well as in chronologically aged cultures. Furthermore, RSV supplementation promoted the polarization of the mitochondrial membrane in both cultures. Finally, RSV decreased the release of H 2 O 2 with high-level glucose and increased it at low-level glucose. Altogether, this data supports the hypothesis that RSV supplementation decreases CLS as a result of mitochondrial dysfunction and this phenotype occurs in a glucose-dependent manner.
Thrailkill, K M; Birky, C W
1980-09-01
We report evidence for random drift of mitochondrial allele frequencies in zygote clones of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Monofactorial and bifactorial crosses were done, using strains resistant or sensitive to erythromycin (alleles Er, Es), oligomycin (Or, Os), or diuron (Dr, Ds). The frequencies of resistant and sensitive cells (and thus the frequencies of the resistant and sensitive alleles) were determined for each of a number of clones of diploid cells arising from individual zygotes. Allele frequencies were extremely variable among these zygote clones; some clones were "uniparental," with mitochondrial alleles from only one parent present. These observations suggest random drift of the allele frequencies in the population of mitochondrial genes within an individual zygote and its diploid progeny. Drift would cease when all the cells in a clone become homoplasmic, due to segregation of the mitochondrial genomes during vegetative cell divisions. To test this, we delayed cell division (and hence segregation) for varying times by starving zygotes in order to give drift more time to operate. As predicted, delaying cell division resulted in an increase in the variance of allele frequencies among the zygote clones and an increase in the proportion of uniparental zygote clones. The changes in form of the allele frequency distributions resembled those seen during random drift in finite Mendelian populations. In bifactorial crosses, genotypes as well as individual alleles were fixed or lost in some zygote clones. However, the mean recombination frequency for a large number of clones did not increase when cell division was delayed. Several possible molecular mechanisms for intracellular random drift are discussed.
He, Xiangyu; Zhu, Xiaoyu; Wang, Xuexiang; Wang, Wei; Dai, Yu; Yan, Qingfeng
2013-01-01
The phenotypic manifestations of mitochondrial DNA (mtDNA) mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R) or P(R) 454) mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R))), the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S)), mto2(P(S)) and MTO2(P(R))). The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R)) strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.
Misonou, Yoshiko; Kikuchi, Maiko; Sato, Hiroshi; Inai, Tomomi; Kuroiwa, Tsuneyoshi; Tanaka, Kenji; Miyakawa, Isamu
2014-01-01
ABSTRACT When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast. PMID:24771619
Gamberi, Tania; Fiaschi, Tania; Modesti, Alessandra; Massai, Lara; Messori, Luigi; Balzi, Manuela; Magherini, Francesca
2015-08-01
Auranofin is a gold based drug in clinical use since 1985 for the treatment of rheumatoid arthritis. Beyond its antinflammatory properties, auranofin exhibits other attractive biological and pharmacological actions such as a potent in vitro cytotoxicity and relevant antimicrobial and antiparasitic effects that make it amenable for new therapeutic indications. For instance, auranofin is currently tested as an anticancer agent in four independent clinical trials; yet, its mode of action is highly controversial. With the present study, we explore the effects of auranofin in Saccharomyces cerevisiae and its likely mechanism. Notably, auranofin is reported to induce remarkable yeast growth inhibition. Solid evidence is provided that growth inhibition is the consequence of a direct cytotoxic insult occurring at the mitochondrial level; a profound depression of cell respiration is indeed clearly documented as the main cause of cell death while induction of ROS plays only a secondary role. More in detail, the mitochondrial NADH kinase Pos5 is identified as a primary target for auranofin. The implications of these results are discussed in the frame of current mechanistic knowledge on the cellular effects of auranofin and of its role as a prospective anticancer drug. Copyright © 2015 Elsevier Ltd. All rights reserved.
Kim, Jong H; Mahoney, Noreen; Chan, Kathleen L; Molyneux, Russell J; Campbell, Bruce C
2004-10-01
Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2- isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2- isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2Delta, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1Delta, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H(2)O(2)) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration.
Stein, Alexis; Kalifa, Lidza; Sia, Elaine A
2015-11-01
Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.
Stein, Alexis; Kalifa, Lidza; Sia, Elaine A.
2015-01-01
Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs. PMID:26540255
Koprowski, P; Fikus, M U; Dzierzbicki, P; Mieczkowski, P; Lazowska, J; Ciesla, Z
2003-08-01
We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.
Waters, R; Moustacchi, E
1975-01-01
The photoreactivability of UV-induced pyrimidine dimers in the nuclear and mitochondrial DNAs of Saccharomyces cerevisiae has been investigated in conjunction with the fate of these photoproducts following postirradiation dark incubation in saline and nutrient media. In all instances, survival and "petite" induction were measured. An attempt has been made to relate these results to present ideas on the repair of UV damages in DNA.
Rubino, L; Di Franco, A; Russo, M
2000-01-01
Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.
Tian, Siqi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi
2015-01-01
To elucidate the mechanism of interorganelle sterol transport, a system to evaluate sterol transport from the endoplasmic reticulum (ER) to the mitochondria was constructed. A bacterial glycerophospholipid: cholesterol acyltransferase fused with a mitochondria-targeting sequence and a membrane-spanning domain of the mitochondrial inner membrane protein Pet100 and enhanced green fluorescent protein was expressed in a Saccharomyces cerevisiae mutant deleted for ARE1 and ARE2 encoding acyl-CoA:sterol acyltransferases. Microscopic observation and subcellular fractionation suggested that this fusion protein, which was named mito-SatA-EGFP, was localized in the mitochondria. Steryl esters were synthesized in the mutant expressing mito-SatA-EGFP. This system will be applicable for evaluations of sterol transport from the ER to the mitochondria in yeast by examining sterol esterification in the mitochondria.
Karanyicz, Edina; Antunovics, Zsuzsa; Kallai, Z; Sipiczki, M
2017-06-01
Saccharomyces strains with chimerical genomes consisting of mosaics of the genomes of different species ("natural hybrids") occur quite frequently among industrial and wine strains. The most widely endorsed hypothesis is that the mosaics are introgressions acquired via hybridisation and repeated backcrosses of the hybrids with one of the parental species. However, the interspecies hybrids are sterile, unable to mate with their parents. Here, we show by analysing synthetic Saccharomyces kudriavzevii x Saccharomyces uvarum hybrids that mosaic (chimeric) genomes can arise without introgressive backcrosses. These species are biologically separated by a double sterility barrier (sterility of allodiploids and F1 sterility of allotetraploids). F1 sterility is due to the diploidisation of the tetraploid meiosis resulting in MAT a /MAT α heterozygosity which suppresses mating in the spores. This barrier can occasionally be broken down by malsegregation of autosyndetically paired chromosomes carrying the MAT loci (loss of MAT heterozygosity). Subsequent malsegregation of additional autosyndetically paired chromosomes and occasional allosyndetic interactions chimerise the hybrid genome. Chromosomes are preferentially lost from the S. kudriavzevii subgenome. The uniparental transmission of the mitochondrial DNA to the hybrids indicates that nucleo-mitochondrial interactions might affect the direction of the genomic changes. We propose the name GARMe (Genome AutoReduction in Meiosis) for this process of genome reduction and chimerisation which involves no introgressive backcrossings. It opens a way to transfer genetic information between species and thus to get one step ahead after hybridisation in the production of yeast strains with beneficial combinations of properties of different species.
Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration.
Zampol, Mariana A; Busso, Cleverson; Gomes, Fernando; Ferreira-Junior, Jose Ribamar; Tzagoloff, Alexander; Barros, Mario H
2010-11-05
COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q(2). Rescue of respiration by Q(2) is a characteristic of mutants blocked in coenzyme Q(6) synthesis. Unlike Q(6) deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q(6). The physiological significance of earlier observations that purified Coq10p contains bound Q(6) was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q(2). This suggests that in vivo binding of Q(6) by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p. Copyright © 2010 Elsevier Inc. All rights reserved.
Pallotta, Maria Luigia
2014-01-01
L-Proline (pyrrolidine-2-carboxylic acid) is a distinctive metabolite both biochemically and biotechnologically and is currently recognized to have a cardinal role in gene expression and cellular signaling pathways in stress response. Proline-fueled mitochondrial metabolism involves the oxidative conversion of L-Proline to L-Glutamate in two enzymatic steps by means of Put1p and Put2p that help Saccharomyces cerevisiae to respond to changes in the nutritional environment by initiating the breakdown of L-Proline as a source for nitrogen, carbon, and energy. Compartmentalization of L-Proline catabolic pathway implies that extensive L-Proline transport must take place between the cytosol where its biogenesis via Pro1p, Pro2p, Pro3p occurs and mitochondria. L-Proline uptake in S. cerevisiae purified and active mitochondria was investigated by swelling experiments, oxygen uptake and fluorimetric measurement of a membrane potential generation (ΔΨ). Our results strongly suggest that L-Proline uptake occurs via a carried-mediated process as demonstrated by saturation kinetics and experiments with N-ethylmaleimide, a pharmacological compound that is a cysteine-modifying reagent in hydrophobic protein domains and that inhibited mitochondrial transport. Plasticity of S. cerevisiae cell biochemistry according to background fluctuations is an important factor of adaptation to stress. Thus L-Proline → Glutamate route feeds Krebs cycle providing energy and anaplerotic carbon for yeast survival.
Donnini, C; Lodi, T; Ferrero, I; Puglisi, P P
1992-02-01
The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.
Malina, Carl; Larsson, Christer; Nielsen, Jens
2018-08-01
Mitochondria are dynamic organelles of endosymbiotic origin that are essential components of eukaryal cells. They contain their own genetic machinery, have multicopy genomes and like their bacterial ancestors they consist of two membranes. However, the majority of the ancestral genome has been lost or transferred to the nuclear genome of the host, preserving only a core set of genes involved in oxidative phosphorylation. Mitochondria perform numerous biological tasks ranging from bioenergetics to production of protein co-factors, including heme and iron-sulfur clusters. Due to the importance of mitochondria in many cellular processes, mitochondrial dysfunction is implicated in a wide variety of human disorders. Much of our current knowledge on mitochondrial function and dysfunction comes from studies using Saccharomyces cerevisiae. This yeast has good fermenting capacity, rendering tolerance to mutations that inactivate oxidative phosphorylation and complete loss of mitochondrial DNA. Here, we review yeast mitochondrial metabolism and function with focus on S. cerevisiae and its contribution in understanding mitochondrial biology. We further review how systems biology studies, including mathematical modeling, has allowed gaining new insight into mitochondrial function, and argue that this approach may enable us to gain a holistic view on how mitochondrial function interacts with different cellular processes.
Foury, F; Goffeau, A
1979-01-01
Five nuclear mutants enhancing the spontaneous mutation rate of mtDNA have been isolated in Saccharomyces cerevisiae. These mutators fall into five complementation groups and are located at five genetic loci different from rad50 to rad57 loci. Three mutants (gam1, gam2, and gam4), insensitive or weakly sensitive to gamma-rays, exhibit increased frequency of spontaneous production of mutants with large deletions of the mtDNA (p-) and of all tested mitochondrial drug-resistant mutants. Two other mutants (gam3 and gam5), highly sensitive to gamma-rays, increase only the mutation rate of particular alleles of the mtDNA. The mutant gam5 enhances only the production of p- and erythromycin-resistant clones. The mutant gam3 exhibits an enhanced rate of oligomycin-resistant clones as well as a collateral increase of nuclear mutability. The existence of gam3 and gam5 mutants indicates that at least two common steps control both nuclear DNA repair and the mutability of particular alleles of the mtDNA. However, the general spontaneous mutability of the mtDNA includes at least three steps not involved in the repair of nuclear DNA, as revealed by the gam1, gam2, and gam4 mutations. PMID:392521
Osman, Christof; Noriega, Thomas R.; Okreglak, Voytek; Fung, Jennifer C.; Walter, Peter
2015-01-01
Mitochondrial DNA (mtDNA) is essential for mitochondrial and cellular function. In Saccharomyces cerevisiae, mtDNA is organized in nucleoprotein structures termed nucleoids, which are distributed throughout the mitochondrial network and are faithfully inherited during the cell cycle. How the cell distributes and inherits mtDNA is incompletely understood although an involvement of mitochondrial fission and fusion has been suggested. We developed a LacO-LacI system to noninvasively image mtDNA dynamics in living cells. Using this system, we found that nucleoids are nonrandomly spaced within the mitochondrial network and observed the spatiotemporal events involved in mtDNA inheritance. Surprisingly, cells deficient in mitochondrial fusion and fission distributed and inherited mtDNA normally, pointing to alternative pathways involved in these processes. We identified such a mechanism, where we observed fission-independent, but F-actin–dependent, tip generation that was linked to the positioning of mtDNA to the newly generated tip. Although mitochondrial fusion and fission were dispensable for mtDNA distribution and inheritance, we show through a combination of genetics and next-generation sequencing that their absence leads to an accumulation of mitochondrial genomes harboring deleterious structural variations that cluster at the origins of mtDNA replication, thus revealing crucial roles for mitochondrial fusion and fission in maintaining the integrity of the mitochondrial genome. PMID:25730886
Yoon, Young Geol; Koob, Michael D; Yoo, Young Hyun
2011-09-15
Mitochondrial transcription factor A (Tfam) binds to and organizes mitochondrial DNA (mtDNA) genome into a mitochondrial nucleoid (mt-nucleoid) structure, which is necessary for mtDNA transcription and maintenance. Here, we demonstrate the mtDNA-organizing activity of mouse Tfam and its transcript isoform (Tfam(iso)), which has a smaller high-mobility group (HMG)-box1 domain, using a yeast model system that contains a deletion of the yeast homolog of mouse Tfam protein, Abf2p. When the mouse Tfam genes were introduced into the ABF2 locus of yeast genome, the corresponding mouse proteins, Tfam and Tfam(iso), can functionally replace the yeast Abf2p and support mtDNA maintenance and mitochondrial biogenesis in yeast. Growth properties, mtDNA content and mitochondrial protein levels of genes encoded in the mtDNA were comparable in the strains expressing mouse proteins and the wild-type yeast strain, indicating that the proteins have robust mtDNA-maintaining and -expressing function in yeast mitochondria. These results imply that the mtDNA-organizing activities of the mouse mt-nucleoid proteins are structurally and evolutionary conserved, thus they can maintain the mtDNA of distantly related and distinctively different species, such as yeast. Copyright © 2011 Elsevier B.V. All rights reserved.
Higuchi-Sanabria, Ryo; Charalel, Joseph K.; Viana, Matheus P.; Garcia, Enrique J.; Sing, Cierra N.; Koenigsberg, Andrea; Swayne, Theresa C.; Vevea, Jason D.; Boldogh, Istvan R.; Rafelski, Susanne M.; Pon, Liza A.
2016-01-01
Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud. PMID:26764088
Mitochondrial inheritance in budding yeasts: towards an integrated understanding.
Solieri, Lisa
2010-11-01
Recent advances in yeast mitogenomics have significantly contributed to our understanding of the diversity of organization, structure and topology in the mitochondrial genome of budding yeasts. In parallel, new insights on mitochondrial DNA (mtDNA) inheritance in the model organism Saccharomyces cerevisiae highlighted an integrated scenario where recombination, replication and segregation of mtDNA are intricately linked to mitochondrial nucleoid (mt-nucleoid) structure and organelle sorting. In addition to this, recent discoveries of bifunctional roles of some mitochondrial proteins have interesting implications on mito-nuclear genome interactions and the relationship between mtDNA inheritance, yeast fitness and speciation. This review summarizes the current knowledge on yeast mitogenomics, mtDNA inheritance with regard to mt-nucleoid structure and organelle dynamics, and mito-nuclear genome interactions. Copyright © 2010 Elsevier Ltd. All rights reserved.
Naumov, Gennadi I; Lee, Ching-Fu; Naumova, Elena S
2013-01-01
Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.
Irc3 is a mitochondrial DNA branch migration enzyme
Gaidutšik, Ilja; Sedman, Tiina; Sillamaa, Sirelin; Sedman, Juhan
2016-01-01
Integrity of mitochondrial DNA (mtDNA) is essential for cellular energy metabolism. In the budding yeast Saccharomyces cerevisiae, a large number of nuclear genes influence the stability of mitochondrial genome; however, most corresponding gene products act indirectly and the actual molecular mechanisms of mtDNA inheritance remain poorly characterized. Recently, we found that a Superfamily II helicase Irc3 is required for the maintenance of mitochondrial genome integrity. Here we show that Irc3 is a mitochondrial DNA branch migration enzyme. Irc3 modulates mtDNA metabolic intermediates by preferential binding and unwinding Holliday junctions and replication fork structures. Furthermore, we demonstrate that the loss of Irc3 can be complemented with mitochondrially targeted RecG of Escherichia coli. We suggest that Irc3 could support the stability of mtDNA by stimulating fork regression and branch migration or by inhibiting the formation of irregular branched molecules. PMID:27194389
Mitochondrial DNA repairs double-strand breaks in yeast chromosomes.
Ricchetti, M; Fairhead, C; Dujon, B
1999-11-04
The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process.
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.
Zyrina, Anna N; Smirnova, Ekaterina A; Markova, Olga V; Severin, Fedor F; Knorre, Dmitry A
2017-02-01
There are two superoxide dismutases in the yeast Saccharomyces cerevisiae-cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2 Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance. Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria. Copyright © 2017 American Society for Microbiology.
Zyrina, Anna N.; Smirnova, Ekaterina A.; Markova, Olga V.; Severin, Fedor F.
2016-01-01
ABSTRACT There are two superoxide dismutases in the yeast Saccharomyces cerevisiae—cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2. Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance. IMPORTANCE Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria. PMID:27864171
Higuchi-Sanabria, Ryo; Charalel, Joseph K; Viana, Matheus P; Garcia, Enrique J; Sing, Cierra N; Koenigsberg, Andrea; Swayne, Theresa C; Vevea, Jason D; Boldogh, Istvan R; Rafelski, Susanne M; Pon, Liza A
2016-03-01
Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud. © 2016 Higuchi-Sanabria et al. 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).
Rigoulet, Michel; Salin, Benedicte; Masneuf-Pomarede, Isabelle; de Vienne, Dominique; Sicard, Delphine; Bely, Marina; Marullo, Philippe
2013-01-01
In eukaryotes, mitochondrial DNA (mtDNA) has high rate of nucleotide substitution leading to different mitochondrial haplotypes called mitotypes. However, the impact of mitochondrial genetic variant on phenotypic variation has been poorly considered in microorganisms because mtDNA encodes very few genes compared to nuclear DNA, and also because mitochondrial inheritance is not uniparental. Here we propose original material to unravel mitotype impact on phenotype: we produced interspecific hybrids between S. cerevisiae and S. uvarum species, using fully homozygous diploid parental strains. For two different interspecific crosses involving different parental strains, we recovered 10 independent hybrids per cross, and allowed mtDNA fixation after around 80 generations. We developed PCR-based markers for the rapid discrimination of S. cerevisiae and S. uvarum mitochondrial DNA. For both crosses, we were able to isolate fully isogenic hybrids at the nuclear level, yet possessing either S. cerevisiae mtDNA (Sc-mtDNA) or S. uvarum mtDNA (Su-mtDNA). Under fermentative conditions, the mitotype has no phenotypic impact on fermentation kinetics and products, which was expected since mtDNA are not necessary for fermentative metabolism. Alternatively, under respiratory conditions, hybrids with Sc-mtDNA have higher population growth performance, associated with higher respiratory rate. Indeed, far from the hypothesis that mtDNA variation is neutral, our work shows that mitochondrial polymorphism can have a strong impact on fitness components and hence on the evolutionary fate of the yeast populations. We hypothesize that under fermentative conditions, hybrids may fix stochastically one or the other mt-DNA, while respiratory environments may increase the probability to fix Sc-mtDNA. PMID:24086452
Trial and error: how the unclonable human mitochondrial genome was cloned in yeast.
Bigger, Brian W; Liao, Ai-Yin; Sergijenko, Ana; Coutelle, Charles
2011-11-01
Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene. We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination. Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated. This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.
ATP Synthase Diseases of Mitochondrial Genetic Origin
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
Kalifa, Lidza; Quintana, Daniel F.; Schiraldi, Laura K.; Phadnis, Naina; Coles, Garry L.; Sia, Rey A.; Sia, Elaine A.
2012-01-01
Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat–mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria. PMID:22214610
Kalifa, Lidza; Quintana, Daniel F; Schiraldi, Laura K; Phadnis, Naina; Coles, Garry L; Sia, Rey A; Sia, Elaine A
2012-03-01
Mitochondrial DNA (mtDNA) deletions are associated with sporadic and inherited diseases and age-associated neurodegenerative disorders. Approximately 85% of mtDNA deletions identified in humans are flanked by short directly repeated sequences; however, mechanisms by which these deletions arise are unknown. A limitation in deciphering these mechanisms is the essential nature of the mitochondrial genome in most living cells. One exception is budding yeast, which are facultative anaerobes and one of the few organisms for which directed mtDNA manipulation is possible. Using this model system, we have developed a system to simultaneously monitor spontaneous direct-repeat-mediated deletions (DRMDs) in the nuclear and mitochondrial genomes. In addition, the mitochondrial DRMD reporter contains a unique KpnI restriction endonuclease recognition site that is not present in otherwise wild-type (WT) mtDNA. We have expressed KpnI fused to a mitochondrial localization signal to induce a specific mitochondrial double-strand break (mtDSB). Here we report that loss of the MRX (Mre11p, Rad50p, Xrs2p) and Ku70/80 (Ku70p, Ku80p) complexes significantly impacts the rate of spontaneous deletion events in mtDNA, and these proteins contribute to the repair of induced mtDSBs. Furthermore, our data support homologous recombination (HR) as the predominant pathway by which mtDNA deletions arise in yeast, and suggest that the MRX and Ku70/80 complexes are partially redundant in mitochondria.
Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae
Wang, Jing; Gammon, Micah G.; Maynard, Margaret K.; White, Olivia L.; Cobine, Jai A.; Mahone, Wilkerson K.
2016-01-01
In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix. PMID:26763345
Sariki, Santhosh Kumar; Sahu, Pushpendra Kumar; Golla, Upendarrao; Singh, Vikash; Azad, Gajendra Kumar; Tomar, Raghuvir S
2016-11-01
Mutations in the Senataxin gene, SETX are known to cause the neurodegenerative disorders, ataxia with oculomotor apraxia type 2 (AOA2), and amyotrophic lateral sclerosis 4 (ALS4). However, the mechanism underlying disease pathogenesis is still unclear. The Senataxin N-terminal protein-interaction and C-terminal RNA/DNA helicase domains are conserved in the Saccharomyces cerevisiae homolog, Sen1p. Using genome-wide expression analysis, we first show alterations in key cellular pathways such as: redox, unfolded protein response, and TOR in the yeast sen1 ΔN mutant (N-terminal truncation). This mutant exhibited growth defects on nonfermentable carbon sources, was sensitive to oxidative stress, and showed severe loss of mitochondrial DNA. The growth defect could be partially rescued upon supplementation with reducing agents and antioxidants. Furthermore, the mutant showed higher levels of reactive oxygen species, lower UPR activity, and alterations in mitochondrial membrane potential, increase in vacuole acidity, free calcium ions in the cytosol, and resistance to rapamycin treatment. Notably, the sen1 ∆N mutant showed increased cell death and shortened chronological life span. Given the strong similarity of the yeast and human Sen1 proteins, our study thus provides a mechanism for the progressive neurological disorders associated with mutations in human senataxin. © 2016 Federation of European Biochemical Societies.
Shima, Jun; Ando, Akira; Takagi, Hiroshi
2008-03-01
Yeasts used in bread making are exposed to air-drying stress during dried yeast production processes. To clarify the genes required for air-drying tolerance, we performed genome-wide screening using the complete deletion strain collection of diploid Saccharomyces cerevisiae. The screening identified 278 gene deletions responsible for air-drying sensitivity. These genes were classified based on their cellular function and on the localization of their gene products. The results showed that the genes required for air-drying tolerance were frequently involved in mitochondrial functions and in connection with vacuolar H(+)-ATPase, which plays a role in vacuolar acidification. To determine the role of vacuolar acidification in air-drying stress tolerance, we monitored intracellular pH. The results showed that intracellular acidification was induced during air-drying and that this acidification was amplified in a deletion mutant of the VMA2 gene encoding a component of vacuolar H(+)-ATPase, suggesting that vacuolar H(+)-ATPase helps maintain intracellular pH homeostasis, which is affected by air-drying stress. To determine the effects of air-drying stress on mitochondria, we analysed the mitochondrial membrane potential under air-drying stress conditions using MitoTracker. The results showed that mitochondria were extremely sensitive to air-drying stress, suggesting that a mitochondrial function is required for tolerance to air-drying stress. We also analysed the correlation between oxidative-stress sensitivity and air-drying-stress sensitivity. The results suggested that oxidative stress is a critical determinant of sensitivity to air-drying stress, although ROS-scavenging systems are not necessary for air-drying stress tolerance. (c) 2008 John Wiley & Sons, Ltd.
Kawai, Shigeyuki; Urban, Jörg; Piccolis, Manuele; Panchaud, Nicolas; De Virgilio, Claudio; Loewith, Robbie
2011-10-01
TORC1-dependent phosphorylation of Saccharomyces cerevisiae Sch9 was dramatically reduced upon exposure to a protonophore or in respiration-incompetent ρ(0) cells but not in respiration-incompetent pet mutants, providing important insight into the molecular mechanisms governing interorganellar signaling in general and retrograde signaling in particular.
Mitochondrial fission proteins regulate programmed cell death in yeast.
Fannjiang, Yihru; Cheng, Wen-Chih; Lee, Sarah J; Qi, Bing; Pevsner, Jonathan; McCaffery, J Michael; Hill, R Blake; Basañez, Gorka; Hardwick, J Marie
2004-11-15
The possibility that single-cell organisms undergo programmed cell death has been questioned in part because they lack several key components of the mammalian cell death machinery. However, yeast encode a homolog of human Drp1, a mitochondrial fission protein that was shown previously to promote mammalian cell death and the excessive mitochondrial fragmentation characteristic of apoptotic mammalian cells. In support of a primordial origin of programmed cell death involving mitochondria, we found that the Saccharomyces cerevisiae homolog of human Drp1, Dnm1, promotes mitochondrial fragmentation/degradation and cell death following treatment with several death stimuli. Two Dnm1-interacting factors also regulate yeast cell death. The WD40 repeat protein Mdv1/Net2 promotes cell death, consistent with its role in mitochondrial fission. In contrast to its fission function in healthy cells, Fis1 unexpectedly inhibits Dnm1-mediated mitochondrial fission and cysteine protease-dependent cell death in yeast. Furthermore, the ability of yeast Fis1 to inhibit mitochondrial fission and cell death can be functionally replaced by human Bcl-2 and Bcl-xL. Together, these findings indicate that yeast and mammalian cells have a conserved programmed death pathway regulated by a common molecular component, Drp1/Dnm1, that is inhibited by a Bcl-2-like function.
Mitochondrial Recombination and Introgression during Speciation by Hybridization.
Leducq, Jean-Baptiste; Henault, Mathieu; Charron, Guillaume; Nielly-Thibault, Lou; Terrat, Yves; Fiumera, Heather L; Shapiro, B Jesse; Landry, Christian R
2017-08-01
Genome recombination is a major source of genotypic diversity and contributes to adaptation and speciation following interspecies hybridization. The contribution of recombination in these processes has been thought to be largely limited to the nuclear genome because organelles are mostly uniparentally inherited in animals and plants, which prevents recombination. Unicellular eukaryotes such as budding yeasts do, however, transmit mitochondria biparentally, suggesting that during hybridization, both parents could provide alleles that contribute to mitochondrial functions such as respiration and metabolism in hybrid populations or hybrid species. We examined the dynamics of mitochondrial genome transmission and evolution during speciation by hybridization in the natural budding yeast Saccharomyces paradoxus. Using population-scale mitochondrial genome sequencing in two endemic North American incipient species SpB and SpC and their hybrid species SpC*, we found that both parental species contributed to the hybrid mitochondrial genome through recombination. We support our findings by showing that mitochondrial recombination between parental types is frequent in experimental crosses that recreate the early step of this speciation event. In these artificial hybrids, we observed that mitochondrial genome recombination enhances phenotypic variation among diploid hybrids, suggesting that it could play a role in the phenotypic differentiation of hybrid species. Like the nuclear genome, the mitochondrial genome can, therefore, also play a role in hybrid speciation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Absence of detectable mitochondrial recombination in Paramecium.
Adoutte, A; Knowles, J K; Sainsard-Chanet, A
1979-12-01
An extensive search for recombination between mitochondrial markers was carried out in Paramecium tetraurelia. Thirty-two combinations, altogether involving 24 different markers, were studied. The markers belonged to the three main categories of mitochondrial mutations presently available in this organism, (a) Spontaneous or UV-induced antibiotic resistance mutations, most probably affecting mitochondrial ribosomes, (b) nitrosoguanidine-induced antibiotic resistance markers displaying thermosensitivity or slow growth, enabling easy selection of possible wild-type recombinants, and (c) mitochondrial partial suppressors of a nuclear gene, probably corresponding to molecular alterations distinct from the preceding two categories. In addition, different genetic configurations were analyzed (i.e., mutant X mutant, double-mutant X wild-type, etc.).--None of the combinations yielded any evidence for the occurrence of recombined genomes despite the fact that: (1) all of them were studied on a large scale involving the screening of at least several thousand mitochondrial genomes (often several millions), (2) in many of them the detection level was sufficiently high to enable the isolation of spontaneous mutants in control cells, and (3) in several of them, reconstitution experiments carried out in parallel show that the conditions were fully adequate to detect recombinant genotypes. The results are in marked contrast with those obtained on the few other organisms in which mitochondrial recombination has been studied, particularly Saccharomyces cerevisiae, in which mitochondrial recombination is intense.--The most likely basis for the various manifestations of mitochondrial genetic autonomy in Paramecium, described in this as well as in previous publications, is that the chondriome of this organism is made up of thousands of structurally discrete, noninteracting units.
Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng
2014-01-01
The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.
Organization and dynamics of yeast mitochondrial nucleoids
MIYAKAWA, Isamu
2017-01-01
Mitochondrial DNA (mtDNA) is packaged by association with specific proteins in compact DNA-protein complexes named mitochondrial nucleoids (mt-nucleoids). The budding yeast Saccharomyces cerevisiae is able to grow either aerobically or anaerobically. Due to this characteristic, S. cerevisiae has been extensively used as a model organism to study genetics, morphology and biochemistry of mitochondria for a long time. Mitochondria of S. cerevisiae frequently fuse and divide, and perform dynamic morphological changes depending on the culture conditions and the stage of life cycle of the yeast cells. The mt-nucleoids also dynamically change their morphology, accompanying morphological changes of mitochondria. The mt-nucleoids have been isolated morphologically intact and functional analyses of mt-nucleoid proteins have been extensively performed. These studies have revealed that the functions of mt-nucleoid proteins are essential for maintenance of mtDNA. The aims of this review are to summarize the history on the research of yeast mt-nucleoids as well as recent findings on the organization of the mt-nucleoids and mitochondrial dynamics. PMID:28496055
Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae
Peters, Theodore W.; Miller, Aaron W.; Tourette, Cendrine; Agren, Hannah; Hubbard, Alan; Hughes, Robert E.
2015-01-01
Adenosine triphosphate (ATP) plays an important role as a primary molecule for the transfer of chemical energy to drive biological processes. ATP also functions as an extracellular signaling molecule in a diverse array of eukaryotic taxa in a conserved process known as purinergic signaling. Given the important roles of extracellular ATP in cell signaling, we sought to comprehensively elucidate the pathways and mechanisms governing ATP efflux from eukaryotic cells. Here, we present results of a genomic analysis of ATP efflux from Saccharomyces cerevisiae by measuring extracellular ATP levels in cultures of 4609 deletion mutants. This screen revealed key cellular processes that regulate extracellular ATP levels, including mitochondrial translation and vesicle sorting in the late endosome, indicating that ATP production and transport through vesicles are required for efflux. We also observed evidence for altered ATP efflux in strains deleted for genes involved in amino acid signaling, and mitochondrial retrograde signaling. Based on these results, we propose a model in which the retrograde signaling pathway potentiates amino acid signaling to promote mitochondrial respiration. This study advances our understanding of the mechanism of ATP secretion in eukaryotes and implicates TOR complex 1 (TORC1) and nutrient signaling pathways in the regulation of ATP efflux. These results will facilitate analysis of ATP efflux mechanisms in higher eukaryotes. PMID:26585826
Global Genetic Determinants of Mitochondrial DNA Copy Number
Zhang, Hengshan; Singh, Keshav K.
2014-01-01
Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role. PMID:25170845
Zelenaya-Troitskaya, O; Newman, S M; Okamoto, K; Perlman, P S; Butow, R A
1998-04-01
Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein. These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.
Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies
Lasserre, Jean-Paul; Dautant, Alain; Aiyar, Raeka S.; Kucharczyk, Roza; Glatigny, Annie; Tribouillard-Tanvier, Déborah; Rytka, Joanna; Blondel, Marc; Skoczen, Natalia; Reynier, Pascal; Pitayu, Laras; Rötig, Agnès; Delahodde, Agnès; Steinmetz, Lars M.; Dujardin, Geneviève; Procaccio, Vincent; di Rago, Jean-Paul
2015-01-01
ABSTRACT Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as ‘petite-positivity’), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast. PMID:26035862
Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation.
Zhou, Jingwen; Liu, Liming; Chen, Jian
2010-05-01
Genetic manipulation of mitochondrial DNA (mtDNA) is the most direct method for investigating mtDNA, but until now, this has been achieved only in the diploid yeast Saccharomyces cerevisiae. In this study, the ATP6 gene on mtDNA of the haploid yeast Candida glabrata (Torulopsis glabrata) was deleted by biolistic transformation of DNA fragments with a recoded ARG8(m) mitochondrial genetic marker, flanked by homologous arms to the ATP6 gene. Transformants were identified by arginine prototrophy. However, in the transformants, the original mtDNA was not lost spontaneously, even under arginine selective pressure. Moreover, the mtDNA transformants selectively lost the transformed mtDNA under aerobic conditions. The mtDNA heteroplasmy in the transformants was characterized by PCR, quantitative PCR, and Southern blotting, showing that the heteroplasmy was relatively stable in the absence of arginine. Aerobic conditions facilitated the loss of the original mtDNA, and anaerobic conditions favored loss of the transformed mtDNA. Moreover, detailed investigations showed that increases in reactive oxygen species in mitochondria lacking ATP6, along with their equal cell division, played important roles in determining the dynamics of heteroplasmy. Based on our analysis of mtDNA heteroplasmy in C. glabrata, we were able to generate homoplasmic Deltaatp6 mtDNA strains.
MITOPRED: a web server for the prediction of mitochondrial proteins
Guda, Chittibabu; Guda, Purnima; Fahy, Eoin; Subramaniam, Shankar
2004-01-01
MITOPRED web server enables prediction of nucleus-encoded mitochondrial proteins in all eukaryotic species. Predictions are made using a new algorithm based primarily on Pfam domain occurrence patterns in mitochondrial and non-mitochondrial locations. Pre-calculated predictions are instantly accessible for proteomes of Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila, Homo sapiens, Mus musculus and Arabidopsis species as well as all the eukaryotic sequences in the Swiss-Prot and TrEMBL databases. Queries, at different confidence levels, can be made through four distinct options: (i) entering Swiss-Prot/TrEMBL accession numbers; (ii) uploading a local file with such accession numbers; (iii) entering protein sequences; (iv) uploading a local file containing protein sequences in FASTA format. Automated updates are scheduled for the pre-calculated prediction database so as to provide access to the most current data. The server, its documentation and the data are available from http://mitopred.sdsc.edu. PMID:15215413
Clavijo, Almudena; Calderón, Isabel L; Paneque, Patricia
2011-03-01
An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hsu, T.; Lee, L.W.; Chang, T.H.
1992-09-01
Saccharomyces cerevisiae is not only a key microorganism in brewing or fermentation processes, it has also been employed for monitoring aquatic pollutants. The major advantage of using Saccharomyces cerevisiae as a bioassay system is that this yeast can be easily obtained as dry pellets from commercial sources at low cost. In addition to its economical aspect, Saccharomyces cerevisiae, like other microorganisms, is easy to handle, grows rapidly, and provides a large number of homogeneous individuals for utilization in toxicity tests. Although cell growth, cell viability, electron transport and mitochondrial respiration of Saccharomyces cerevisiaes have all been selected as parameters formore » toxicity assessment, measuring cell growth by absorbance is by farm the most convenient and rapid method when large amounts of water samples are to be tested. Mochida et al. (1988), however, reported that Saccharomyces cerevisiae was five to ten times less sensitive than cell culture systems to cadmium, mercury and nickel, when cell growth of both systems was monitored. This relative insensitivity to heavy metals might handicap the practical use of this yeast strain for bioassays. Since previous studies indicated that the susceptibility of microorganisms to environmental toxicants can be influenced by incubation temperature and nutrient strength, we attempted to examine the effect of incubation temperature and nutrient strength on the susceptibility of Saccharomyces cerevisiae to heavy metals in order to obtain the optimum bioassay sensitivity. In this study, we used cadmium and mercury as model toxicants. 9 refs., 2 figs., 1 tab.« less
Kobayashi, Jyumpei; Sasaki, Daisuke; Hara, Kiyotaka Y; Hasunuma, Tomohisa; Kondo, Akihiko
2017-03-15
Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes. We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 S32A gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene. This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.
Mitochondrial depolarization in yeast zygotes inhibits clonal expansion of selfish mtDNA.
Karavaeva, Iuliia E; Golyshev, Sergey A; Smirnova, Ekaterina A; Sokolov, Svyatoslav S; Severin, Fedor F; Knorre, Dmitry A
2017-04-01
Non-identical copies of mitochondrial DNA (mtDNA) compete with each other within a cell and the ultimate variant of mtDNA present depends on their relative replication rates. Using yeast Saccharomyces cerevisiae cells as a model, we studied the effects of mitochondrial inhibitors on the competition between wild-type mtDNA and mutant selfish mtDNA in heteroplasmic zygotes. We found that decreasing mitochondrial transmembrane potential by adding uncouplers or valinomycin changes the competition outcomes in favor of the wild-type mtDNA. This effect was significantly lower in cells with disrupted mitochondria fission or repression of the autophagy-related genes ATG8 , ATG32 or ATG33 , implying that heteroplasmic zygotes activate mitochondrial degradation in response to the depolarization. Moreover, the rate of mitochondrially targeted GFP turnover was higher in zygotes treated with uncoupler than in haploid cells or untreated zygotes. Finally, we showed that vacuoles of zygotes with uncoupler-activated autophagy contained DNA. Taken together, our data demonstrate that mitochondrial depolarization inhibits clonal expansion of selfish mtDNA and this effect depends on mitochondrial fission and autophagy. These observations suggest an activation of mitochondria quality control mechanisms in heteroplasmic yeast zygotes. © 2017. Published by The Company of Biologists Ltd.
Medkour, Younes; Dakik, Paméla; McAuley, Mélissa; Mohammad, Karamat; Mitrofanova, Darya
2017-01-01
The functional state of mitochondria is vital to cellular and organismal aging in eukaryotes across phyla. Studies in the yeast Saccharomyces cerevisiae have provided evidence that age-related changes in some aspects of mitochondrial functionality can create certain molecular signals. These signals can then define the rate of cellular aging by altering unidirectional and bidirectional communications between mitochondria and other organelles. Several aspects of mitochondrial functionality are known to impact the replicative and/or chronological modes of yeast aging. They include mitochondrial electron transport, membrane potential, reactive oxygen species, and protein synthesis and proteostasis, as well as mitochondrial synthesis of iron-sulfur clusters, amino acids, and NADPH. Our recent findings have revealed that the composition of mitochondrial membrane lipids is one of the key aspects of mitochondrial functionality affecting yeast chronological aging. We demonstrated that exogenously added lithocholic bile acid can delay chronological aging in yeast because it elicits specific changes in mitochondrial membrane lipids. These changes allow mitochondria to operate as signaling platforms that delay yeast chronological aging by orchestrating an institution and maintenance of a distinct cellular pattern. In this review, we discuss molecular and cellular mechanisms underlying the essential role of mitochondrial membrane lipids in yeast chronological aging. PMID:28593023
Kobayashi, Shingo; Mizuike, Aya; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori
2014-09-01
In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3×HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations. Copyright © 2014 Elsevier B.V. All rights reserved.
Gergondey, Rachel; Garcia, Camille; Marchand, Christophe H; Lemaire, Stephane D; Camadro, Jean-Michel; Auchère, Françoise
2017-03-15
The potential biological consequences of oxidative stress and changes in glutathione levels include the oxidation of susceptible protein thiols and reversible covalent binding of glutathione to the -SH groups of proteins by S-glutathionylation. Mitochondria are central to the response to oxidative stress and redox signaling. It is therefore crucial to explore the adaptive response to changes in thiol-dependent redox status in these organelles. We optimized the purification protocol of glutathionylated proteins in the yeast Saccharomyces cerevisiae and present a detailed proteomic analysis of the targets of protein glutathionylation in cells undergoing constitutive metabolism and after exposure to various stress conditions. This work establishes the physiological importance of the glutathionylation process in S. cerevisiae under basal conditions and provides evidence for an atypical and unexpected cellular distribution of the process between the cytosol and mitochondria. In addition, our data indicate that each oxidative condition (diamide, GSSG, H 2 O 2 , or the presence of iron) elicits an adaptive metabolic response affecting specific mitochondrial metabolic pathways, mainly involved in the energetic maintenance of the cells. The correlation of protein modifications with intracellular glutathione levels suggests that protein deglutathionylation may play a role in protecting mitochondria from oxidative stress. This work provides further insights into the diversity of proteins undergoing glutathionylation and the role of this post-translational modification as a regulatory process in the adaptive response of the cell. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast
Fritsch, Emilie S.; Chabbert, Christophe D.; Klaus, Bernd; Steinmetz, Lars M.
2014-01-01
In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome
A genome-wide map of mitochondrial DNA recombination in yeast.
Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M
2014-10-01
In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome
Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance
Sedman, Tiina; Gaidutšik, Ilja; Villemson, Karin; Hou, YingJian; Sedman, Juhan
2014-01-01
Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability. PMID:25389272
Nishimura, Akira; Nasuno, Ryo; Takagi, Hiroshi
2012-07-30
The proline metabolism intermediate Δ(1)-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
A novel class of Saccharomyces cerevisiae mutants specifically UV-sensitive to "petite" induction.
Moustacchi, E; Perlman, P S; Mahler, H R
1976-11-17
A mutant of Saccharomyces cerevisiae has been isolated which, though exhibiting a normal response to nuclear genetic damage by ultraviolet light (UV), is more sensitive than its wild type specifically in the production of the cytoplasmic (rho-) mutation by this agent. Some of the features of this mutation which has been designated uvsrho 5 are: i) The mutation is recessive, it exhibits a Mendelian, and hence presumably nuclear, pattern of segregation, but manifests its effects specifically and pleiotropically on mitochondrial functions. ii) Mutant cells resemble their wild type parents in a) growth characteristics on glucose; b) in their UV induced dose response to lethality or nuclear mutation and c) the ability of their mitochondrial genome, upon mating with appropriate testers, of transmitting and recombining various markers, albeit with enhanced efficiency. Similarly, d) they are able to modulate the expression of mitochondrial mutagenesis by ethidium bromide. Thus their mitochondrial DNA appears genetically as competent as that of the wild type. iii) Mutant cells differ from their wild type parents in a) growth characteristics on glycerol; b) susceptibility to induction of the mitochondrial (rho-) mutation by various mutagens, in that the rate of spontaneous mutation is slightly and that by UV is significantly enhanced, whild that by ethidium bromide is greatly diminished. Conversely, c) modulating influences resulting in the repair of initial damage are diminished fro UV and stimulated in the case of Berenil. iv) The amount of mitochondrial DNA per cell appears elevated in the mutant, relative to wild type, and its rate of degradation subsequent to a mutagenic exposure to either UV or ethidium bromide is diminished. v) A self-consistent scheme to account for this and all other information so far available for the induction and modulation of the (rho-) mutation is presented. In a previous study it was shown that some nuclear mutants of Saccharomyces cerevisiae
Martí, María C; Olmos, Enrique; Calvete, Juan J; Díaz, Isabel; Barranco-Medina, Sergio; Whelan, James; Lázaro, Juan J; Sevilla, Francisca; Jiménez, Ana
2009-06-01
Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.
Evidence for a Role of FEN1 in Maintaining Mitochondrial DNA Integrity
Kalifa, Lidza; Beutner, Gisela; Phadnis, Naina; Sheu, Shey-Shing; Sia, Elaine A.
2009-01-01
Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle’s high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5′ flap structures generated during DNA synthesis. Furthermore, removal of 5′ flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity. PMID:19699691
Acevedo-Torres, Karina; Fonseca-Williams, Sharon; Ayala-Torres, Sylvette; Torres-Ramos, Carlos A.
2010-01-01
The Saccharomyces cerevisiae APN1 gene that participates in base excision repair has been localized both in the nucleus and the mitochondria. APN1 deficient cells (apn1Δ) show increased mutation frequencies in mitochondrial DNA (mtDNA) suggesting that APN1 is also important for mtDNA stability. To understand APN1-dependent mtDNA repair processes we studied the formation and repair of mtDNA lesions in cells exposed to methyl methanesulfonate (MMS). We show that MMS induces mtDNA damage in a dose-dependent fashion and that deletion of the APN1 gene enhances the susceptibility of mtDNA to MMS. Repair kinetic experiments demonstrate that in wild-type cells (WT) it takes 4 hr to repair the damage induced by 0.1% MMS, whereas in the apn1Δ strain there is a lag in mtDNA repair that results in significant differences in the repair capacity between the two yeast strains. Analysis of lesions in nuclear DNA (nDNA) after treatment with 0.1% MMS shows a significant difference in the amount of nDNA lesions between WT and apn1Δ cells. Interestingly, comparisons between nDNA and mtDNA damage show that nDNA is more sensitive to the effects of MMS treatment. However, both strains are able to repair the nDNA lesions, contrary to mtDNA repair, which is compromised in the apn1Δ mutant strain. Therefore, although nDNA is more sensitive than mtDNA to the effects of MMS, deletion of APN1 has a stronger phenotype in mtDNA repair than in nDNA. These results highlight the prominent role of APN1 in the repair of environmentally induced mtDNA damage. PMID:19197988
DOE Office of Scientific and Technical Information (OSTI.GOV)
Choi, Joon-Seok; Choi, Kyung-Mi; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr
2011-06-03
Highlights: {yields} Calorie restriction (CR) increases electron transport chain (ETC) at both RNA and protein level. {yields} CR enhances mitochondrial membrane potential, and, regardless of ages, reduces reactive oxygen species. {yields} CR increases both efficiency and capacity of the ETC. {yields} CR induces intensive modulation at mitochondrial ETC where might be a major site leading to extension of lifespan. -- Abstract: Caloric restriction (CR) is known to extend lifespan in a variety of species; however, the mechanism remains unclear. In this study, we found that CR potentiated the mitochondrial electron transport chain (ETC) at both the transcriptional and translational levels.more » Indeed, mitochondrial membrane potential (MMP) was increased by CR, and, regardless of ages, overall reactive oxygen species (ROS) generation was decreased by CR. With these changes, overall growth rate of cells was maintained under various CR conditions, just like cells under a non-restricted condition. All of these data support increased efficiency and capacity of the ETC by CR, and this change might lead to extension of lifespan.« less
Kucejová, B; Foury, F
2003-01-01
RIM1 is a nuclear gene of the yeast Saccharomyces cerevisiae coding for a protein with single-stranded DNA-binding activity that is essential for mitochondrial genome maintenance. No protein partners of Rim1p have been described so far in yeast. To better understand the role of this protein in mitochondrial DNA replication and recombination, a search for protein interactors by the yeast two-hybrid system was performed. This approach led to the identification of several candidates, including a putative transcription factor, Azf1p, and Mph1p, a protein with an RNA helicase domain which is known to influence the mutation rate of nuclear and mitochondrial genomes.
Mitochondrial Recombination Reveals Mito-Mito Epistasis in Yeast.
Wolters, John F; Charron, Guillaume; Gaspary, Alec; Landry, Christian R; Fiumera, Anthony C; Fiumera, Heather L
2018-05-01
Genetic variation in mitochondrial DNA (mtDNA) provides adaptive potential although the underlying genetic architecture of fitness components within mtDNAs is not known. To dissect functional variation within mtDNAs, we first identified naturally occurring mtDNAs that conferred high or low fitness in Saccharomyces cerevisiae by comparing growth in strains containing identical nuclear genotypes but different mtDNAs. During respiratory growth under temperature and oxidative stress conditions, mitotype effects were largely independent of nuclear genotypes even in the presence of mito-nuclear interactions. Recombinant mtDNAs were generated to determine fitness components within high- and low-fitness mtDNAs. Based on phenotypic distributions of isogenic strains containing recombinant mtDNAs, we found that multiple loci contributed to mitotype fitness differences. These mitochondrial loci interacted in epistatic, nonadditive ways in certain environmental conditions. Mito-mito epistasis ( i.e. , nonadditive interactions between mitochondrial loci) influenced fitness in progeny from four different crosses, suggesting that mito-mito epistasis is a widespread phenomenon in yeast and other systems with recombining mtDNAs. Furthermore, we found that interruption of coadapted mito-mito interactions produced recombinant mtDNAs with lower fitness. Our results demonstrate that mito-mito epistasis results in functional variation through mitochondrial recombination in fungi, providing modes for adaptive evolution and the generation of mito-mito incompatibilities. Copyright © 2018 by the Genetics Society of America.
Mitochondrial-morphology-targeted breeding of industrial yeast strains for alcohol fermentation.
Kitagaki, Hiroshi
2009-05-29
Since mitochondrial genes are repressed under high glucose and low O2, and these conditions correspond to the conditions in which yeast cells are exposed during alcohol fermentation, the existence and structure of yeast mitochondria during alcohol fermentation have not been elucidated. Yeast mitochondria can be observed throughout brewing of sake (Japanese rice wine) and fragment during brewing. Furthermore, it has been revealed that Fis1 [fission 1 (mitochondrial outer membrane) homologue (Saccharomyces cerevisiae)], which is a transmembrane protein with its C-terminal anchor embedded in the outer membrane of mitochondria, is required for fragmentation of yeast mitochondria during sake brewing. By utilizing this knowledge, a fis1 disruptant of a sake yeast strain has been generated that has a networked mitochondrial structure throughout sake brewing. It transpired that this strain produces a high content of malate, which imparts a crisp acidic taste, during sake brewing. This strategy is a useful and a completely novel strategy towards developing a new yeast strain which produces a high content of malate in sake, and mitochondrial morphology has now emerged as a promising target for the breeding of practical industrial strains.
NASA Technical Reports Server (NTRS)
Jahnke, L.; Klein, H. P.
1983-01-01
The effect of oxygen on squalene epoxidase activity in Saccharomyces cerevisiae was investigated. In cells grown in standing cultures, the epoxidase was localized mainly in the 'mitochondrial' fraction. Upon aeration, enzyme activity increased and the newly formed enzyme was associated with the 'microsomal' fraction. At 0.03 percent (vol/vol) oxygen, epoxidase levels doubled, whereas the ergosterol level was only slightly increased. Cycloheximide inhibited the increase in epoxidase under these conditions. An apparent K sub m for oxygen of 0.38 percent (vol/vol) was determined from a crude particulate preparation for the epoxidase.
Role of the putative structural protein Sed1p in mitochondrial genome maintenance.
Phadnis, Naina; Ayres Sia, Elaine
2004-09-24
The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae. A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified. Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p. Sed1p is a cell surface protein highly expressed in the stationary phase. We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro. Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration. In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability. Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria. Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K. Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p. Our results point towards a role for Sed1p in mitochondrial genome maintenance.
Charizanis, C; Juhnke, H; Krems, B; Entian, K D
1999-10-01
In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.
Flor yeasts of Saccharomyces cerevisiae--their ecology, genetics and metabolism.
Alexandre, Hervé
2013-10-15
The aging of certain white wines is dependent on the presence of yeast strains that develop a biofilm on the wine surface after the alcoholic fermentation. These strains belong to the genus Saccharomyces and are called flor yeasts. These strains possess distinctive characteristics compared with Saccharomyces cerevisiae fermenting strain. The most important one is their capacity to form a biofilm on the air-liquid interface of the wine. The major gene involved in this phenotype is FLO11, however other genes are also involved in velum formation by these yeast and will be detailed. Other striking features presented in this review are their aneuploidy, and their mitochondrial DNA polymorphism which seems to reflect adaptive evolution of the yeast to a stressful environment where acetaldehyde and ethanol are present at elevated concentration. The biofilm assures access to oxygen and therefore permits continued growth on non-fermentable ethanol. This specific metabolism explains the peculiar organoleptic profile of these wines, especially their content in acetaldehyde and sotolon. This review deals with these different specificities of flor yeasts and will also underline the existing gaps regarding these astonishing yeasts. © 2013.
Callegari, Sylvie; McKinnon, Ross A; Andrews, Stuart; de Barros Lopes, Miguel A
2011-04-20
The Saccharomyces cerevisiae MEF2 gene is a mitochondrial protein translation factor. Formerly believed to catalyze peptide elongation, evidence now suggests its involvement in ribosome recycling. This study confirms the role of the MEF2 gene for cell respiration and further uncovers a slow growth phenotype and reduced chronological lifespan. Furthermore, in comparison with cytoplasmic ρ(0) strains, mef2Δ strains have a marked reduction of the inner mitochondrial membrane potential and mitochondria show a tendency to aggregate, suggesting an additional role for the MEF2 gene in maintenance of mitochondrial health, a role that may also be shared by other mitochondrial protein synthesis factors. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
O'Rourke, Thomas W; Doudican, Nicole A; Mackereth, Melinda D; Doetsch, Paul W; Shadel, Gerald S
2002-06-01
The mitochondrial genome is a significant target of exogenous and endogenous genotoxic agents; however, the determinants that govern this susceptibility and the pathways available to resist mitochondrial DNA (mtDNA) damage are not well characterized. Here we report that oxidative mtDNA damage is elevated in strains lacking Ntg1p, providing the first direct functional evidence that this mitochondrion-localized, base excision repair enzyme functions to protect mtDNA. However, ntg1 null strains did not exhibit a mitochondrial respiration-deficient (petite) phenotype, suggesting that mtDNA damage is negotiated by the cooperative actions of multiple damage resistance pathways. Null mutations in ABF2 or PIF1, two genes implicated in mtDNA maintenance and recombination, exhibit a synthetic-petite phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strains. This phenotype was partially rescued by malonic acid, indicating that reactive oxygen species generated by the electron transport chain contribute to mitochondrial dysfunction in abf2 Delta strains. In contrast, when two other genes involved in mtDNA recombination, CCE1 and NUC1, were inactivated a strong synthetic-petite phenotype was not observed, suggesting that the effects mediated by Abf2p and Pif1p are due to novel activities of these proteins other than recombination. These results document the existence of recombination-independent mechanisms in addition to base excision repair to cope with oxidative mtDNA damage in Saccharomyces cerevisiae. Such systems are likely relevant to those operating in human cells where mtDNA recombination is less prevalent, validating yeast as a model system in which to study these important issues.
Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya
2011-01-01
To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634
Kaniak, Aneta; Dzierzbicki, Piotr; Rogowska, Agata T; Malc, Ewa; Fikus, Marta; Ciesla, Zygmunt
2009-03-01
The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli(r) mutants in an ogg1Delta strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli(r) mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8(m) recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial
Demuyser, Liesbeth; Swinnen, Erwin; Fiori, Alessandro; Herrera-Malaver, Beatriz; Vestrepen, Kevin; Van Dijck, Patrick
2017-07-18
MGE1 encodes a yeast chaperone involved in Fe-S cluster metabolism and protein import into the mitochondria. In this study, we identified MGE1 as a multicopy suppressor of susceptibility to the antifungal fluconazole in the model yeast Saccharomyces cerevisiae We demonstrate that this phenomenon is not exclusively dependent on the integrity of the mitochondrial DNA or on the presence of the drug efflux pump Pdr5. Instead, we show that the increased dosage of Mge1 plays a protective role by retaining increased amounts of ergosterol upon fluconazole treatment. Iron metabolism and, more particularly, Fe-S cluster formation are involved in regulating this process, since the responsible Hsp70 chaperone, Ssq1, is required. Additionally, we show the necessity but, by itself, insufficiency of activating the iron regulon in establishing the Mge1-related effect on drug susceptibility. Finally, we confirm a similar role for Mge1 in fluconazole susceptibility in the pathogenic fungi Candida glabrata and Candida albicans IMPORTANCE Although they are mostly neglected compared to bacterial infections, fungal infections pose a serious threat to the human population. While some of them remain relatively harmless, infections that reach the bloodstream often become lethal. Only a few therapies are available, and resistance of the pathogen to these drugs is a frequently encountered problem. It is thus essential that more research is performed on how these pathogens cope with the treatment and cause recurrent infections. Baker's yeast is often used as a model to study pathogenic fungi. We show here, by using this model, that iron metabolism and the formation of the important iron-sulfur clusters are involved in regulating susceptibility to fluconazole, the most commonly used antifungal drug. We show that the same process likely also occurs in two of the most regularly isolated pathogenic fungi, Candida glabrata and Candida albicans . Copyright © 2017 Demuyser et al.
A nuclear mutation defective in mitochondrial recombination in yeast.
Ling, F; Makishima, F; Morishima, N; Shibata, T
1995-08-15
Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.
Dequard-Chablat, Michelle; Allandt, Cynthia
2002-08-01
In the filamentous fungus Podospora anserina, two degenerative processes which result in growth arrest are associated with mitochondrial genome (mitochondrial DNA [mtDNA]) instability. Senescence is correlated with mtDNA rearrangements and amplification of specific regions (senDNAs). Premature death syndrome is characterized by the accumulation of specific mtDNA deletions. This accumulation is due to indirect effects of the AS1-4 mutation, which alters a cytosolic ribosomal protein gene. The mthmg1 gene has been identified as a double-copy suppressor of premature death. It greatly delays premature death and the accumulation of deletions when it is present in two copies in an ASI-4 context. The duplication of mthmg1 has no significant effect on the wild-type life span or on senDNA patterns. In anAS1+ context, deletion of the mthmg1 gene alters germination, growth, and fertility and reduces the life span. The deltamthmg1 senescent strains display a particular senDNA pattern. This deletion is lethal in an AS1-4 context. According to its physical properties (very basic protein with putative mitochondrial targeting sequence and HMG-type DNA-binding domains) and the cellular localization of an mtHMG1-green fluorescent protein fusion, mtHMG1 appears to be a mitochondrial protein possibly associated with mtDNA. It is noteworthy that it is the first example of a protein combining the two DNA-binding domains, AT-hook motif and HMG-1 boxes. It may be involved in the stability and/or transmission of the mitochondrial genome. To date, no structural homologues have been found in other organisms. However, mtHMG1 displays functional similarities with the Saccharomyces cerevisiae mitochondrial HMG-box protein Abf2.
Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae
2017-01-01
Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell. PMID:28221768
Xu, Chunling; Wang, Jiafeng; Gao, Ye; Lin, Huangyu; Du, Lin; Yang, Shanshan; Long, Simei; She, Zhigang; Cai, Xiaoling; Zhou, Shining; Lu, Yongjun
2010-05-01
Bostrycin is an anthracenedione with phytotoxic and antibacterial activity that belongs to the large family of quinones. We have isolated bostrycin from the secondary metabolites of a mangrove endophytic fungus, no. 1403, collected from the South China Sea. Using the yeast Saccharomyces cerevisiae as a model, we show that bostrycin inhibits cell proliferation by blocking the cell cycle at G1 phase and ultimately leads to cell death in a time- and dose-dependent manner. Bostrycin-induced lethal cytotoxicity is accompanied with increased levels of intracellular reactive oxygen species and hallmarks of apoptosis such as chromatin condensation, DNA fragmentation and externalization of phosphatidylserine. We further show that bostrycin decreases mitochondrial membrane electric potential and causes mitochondrial destruction during the progression of cell death. Bostrycin-induced cell death was promoted in YCA1 null yeast strain but was partially rescued in AIF1 null mutant both in fermentative and respiratory media, strongly indicating that bostrycin induces apoptosis in yeast cells through a mitochondria-mediated but caspase-independent pathway.
Rea, Shane L.; Graham, Brett H.; Nakamaru-Ogiso, Eiko; Kar, Adwitiya; Falk, Marni J.
2013-01-01
The extensive conservation of mitochondrial structure, composition, and function across evolution offers a unique opportunity to expand our understanding of human mitochondrial biology and disease. By investigating the biology of much simpler model organisms, it is often possible to answer questions that are unreachable at the clinical level. Here, we review the relative utility of four different model organisms, namely the bacteria Escherichia coli, the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, in studying the role of mitochondrial proteins relevant to human disease. E. coli are single cell, prokaryotic bacteria that have proven to be a useful model system in which to investigate mitochondrial respiratory chain protein structure and function. S. cerevisiae is a single-celled eukaryote that can grow equally well by mitochondrial-dependent respiration or by ethanol fermentation, a property that has proven to be a veritable boon for investigating mitochondrial functionality. C. elegans is a multi-cellular, microscopic worm that is organized into five major tissues and has proven to be a robust model animal for in vitro and in vivo studies of primary respiratory chain dysfunction and its potential therapies in humans. Studied for over a century, D. melanogaster is a classic metazoan model system offering an abundance of genetic tools and reagents that facilitates investigations of mitochondrial biology using both forward and reverse genetics. The respective strengths and limitations of each species relative to mitochondrial studies are explored. In addition, an overview is provided of major discoveries made in mitochondrial biology in each of these four model systems. PMID:20818735
Baron, J. Allen; Laws, Kaitlin M.; Chen, Janice S.; Culotta, Valeria C.
2013-01-01
Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this “acid burst” were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress. PMID:23281478
Smith, Christopher P; Thorsness, Peter E
2008-07-01
AAC2 is one of three paralogs encoding mitochondrial ADP/ATP carriers in the yeast Saccharomyces cerevisiae, and because it is required for respiratory growth it has been the most extensively studied. To comparatively examine the relative functionality of Aac1, Aac2, and Aac3 in vivo, the gene encoding each isoform was expressed from the native AAC2 locus in aac1Delta aac3Delta yeast. Compared to Aac2, Aac1 exhibited reduced capacity to support growth of yeast lacking mitochondrial DNA or of yeast lacking the ATP/Mg-P(i) carrier, both conditions requiring ATP import into the mitochondrial matrix through the ADP/ATP carrier. Sixteen AAC1/AAC2 chimeric genes were constructed and analyzed to determine the key differences between residues or sections of Aac1 and Aac2. On the basis of the growth rate differences of yeast expressing different chimeras, the C1 and M2 loops of the ADP/ATP carriers contain divergent residues that are responsible for the difference(s) between Aac1 and Aac2. One chimeric gene construct supported growth on nonfermentable carbon sources but failed to support growth of yeast lacking mitochondrial DNA. We identified nine independent intragenic mutations in this chimeric gene that suppressed the growth phenotype of yeast lacking mitochondrial DNA, identifying regions of the carrier important for nucleotide exchange activities.
Marullo, Rossella; Werner, Erica; Degtyareva, Natalya; Moore, Bryn; Altavilla, Giuseppe; Ramalingam, Suresh S.; Doetsch, Paul W.
2013-01-01
Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy. PMID:24260552
Guo, Xiao; Niemi, Natalie M; Coon, Joshua J; Pagliarini, David J
2017-07-14
The pyruvate dehydrogenase complex (PDC) is the primary metabolic checkpoint connecting glycolysis and mitochondrial oxidative phosphorylation and is important for maintaining cellular and organismal glucose homeostasis. Phosphorylation of the PDC E1 subunit was identified as a key inhibitory modification in bovine tissue ∼50 years ago, and this regulatory process is now known to be conserved throughout evolution. Although Saccharomyces cerevisiae is a pervasive model organism for investigating cellular metabolism and its regulation by signaling processes, the phosphatase(s) responsible for activating the PDC in S. cerevisiae has not been conclusively defined. Here, using comparative mitochondrial phosphoproteomics, analyses of protein-protein interactions by affinity enrichment-mass spectrometry, and in vitro biochemistry, we define Ptc6p as the primary PDC phosphatase in S. cerevisiae Our analyses further suggest additional substrates for related S. cerevisiae phosphatases and describe the overall phosphoproteomic changes that accompany mitochondrial respiratory dysfunction. In summary, our quantitative proteomics and biochemical analyses have identified Ptc6p as the primary-and likely sole- S. cerevisiae PDC phosphatase, closing a key knowledge gap about the regulation of yeast mitochondrial metabolism. Our findings highlight the power of integrative omics and biochemical analyses for annotating the functions of poorly characterized signaling proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
New features of mitochondrial DNA replication system in yeast and man.
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.
MitBASE : a comprehensive and integrated mitochondrial DNA database. The present status
Attimonelli, M.; Altamura, N.; Benne, R.; Brennicke, A.; Cooper, J. M.; D’Elia, D.; Montalvo, A. de; Pinto, B. de; De Robertis, M.; Golik, P.; Knoop, V.; Lanave, C.; Lazowska, J.; Licciulli, F.; Malladi, B. S.; Memeo, F.; Monnerot, M.; Pasimeni, R.; Pilbout, S.; Schapira, A. H. V.; Sloof, P.; Saccone, C.
2000-01-01
MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc. The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE. Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities. The database is resident at the EBI and is available at the following site: http://www3.ebi.ac.uk/Research/Mitbase/mitbase.pl . The impact of this project is intended for both basic and applied research. The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields. The database has been funded within the EU Biotechnology programme. PMID:10592207
Lipinski, Kamil A; Kaniak-Golik, Aneta; Golik, Pawel
2010-01-01
As a legacy of their endosymbiotic eubacterial origin, mitochondria possess a residual genome, encoding only a few proteins and dependent on a variety of factors encoded by the nuclear genome for its maintenance and expression. As a facultative anaerobe with well understood genetics and molecular biology, Saccharomyces cerevisiae is the model system of choice for studying nucleo-mitochondrial genetic interactions. Maintenance of the mitochondrial genome is controlled by a set of nuclear-coded factors forming intricately interconnected circuits responsible for replication, recombination, repair and transmission to buds. Expression of the yeast mitochondrial genome is regulated mostly at the post-transcriptional level, and involves many general and gene-specific factors regulating splicing, RNA processing and stability and translation. A very interesting aspect of the yeast mitochondrial system is the relationship between genome maintenance and gene expression. Deletions of genes involved in many different aspects of mitochondrial gene expression, notably translation, result in an irreversible loss of functional mtDNA. The mitochondrial genetic system viewed from the systems biology perspective is therefore very fragile and lacks robustness compared to the remaining systems of the cell. This lack of robustness could be a legacy of the reductive evolution of the mitochondrial genome, but explanations involving selective advantages of increased evolvability have also been postulated. Copyright © 2009 Elsevier B.V. All rights reserved.
Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D; Krejci, Lumir; Tomaska, Lubomir
2015-12-08
Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. © 2016 Authors.
Stribny, Jiri; Gamero, Amparo; Pérez-Torrado, Roberto; Querol, Amparo
2015-07-16
Higher alcohols and acetate esters are important flavour and aroma components in the food industry. In alcoholic beverages these compounds are produced by yeast during fermentation. Although Saccharomyces cerevisiae is one of the most extensively used species, other species of the Saccharomyces genus have become common in fermentation processes. This study analyses and compares the production of higher alcohols and acetate esters from their amino acidic precursors in three Saccharomyces species: Saccharomyces kudriavzevii, Saccharomyces uvarum and S. cerevisiae. The global volatile compound analysis revealed that S. kudriavzevii produced large amounts of higher alcohols, whereas S. uvarum excelled in the production of acetate esters. Particularly from phenylalanine, S. uvarum produced the largest amounts of 2-phenylethyl acetate, while S. kudriavzevii obtained the greatest 2-phenylethanol formation from this precursor. The present data indicate differences in the amino acid metabolism and subsequent production of flavour-active higher alcohols and acetate esters among the closely related Saccharomyces species. This knowledge will prove useful for developing new enhanced processes in fragrance, flavour, and food industries. Copyright © 2015. Published by Elsevier B.V.
Mitochondrial Dynamics in Mitochondrial Diseases
Suárez-Rivero, Juan M.; Villanueva-Paz, Marina; de la Cruz-Ojeda, Patricia; de la Mata, Mario; Cotán, David; Oropesa-Ávila, Manuel; de Lavera, Isabel; Álvarez-Córdoba, Mónica; Luzón-Hidalgo, Raquel; Sánchez-Alcázar, José A.
2016-01-01
Mitochondria are very versatile organelles in continuous fusion and fission processes in response to various cellular signals. Mitochondrial dynamics, including mitochondrial fission/fusion, movements and turnover, are essential for the mitochondrial network quality control. Alterations in mitochondrial dynamics can cause neuropathies such as Charcot-Marie-Tooth disease in which mitochondrial fusion and transport are impaired, or dominant optic atrophy which is caused by a reduced mitochondrial fusion. On the other hand, mitochondrial dysfunction in primary mitochondrial diseases promotes reactive oxygen species production that impairs its own function and dynamics, causing a continuous vicious cycle that aggravates the pathological phenotype. Mitochondrial dynamics provides a new way to understand the pathophysiology of mitochondrial disorders and other diseases related to mitochondria dysfunction such as diabetes, heart failure, or Hungtinton’s disease. The knowledge about mitochondrial dynamics also offers new therapeutics targets in mitochondrial diseases. PMID:28933354
Mechanisms of iron sensing and regulation in the yeast Saccharomyces cerevisiae.
Martínez-Pastor, María Teresa; Perea-García, Ana; Puig, Sergi
2017-04-01
Iron is a redox active element that functions as an essential cofactor in multiple metabolic pathways, including respiration, DNA synthesis and translation. While indispensable for eukaryotic life, excess iron can lead to oxidative damage of macromolecules. Therefore, living organisms have developed sophisticated strategies to optimally regulate iron acquisition, storage and utilization in response to fluctuations in environmental iron bioavailability. In the yeast Saccharomyces cerevisiae, transcription factors Aft1/Aft2 and Yap5 regulate iron metabolism in response to low and high iron levels, respectively. In addition to producing and assembling iron cofactors, mitochondrial iron-sulfur (Fe/S) cluster biogenesis has emerged as a central player in iron sensing. A mitochondrial signal derived from Fe/S synthesis is exported and converted into an Fe/S cluster that interacts directly with Aft1/Aft2 and Yap5 proteins to regulate their transcriptional function. Various conserved proteins, such as ABC mitochondrial transporter Atm1 and, for Aft1/Aft2, monothiol glutaredoxins Grx3 and Grx4 are implicated in this iron-signaling pathway. The analysis of a wide range of S. cerevisiae strains of different geographical origins and sources has shown that yeast strains adapted to high iron display growth defects under iron-deficient conditions, and highlighted connections that exist in the response to both opposite conditions. Changes in iron accumulation and gene expression profiles suggest differences in the regulation of iron homeostasis genes.
The genetic architecture of low-temperature adaptation in the wine yeast Saccharomyces cerevisiae.
García-Ríos, Estéfani; Morard, Miguel; Parts, Leopold; Liti, Gianni; Guillamón, José M
2017-02-14
Low-temperature growth and fermentation of wine yeast can enhance wine aroma and make them highly desirable traits for the industry. Elucidating response to cold in Saccharomyces cerevisiae is, therefore, of paramount importance to select or genetically improve new wine strains. As most enological traits of industrial importance in yeasts, adaptation to low temperature is a polygenic trait regulated by many interacting loci. In order to unravel the genetic determinants of low-temperature fermentation, we mapped quantitative trait loci (QTLs) by bulk segregant analyses in the F13 offspring of two Saccharomyces cerevisiae industrial strains with divergent performance at low temperature. We detected four genomic regions involved in the adaptation at low temperature, three of them located in the subtelomeric regions (chromosomes XIII, XV and XVI) and one in the chromosome XIV. The QTL analysis revealed that subtelomeric regions play a key role in defining individual variation, which emphasizes the importance of these regions' adaptive nature. The reciprocal hemizygosity analysis (RHA), run to validate the genes involved in low-temperature fermentation, showed that genetic variation in mitochondrial proteins, maintenance of correct asymmetry and distribution of phospholipid in the plasma membrane are key determinants of low-temperature adaptation.
Crosstalk between mitochondrial stress signals regulates yeast chronological lifespan.
Schroeder, Elizabeth A; Shadel, Gerald S
2014-01-01
Mitochondrial DNA (mtDNA) exists in multiple copies per cell and is essential for oxidative phosphorylation. Depleted or mutated mtDNA promotes numerous human diseases and may contribute to aging. Reduced TORC1 signaling in the budding yeast, Saccharomyces cerevisiae, extends chronological lifespan (CLS) in part by generating a mitochondrial ROS (mtROS) signal that epigenetically alters nuclear gene expression. To address the potential requirement for mtDNA maintenance in this response, we analyzed strains lacking the mitochondrial base-excision repair enzyme Ntg1p. Extension of CLS by mtROS signaling and reduced TORC1 activity, but not caloric restriction, was abrogated in ntg1Δ strains that exhibited mtDNA depletion without defects in respiration. The DNA damage response (DDR) kinase Rad53p, which transduces pro-longevity mtROS signals, is also activated in ntg1Δ strains. Restoring mtDNA copy number alleviated Rad53p activation and re-established CLS extension following mtROS signaling, indicating that Rad53p senses mtDNA depletion directly. Finally, DDR kinases regulate nucleus-mitochondria localization dynamics of Ntg1p. From these results, we conclude that the DDR pathway senses and may regulate Ntg1p-dependent mtDNA stability. Furthermore, Rad53p senses multiple mitochondrial stresses in a hierarchical manner to elicit specific physiological outcomes, exemplified by mtDNA depletion overriding the ability of Rad53p to transduce an adaptive mtROS longevity signal. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
MPIC: a mitochondrial protein import components database for plant and non-plant species.
Murcha, Monika W; Narsai, Reena; Devenish, James; Kubiszewski-Jakubiak, Szymon; Whelan, James
2015-01-01
In the 2 billion years since the endosymbiotic event that gave rise to mitochondria, variations in mitochondrial protein import have evolved across different species. With the genomes of an increasing number of plant species sequenced, it is possible to gain novel insights into mitochondrial protein import pathways. We have generated the Mitochondrial Protein Import Components (MPIC) Database (DB; http://www.plantenergy.uwa.edu.au/applications/mpic) providing searchable information on the protein import apparatus of plant and non-plant mitochondria. An in silico analysis was carried out, comparing the mitochondrial protein import apparatus from 24 species representing various lineages from Saccharomyces cerevisiae (yeast) and algae to Homo sapiens (human) and higher plants, including Arabidopsis thaliana (Arabidopsis), Oryza sativa (rice) and other more recently sequenced plant species. Each of these species was extensively searched and manually assembled for analysis in the MPIC DB. The database presents an interactive diagram in a user-friendly manner, allowing users to select their import component of interest. The MPIC DB presents an extensive resource facilitating detailed investigation of the mitochondrial protein import machinery and allowing patterns of conservation and divergence to be recognized that would otherwise have been missed. To demonstrate the usefulness of the MPIC DB, we present a comparative analysis of the mitochondrial protein import machinery in plants and non-plant species, revealing plant-specific features that have evolved. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Mitochondrial Copper Metabolism and Delivery to Cytochrome c Oxidase
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
Murashchenko, Lidiia; Abbas, Charles; Dmytruk, Kostyantyn; Sibirny, Andriy
2016-08-01
Acetolactate synthase is a mitochondrial enzyme that catalyses the conversion of two pyruvate molecules to an acetolactate molecule with release of carbon dioxide. The overexpression of the truncated version of the corresponding gene, ILV2, that codes for presumably cytosolic acetolactate synthase in the yeast Saccharomyces cerevisiae, led to a decrease in intracellular pyruvate concentration. This recombinant strain was also characterized by a four-fold increase in glycerol production, with a concomitant 1.8-fold reduction in ethanol production, when compared to that of the wild-type strain under anaerobic conditions in a glucose alcoholic fermentation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Central roles of iron in the regulation of oxidative stress in the yeast Saccharomyces cerevisiae.
Matsuo, Ryo; Mizobuchi, Shogo; Nakashima, Maya; Miki, Kensuke; Ayusawa, Dai; Fujii, Michihiko
2017-10-01
Oxygen is essential for aerobic organisms but causes cytotoxicity probably through the generation of reactive oxygen species (ROS). In this study, we screened for the genes that regulate oxidative stress in the yeast Saccharomyces cerevisiae, and found that expression of CTH2/TIS11 caused an increased resistance to ROS. CTH2 is up-regulated upon iron starvation and functions to remodel metabolism to adapt to iron starvation. We showed here that increased resistance to ROS by CTH2 would likely be caused by the decreased ROS production due to the decreased activity of mitochondrial respiration, which observation is consistent with the fact that CTH2 down-regulates the mitochondrial respiratory proteins. We also found that expression of CTH1, a paralog of CTH2, also caused an increased resistance to ROS. This finding supported the above view, because mitochondrial respiratory proteins are the common targets of CTH1 and CTH2. We further showed that supplementation of iron in medium augmented the growth of S. cerevisiae under oxidative stress, and expression of CTH2 and supplementation of iron collectively enhanced its growth under oxidative stress. Since CTH2 is regulated by iron, these findings suggested that iron played crucial roles in the regulation of oxidative stress in S. cerevisiae.
García-Ríos, Estéfani; Querol, Amparo; Guillamón, José Manuel
2016-09-02
Temperature is one of the most important parameters to affect the duration and rate of alcoholic fermentation and final wine quality. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae, which was the case of cryotolerant yeasts Saccharomyces uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the proteomic landscape of these cryotolerant species grown at 12°C and 28°C, which we compared with the proteome of S. cerevisiae (poorly adapted at low temperature). Our results showed that the main differences among the proteomic profiling of the three Saccharomyces strains grown at 12°C and 28°C lay in translation, glycolysis and amino acid metabolism. Our data corroborate previous transcriptomic results, which suggest that S. kudriavzevii is better adapted to grow at low temperature as a result of enhanced more efficient translation. Fitter amino acid biosynthetic pathways can also be mechanisms that better explain biomass yield in cryotolerant strains. Yet even at low temperature, S. cerevisiae is the most fermentative competitive species. A higher concentration of glycolytic and alcoholic fermentation enzymes in the S. cerevisiae strain might explain such greater fermentation activity. Temperature is one of the main relevant environmental variables that microorganisms have to cope with and it is also a key factor in some industrial processes that involve microorganisms. However, we are still far from understanding the molecular and physiological mechanisms of adaptation at low temperatures. The results obtained in this study provided a global atlas of the proteome changes triggered by temperature in three different species of the genus Saccharomyces with different degree of cryotolerance. These results would facilitate a better understanding of mechanisms for how yeast could adapt at the low temperature of growth. Copyright © 2016
Tzagoloff, A; Shtanko, A
1995-06-01
Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.
Yor022c protein is a phospholipase A{sub 1} that localizes to the mitochondrial matrix
DOE Office of Scientific and Technical Information (OSTI.GOV)
Urafuji, Kyosei; Arioka, Manabu
In mammals, three types of intracellular phospholipase A{sub 1} (iPLA{sub 1}) enzymes have been characterized and are thought to be involved in various cellular processes such as phospholipid metabolism, organelle biogenesis, and membrane trafficking. In this study we analyzed the unique iPLA{sub 1}-like protein, Yor022c, in the budding yeast Saccharomyces cerevisiae. By the mass spectrometry analysis, we demonstrate that Yor022c is actually a phospholipase displaying sn-1-specific activity toward phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid, generating 2-acyl lysophospholipids. GFP-fused Yor022c co-stained with the mitochondrial dye MitoTracker, indicating that, unlike its mammalian counterparts, it is a mitochondrial protein. Further biochemical fractionation experiment combinedmore » with protease sensitivity assay showed that Yor022c localizes to the mitochondrial matrix. Thus Yor022c is the first PLA{sub 1} putatively involved in the maintenance of sn-1 acyl chains of phospholipids in the mitochondrial inner membrane. - Highlights: • Yeast Yor022c protein displays phospholipase A{sub 1} activity to various phospholipids. • Yor022c-GFP fusion protein localizes to mitochondria. • Biochemical fractionation showed that Yor022c localizes to the mitochondrial matrix.« less
Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.
Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H
2017-03-01
Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.
Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate.
Palmieri, L; Vozza, A; Agrimi, G; De Marco, V; Runswick, M J; Palmieri, F; Walker, J E
1999-08-06
Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase.
Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio
2015-11-01
During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.
Mookerjee, Shona A; Lyon, Hiram D; Sia, Elaine A
2005-02-01
Mitochondrial DNA (mtDNA) repair occurs in all eukaryotic organisms and is essential for the maintenance of mitochondrial function. Evidence from both humans and yeast suggests that mismatch repair is one of the pathways that functions in overall mtDNA stability. In the mitochondria of the yeast Saccharomyces cerevisiae, the presence of a homologue to the bacterial MutS mismatch repair protein, MSH1, has long been known to be essential for mitochondrial function. The mechanisms for which it is essential are unclear, however. Here, we analyze the effects of two point mutations, msh1-F105A and msh1-G776D, both predicted to be defective in mismatch repair; and we show that they are both able to maintain partial mitochondrial function. Moreover, there are significant differences in the severity of mitochondrial disruption between the two mutants that suggest multiple roles for Msh1p in addition to mismatch repair. Our overall findings suggest that these additional predicted functions of Msh1p, including recombination surveillance and heteroduplex rejection, may be primarily responsible for its essential role in mtDNA stability.
The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome.
Pevala, Vladimír; Truban, Dominika; Bauer, Jacob A; Košťan, Július; Kunová, Nina; Bellová, Jana; Brandstetter, Marlene; Marini, Victoria; Krejčí, Lumír; Tomáška, Ľubomír; Nosek, Jozef; Kutejová, Eva
2016-03-18
To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101-1(ts) mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome
Pevala, Vladimír; Truban, Dominika; Bauer, Jacob A.; Košťan, Július; Kunová, Nina; Bellová, Jana; Brandstetter, Marlene; Marini, Victoria; Krejčí, Lumír; Tomáška, Ľubomír; Nosek, Jozef; Kutejová, Eva
2016-01-01
To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101–1ts mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres. PMID:26743001
Cao, Juxiang; Barbosa, Jose M; Singh, Narendra; Locy, Robert D
2013-07-01
GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.
Klöppel, Christine; Michels, Christine; Zimmer, Julia; Herrmann, Johannes M; Riemer, Jan
2010-12-03
The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1(G93A) becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1(G85R). Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS. Copyright © 2010 Elsevier Inc. All rights reserved.
Tigano, Marco; Ruotolo, Roberta; Dallabona, Cristina; Fontanesi, Flavia; Barrientos, Antoni; Donnini, Claudia; Ottonello, Simone
2015-01-01
To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNALysUUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease. PMID:26240381
Lleixà, Jessica; Manzano, Maria; Mas, Albert; Portillo, María del C.
2016-01-01
The inoculation of wines with autochthonous yeast allows obtaining complex wines with a peculiar microbial footprint characteristic from a wine region. Mixed inoculation of non-Saccharomyces yeasts and S. cerevisiae is of interest for the wine industry for technological and sensory reasons. However, the interactions between these yeasts are not well understood, especially those regarding the availability of nutrients. The aim of the present study was to analyze the effect of nitrogen and sugar concentration on the evolution of mixed yeast populations on controlled laboratory-scale fermentations monitored by density, plate culturing, PCR-DGGE and sugar and nitrogen consumption. Furthermore, the effect of the time of inoculation of Saccharomyces cerevisiae respect the initial co-inoculation of three non-Saccharomyces yeasts was evaluated over the evolution of fermentation. Our results have shown that S. cerevisiae inoculation during the first 48 h conferred a stabilizing effect over the fermentations with non-Saccharomyces strains tested and, generally, reduced yeast diversity at the end of the fermentation. On the other hand, nitrogen limitation increased the time of fermentation and also the proportion of non-Saccharomyces yeasts at mid and final fermentation. High sugar concentration resulted in different proportions of the inoculated yeast depending on the time of S. cerevisiae inoculation. This work emphasizes the importance of the concentration of nutrients on the evolution of mixed fermentations and points to the optimal conditions for a stable fermentation in which the inoculated yeasts survived until the end. PMID:27994585
Samoilova, E O; Krasheninnikov, I A; Levitskii, S A
2016-10-01
Mitochondrial DNA is susceptible to the action of reactive oxygen species generated by the reactions of oxidative phosphorylation. Homologous recombination is one of the mechanisms providing integrity of the mitochondrial genome. Some proteins that take part in this process in budding yeast mitochondria have been identified. These include Abf2p, the major protein of the mt-nucleoid that specifically binds cruciform DNA, and Cce1p - Holliday junction resolvase. Here we show that Abf2p does not significantly affect either binding of Cce1p to branched DNA or rate and specificity of Holliday junction resolution. These data suggest the existence of an alternative homologous recombination pathway in yeast mitochondria.
Extension of chronological lifespan by ScEcl1 depends on mitochondria in Saccharomyces cerevisiae.
Azuma, Kenko; Ohtsuka, Hokuto; Murakami, Hiroshi; Aiba, Hirofumi
2012-01-01
Ecl1, a product of the YGR146C gene in Saccharomyces cerevisiae, was identified as a factor involved in chronological lifespan. In this study we found evidence that the function of Ecl1 in the extension of chronological lifespan is dependent on mitochondrial function. The respiratory activity of cells increased when Ecl1 was overexpressed or cells were grown under calorie restriction, but there was no additive effect of calorie restriction and Ecl1 overexpression on increases in respiratory activity or on the extension of chronological lifespan. Based on these results, we propose that overexpression of Ecl1 has same effect as caloric restriction and that its function also depends on mitochondria, just like caloric restriction.
Cells with impaired mitochondrial H2O2 sensing generate less •OH radicals and live longer.
Martins, Dorival; Titorenko, Vladimir I; English, Ann M
2014-10-01
Mitochondria are major sites of reactive oxygen species (ROS) generation, and adaptive mitochondrial ROS signaling extends longevity. We aim at linking the genetic manipulation of mitochondrial H2O2 sensing in live cells to mechanisms driving aging in the model organism, Saccharomyces cerevisiae. To this end, we compare in vivo ROS (O2(•-), H2O2 and (•)OH) accumulation, antioxidant enzyme activities, labile iron levels, GSH depletion, and protein oxidative damage during the chronological aging of three yeast strains: ccp1Δ that does not produce the mitochondrial H2O2 sensor protein, cytochrome c peroxidase (Ccp1); ccp1(W191F) that produces a hyperactive variant of this sensor protein (Ccp1(W191F)); and the isogenic wild-type strain. Since they possess elevated manganese superoxide dismutase (Sod2) activity, young ccp1Δ cells accumulate low mitochondrial superoxide (O2(•-)) levels but high H2O2 levels. These cells exhibit stable aconitase activity and contain low amounts of labile iron and hydroxyl radicals ((•)OH). Furthermore, they undergo late glutathione (GSH) depletion, less mitochondrial protein oxidative damage and live longer than wild-type cells. In contrast, young ccp1(W191F) cells accumulate little H2O2, possess depressed Sod2 activity, enabling their O2(•-) level to spike and deactivate aconitase, which, ultimately, leads to greater mitochondrial oxidative damage, early GSH depletion, and a shorter lifespan than wild-type cells. Modulation of mitochondrial H2O2 sensing offers a novel interventional approach to alter mitochondrial H2O2 levels in live cells and probe the pro- versus anti-aging effects of ROS. The strength of mitochondrial H2O2 sensing modulates adaptive mitochondrial ROS signaling and, hence, lifespan.
Vasicova, Pavla; Lejskova, Renata; Malcova, Ivana; Hasek, Jiri
2015-11-01
Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Lejskova, Renata; Malcova, Ivana
2015-01-01
Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. PMID:26351139
Barnard, Emma; McFerran, Neil V; Trudgett, Alan; Nelson, John; Timson, David J
2008-05-01
An alternative method for monitoring protein-protein interactions in Saccharomyces cerevisiae has been developed. It relies on the ability of two fragments of enhanced green fluorescent protein (EGFP) to reassemble and fluoresce when fused to interacting proteins. Since this fluorescence can be detected in living cells, simultaneous detection and localisation of interacting pairs is possible. DNA sequences encoding N- and C-terminal EGFP fragments flanked by sequences from the genes of interest were transformed into S. cerevisiae JPY5 cells and homologous recombination into the genome verified by PCR. The system was evaluated by testing known interacting proteins: labelling of the phosphofructokinase subunits, Pfk1p and Pfk2p, with N- and C-terminal EGFP fragments, respectively, resulted in green fluorescence in the cytoplasm. The system works in other cellular compartments: labelling of Idh1p and Idh2p (mitochondrial matrix), Sdh3p and Sdh4p (mitochondrial membrane) and Pap2p and Mtr4p (nucleus) all resulted in fluorescence in the appropriate cellular compartment.
Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.
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).
Rayko, E; Goursot, R; Cherif-Zahar, B; Melis, R; Bernardi, G
1988-03-31
The mitochondrial genomes of progenies from 26 crosses between 17 cytoplasmic, spontaneous, suppressive, ori+ petite mutants of Saccharomyces cerevisiae have been studied by electrophoresis of restriction fragments. Only parental genomes (or occasionally, genomes derived from them by secondary excisions) were found in the progenies of the almost 500 diploids investigated; no evidence for illegitimate, site-specific mitochondrial recombination was detected. One of the parental genomes was always found to be predominate over the other one, although to different extents in different crosses. This predominance appears to be due to a higher replication efficiency, which is correlated with a greater density of ori sequences on the mitochondrial genome (and with a shorter repeat unit size of the latter). Exceptions to the 'repeat-unit-size rule' were found, however, even when the parental mitochondrial genomes carried the same ori sequence. This indicates that noncoding, intergenic sequences outside ori sequences also play a role in modulating replication efficiency. Since in different petites such sequences differ in primary structure, size, and position relative to ori sequences, this modulation is likely to take place through an indirect effect on DNA and nucleoid structure.
Shekhawat, Kirti; Bauer, Florian F; Setati, Mathabatha E
2017-03-01
The sequential or co-inoculation of grape must with non-Saccharomyces yeast species and Saccharomyces cerevisiae wine yeast strains has recently become a common practice in winemaking. The procedure intends to enhance unique aroma and flavor profiles of wine. The extent of the impact of non-Saccharomyces strains depends on their ability to produce biomass and to remain metabolically active for a sufficiently long period. However, mixed-culture wine fermentations tend to become rapidly dominated by S. cerevisiae, reducing or eliminating the non-Saccharomyces yeast contribution. For an efficient application of these yeasts, it is therefore essential to understand the environmental factors that modulate the population dynamics of such ecosystems. Several environmental parameters have been shown to influence population dynamics, but their specific effect remains largely uncharacterized. In this study, the population dynamics in co-fermentations of S. cerevisiae and three non-Saccharomyces yeast species: Torulaspora delbrueckii, Lachancea thermotolerans, and Metschnikowia pulcherrima, was investigated as a function of oxygen availability. In all cases, oxygen availability strongly influenced population dynamics, but clear species-dependent differences were observed. Our data show that L. thermotolerans required the least oxygen, followed by T. delbrueckii and M. pulcherrima. Distinct species-specific chemical volatile profiles correlated in all cases with increased persistence of non-Saccharomyces yeasts, in particular increases in some higher alcohols and medium chain fatty acids. The results highlight the role of oxygen in regulating the succession of yeasts during wine fermentations and suggests that more stringent aeration strategies would be necessary to support the persistence of non-Saccharomyces yeasts in real must fermentations.
... blisters. High cholesterol. Hives. Lactose intolerance. Lyme disease. Urinary tract infections (UTIs). Yeast infections. Other conditions. More evidence is needed to rate Saccharomyces boulardii for these uses.
Cartledge, T. G.; Lloyd, D.
1972-01-01
1. Homogenates were prepared from sphaeroplasts of aerobically grown glucose-de-repressed Saccharomyces carlsbergensis and the distributions of marker enzymes were investigated after differential centrifugation. Cytochrome c oxidase and cytochrome c were sedimented almost completely at 105g-min, and this fraction also contained 37% of the catalase, 27% of the acid p-nitrophenyl phosphatase, 53 and 54% respectively of the NADH– and NADPH–cytochrome c oxidoreductases. 2. Zonal centrifugation indicated complex density distributions of the sedimentable portions of these enzymes and of adenosine triphosphatases and suggested the presence of two mitochondrial populations, as well as a bimodal distribution of peroxisomes and heterogeneity of the acid p-nitrophenyl phosphatase-containing particles. 3. Several different adenosine triphosphatases were distinguished in a post-mitochondrial supernatant that contained no mitochondrial fragments; these enzymes varied in their sensitivities to oligomycin and ouabain and their distributions were different from those of pyrophosphatase, adenosine phosphatase and adenosine pyrophosphatase. 4. The distribution of NADPH–cytochrome c oxidoreductase demonstrated that it cannot be used in S. carlsbergensis as a specific marker enzyme for the microsomal fraction. Glucose 6-phosphatase, inosine pyrophosphatase, cytochrome P-450 and five other enzymes frequently assigned to microsomal fractions of mammalian origin were not detected in yeast under these growth conditions. ImagesPLATE 2PLATE 1 (cont.)PLATE 1PLATE 2 (cont.) PMID:4400904
Ultraviolet mutagenesis studies of [psi], a cytoplasmic determinant of Saccharomyces cerevisiae.
Tuite, M F; Cox, B S
1980-07-01
UV mutagenesis was used to probe the molecular nature of [psi], a nonmitochondrial cytoplasmic determinant of Saccharomyces cerevisiae involved in the control of nonsense suppression. The UV-induced mutation from [psi+] to [psi-] showed characteristics of forward nuclear gene mutation in terms of frequency, induction kinetics, occurrence of whole and sectored mutant clones and the effect of the stage in the growth cycle on mutation frequency. The involvement of pyrimidine dimers in the premutational lesion giving the [psi-] mutation was demonstrated by photoreactivation. UV-induced damage to the [psi] genetic determinant was shown to be repaired by nuclear-coded repair enzymes that are responsible for the repair of nuclear DNA damage. UV-induced damage to mitochondrial DNA appeared to be, at least partly, under the control of different repair processes. The evidence obtained suggests that the [psi] determinant is DNA.
Toxicity of nalidixic acid on candida albicans, Saccharomyces cerevisiae, and Kluyveromyces lactis.
Sobieski, R J; Brewer, A R
1976-03-01
The antibacterial drug nalidixic acid (Nal) can suppress the growth of Candida albicans at levels of the drug normally found in urine. Growth suppression increases as drug levels are increased, and Nal also causes a similar proportional inhibition of the synthesis of all cellular macromolecules. However, growth temperature (25 versus 37 C) and the divalent cations Mg(2+) and Mn(2+) can increase C. albicans resistance to Nal. Also, nitrogen depletion of Candida shows that Nal-treated and untreated cells exhibit no difference in leucine uptake during readaptation to nitrogen. In Nal-treated, nitrogen-starved cells, ribonucleic acid and deoxyribonucleic acid (DNA) biosynthesis are less affected than in unstarved Nal-treated cells, but of the two nucleic acids DNA synthesis is the most affected. Nal-resistant strains of C. albicans exhibit a slight toxicity for macromolecular synthesis. Nal treatment of a synchronized population of Saccharomyces cerevisiae results in an increase in the culture mean doubling time of, at most, 20%, but Nal causes the loss of synchronous cell division. With a synchronized population of Kluyveromyces lactis, Nal causes an increase in the mean doubling time of upwards of 300%, with synchrony of cell division being maintained. It is known that S. cerevisiae asynchronously synthesizes mitochondrial DNA during the cell cycle, whereas with K. lactis it is synchronous. Thus, with C. albicans Nal toxicity is dependent both on the dose and the physiological state of the cell. Furthermore, Nal inhibits growth of yeast with synchronous mitochondrial DNA synthesis more adversely than yeast with asynchronous mitochondrial DNA synthesis.
Zim17/Tim15 links mitochondrial iron-sulfur cluster biosynthesis to nuclear genome stability.
Díaz de la Loza, María Del Carmen; Gallardo, Mercedes; García-Rubio, María Luisa; Izquierdo, Alicia; Herrero, Enrique; Aguilera, Andrés; Wellinger, Ralf Erik
2011-08-01
Genomic instability is related to a wide-range of human diseases. Here, we show that mitochondrial iron-sulfur cluster biosynthesis is important for the maintenance of nuclear genome stability in Saccharomyces cerevisiae. Cells lacking the mitochondrial chaperone Zim17 (Tim15/Hep1), a component of the iron-sulfur biosynthesis machinery, have limited respiration activity, mimic the metabolic response to iron starvation and suffer a dramatic increase in nuclear genome recombination. Increased oxidative damage or deficient DNA repair do not account for the observed genomic hyperrecombination. Impaired cell-cycle progression and genetic interactions of ZIM17 with components of the RFC-like complex involved in mitotic checkpoints indicate that replicative stress causes hyperrecombination in zim17Δ mutants. Furthermore, nuclear accumulation of pre-ribosomal particles in zim17Δ mutants reinforces the importance of iron-sulfur clusters in normal ribosome biosynthesis. We propose that compromised ribosome biosynthesis and cell-cycle progression are interconnected, together contributing to replicative stress and nuclear genome instability in zim17Δ mutants.
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
Gayevskiy, Velimir; Goddard, Matthew R
2016-04-01
Saccharomyces is one of the best-studied microbial genera, but our understanding of the global distributions and evolutionary histories of its members is relatively poor. Recent studies have altered our view of Saccharomyces' origin, but a lack of sampling from the vast majority of the world precludes a holistic perspective. We evaluate alternate Gondwanan and Far East Asian hypotheses concerning the origin of these yeasts. Being part of Gondwana, and only colonized by humans in the last ∼1000 years, New Zealand represents a unique environment for testing these ideas. Genotyping and ribosomal sequencing of samples from North Island native forest parks identified a widespread population of Saccharomyces. Whole genome sequencing identified the presence of S. arboricola and S. eubayanus in New Zealand, which is the first report of S. arboricola outside Far East Asia, and also expands S. eubayanus' known distribution to include the Oceanic region. Phylogenomic approaches place the S. arboricola population as significantly diverged from the only other sequenced Chinese isolate but indicate that S. eubayanus might be a recent migrant from South America. These data tend to support the Far East Asian origin of the Saccharomyces, but the history of this group is still far from clear. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.
NASA Technical Reports Server (NTRS)
Jahnke, L.; Klein, H. P.
1979-01-01
A comparative study of the effects of varying levels of oxygen on some of the metabolic functions of the primitive eukaryote, Saccharomyces cerevisiae, has shown that these cells are responsive to very low levels of oxygen: the level of palmitoyl-Co A desaturase was greatly enhanced by only 0.03 vol % oxygen. Similarly, an acetyl-CoA synthetase associated predominantly with anaerobic growth was stimulated by as little as 0.1% oxygen, while an isoenzyme correlated with aerobic growth was maximally active at much higher oxygen levels (greater than 1%). Closely following this latter pattern were three mitochondrial enzymes that attained maximal activity only under atmospheric levels of oxygen.
Tigano, Marco; Ruotolo, Roberta; Dallabona, Cristina; Fontanesi, Flavia; Barrientos, Antoni; Donnini, Claudia; Ottonello, Simone
2015-09-30
To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA(Lys) UUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
The ecology and evolution of non-domesticated Saccharomyces species.
Boynton, Primrose J; Greig, Duncan
2014-12-01
Yeast researchers need model systems for ecology and evolution, but the model yeast Saccharomyces cerevisiae is not ideal because its evolution has been affected by domestication. Instead, ecologists and evolutionary biologists are focusing on close relatives of S. cerevisiae, the seven species in the genus Saccharomyces. The best-studied Saccharomyces yeast, after S. cerevisiae, is S. paradoxus, an oak tree resident throughout the northern hemisphere. In addition, several more members of the genus Saccharomyces have recently been discovered. Some Saccharomyces species are only found in nature, while others include both wild and domesticated strains. Comparisons between domesticated and wild yeasts have pinpointed hybridization, introgression and high phenotypic diversity as signatures of domestication. But studies of wild Saccharomyces natural history, biogeography and ecology are only beginning. Much remains to be understood about wild yeasts' ecological interactions and life cycles in nature. We encourage researchers to continue to investigate Saccharomyces yeasts in nature, both to place S. cerevisiae biology into its ecological context and to develop the genus Saccharomyces as a model clade for ecology and evolution. © 2014 The Authors. Yeast published by John Wiley & Sons, Ltd.
The ecology and evolution of non-domesticated Saccharomyces species
Boynton, Primrose J; Greig, Duncan
2014-01-01
Yeast researchers need model systems for ecology and evolution, but the model yeast Saccharomyces cerevisiae is not ideal because its evolution has been affected by domestication. Instead, ecologists and evolutionary biologists are focusing on close relatives of S. cerevisiae, the seven species in the genus Saccharomyces. The best-studied Saccharomyces yeast, after S. cerevisiae, is S. paradoxus, an oak tree resident throughout the northern hemisphere. In addition, several more members of the genus Saccharomyces have recently been discovered. Some Saccharomyces species are only found in nature, while others include both wild and domesticated strains. Comparisons between domesticated and wild yeasts have pinpointed hybridization, introgression and high phenotypic diversity as signatures of domestication. But studies of wild Saccharomyces natural history, biogeography and ecology are only beginning. Much remains to be understood about wild yeasts' ecological interactions and life cycles in nature. We encourage researchers to continue to investigate Saccharomyces yeasts in nature, both to place S. cerevisiae biology into its ecological context and to develop the genus Saccharomyces as a model clade for ecology and evolution. © 2014 The Authors. Yeast published by John Wiley & Sons Ltd. PMID:25242436
Ward, Diane M; Chen, Opal S; Li, Liangtao; Kaplan, Jerry; Bhuiyan, Shah Alam; Natarajan, Selvamuthu K; Bard, Martin; Cox, James E
2018-05-17
Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial Fe metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increase mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
Induction and characterization of morphologic mutants in a natural Saccharomyces cerevisiae strain.
Barberio, Claudia; Bianchi, Lucia; Pinzauti, Francesca; Lodi, Tiziana; Ferrero, Iliana; Polsinelli, Mario; Casalone, Enrico
2007-02-01
Saccharomyces cerevisiae is a good model with which to study the effects of morphologic differentiation on the ecological behaviour of fungi. In this work, 33 morphologic mutants of a natural strain of S. cerevisiae, obtained with UV mutagenesis, were selected for their streak shape and cell shape on rich medium. Two of them, showing both high sporulation proficiency and constitutive pseudohyphal growth, were analysed from a genetic and physiologic point of view. Each mutant carries a recessive monogenic mutation, and the two mutations reside in unlinked genes. Flocculation ability and responsiveness to different stimuli distinguished the two mutants. Growth at 37 degrees C affected the cell but not the colony morphology, suggesting that these two phenotypes are regulated differently. The effect of ethidium bromide, which affects mitochondrial DNA replication, suggested a possible "retrograde action" of mitochondria in pseudohyphal growth.
Inhibition of DNA replication in Saccharomyces cerevisiae by araCMP.
McIntosh, E M; Kunz, B A; Haynes, R H
1986-01-01
Cytosine arabinoside (araC), a potent inhibitor of DNA replication in mammalian cells, was found to be completely ineffective in Saccharomyces cerevisiae. The 5' monophosphate derivative, araCMP, is toxic and effectively inhibits both nuclear and mitochondrial DNA synthesis in this organism. Although wild-type strains can be inhibited by araCMP, dTMP permeable (tup-) strains were found to be much more sensitive to the analogue. In vivo labelling experiments indicate that araC enters yeast cells; however, it is extensively catabolized by deamination and breakage of the glycosidic bond. In addition, the analogue is not efficiently phosphorylated in S. cerevisiae owing to an apparent lack of deoxynucleoside kinase activity. These results provide further evidence that deoxyribonucleotides can be synthesized only through de novo pathways in this organism. Finally, araCMP was found to be recombinagenic in S. cerevisiae which suggests, together with other previous studies, that, in general, inhibition of DNA synthesis in yeast promotes mitotic recombination events.
Dickinson, Quinn; Bottoms, Scott; Hinchman, Li; ...
2016-01-20
In this study, imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae. As a result, we found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effectsmore » of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2Δ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance. In conclusion, this work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.« less
Rodriguez-Colman, Maria José; Reverter-Branchat, Gemma; Sorolla, M. Alba; Tamarit, Jordi; Ros, Joaquim; Cabiscol, Elisa
2010-01-01
In Saccharomyces cerevisiae, the forkhead transcription factor Hcm1 is involved in chromosome segregation, spindle pole dynamics, and budding. We found that Hcm1 interacts with the histone deacetylase Sir2 and shifts from cytoplasm to the nucleus in the G1/S phase or in response to oxidative stress stimuli. The nuclear localization of Hcm1 depends on the activity of Sir2 as revealed by activators and inhibitors of the sirtuins and the Δsir2 mutant. Hcm1-overexpressing cells display more mitochondria that can be attributed to increased amounts of Abf2, a protein involved in mitochondrial biogenesis. These cells also show higher rates of oxygen consumption and improved resistance to oxidative stress that would be explained by increased catalase and Sod2 activities and molecular chaperones such as Hsp26, Hsp30, and members of Hsp70 family. Microarray analyses also reveal increased expression of genes involved in mitochondrial energy pathways and those allowing the transition from the exponential to the stationary phase. Taken together, these results describe a new and relevant role of Hcm1 for mitochondrial functions, suggesting that this transcription factor would participate in the adaptation of cells from fermentative to respiratory metabolism. PMID:20847055
Mitochondrial Aging: Is There a Mitochondrial Clock?
Zorov, Dmitry B; Popkov, Vasily A; Zorova, Ljubava D; Vorobjev, Ivan A; Pevzner, Irina B; Silachev, Denis N; Zorov, Savva D; Jankauskas, Stanislovas S; Babenko, Valentina A; Plotnikov, Egor Y
2017-09-01
Fragmentation (fission) of mitochondria, occurring in response to oxidative challenge, leads to heterogeneity in the mitochondrial population. It is assumed that fission provides a way to segregate mitochondrial content between the "young" and "old" phenotype, with the formation of mitochondrial "garbage," which later will be disposed. Fidelity of this process is the basis of mitochondrial homeostasis, which is disrupted in pathological conditions and aging. The asymmetry of the mitochondrial fission is similar to that of their evolutionary ancestors, bacteria, which also undergo an aging process. It is assumed that mitochondrial markers of aging are recognized by the mitochondrial quality control system, preventing the accumulation of dysfunctional mitochondria, which normally are subjected to disposal. Possibly, oncocytoma, with its abnormal proliferation of mitochondria occupying the entire cytoplasm, represents the case when segregation of damaged mitochondria is impaired during mitochondrial division. It is plausible that mitochondria contain a "clock" which counts the degree of mitochondrial senescence as the extent of flagging (by ubiquitination) of damaged mitochondria. Mitochondrial aging captures the essence of the systemic aging which must be analyzed. We assume that the mitochondrial aging mechanism is similar to the mechanism of aging of the immune system which we discuss in detail. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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
Hermann, Greg J.; King, Edward J.; Shaw, Janet M.
1997-01-01
In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton. PMID:9105043
Basu, Somsuvro; Leonard, Joanne C.; Desai, Nishal; Mavridou, Despoina A. I.; Tang, Kong Ho; Goddard, Alan D.
2013-01-01
In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O2, unexpectedly find O2 and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O2 by TbERV1 is not dependent upon a simple O2 channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes. PMID:23264646
Lange, Heike; Lisowsky, Thomas; Gerber, Jana; Mühlenhoff, Ulrich; Kispal, Gyula; Lill, Roland
2001-01-01
Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process. PMID:11493598
Lange, H; Lisowsky, T; Gerber, J; Mühlenhoff, U; Kispal, G; Lill, R
2001-08-01
Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR ('augmenter of liver regeneration'), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.
Origone, Andrea Cecilia; Rodríguez, María Eugenia; Oteiza, Juan Martín; Querol, Amparo; Lopes, Christian Ariel
2018-01-01
Interspecific hybrids among species in the Saccharomyces genus are frequently detected in anthropic habitats and can also be obtained easily in the laboratory. This occurs because the most important genetic barriers among Saccharomyces species are post-zygotic. Depending on several factors, including the involved strains, the hybridization mechanism and stabilization conditions, hybrids that bear differential genomic constitutions, and hence phenotypic variability, can be obtained. In the present study, Saccharomyces cerevisiae × Saccharomyces uvarum hybrids were constructed using genetically and physiologically different S. uvarum parents at distinct temperatures (13 and 20°C). The effect of those variables on the main oenological features of the wines obtained with these hybrids was evaluated. Hybrids were successfully obtained in all cases. However, genetic stabilization based on successive fermentations in white wine at 13°C was significantly longer than that at 20°C. Our results demonstrated that, irrespective of the S. uvarum parent and temperature used for hybrid generation and stabilization, similar physicochemical and aromatic features were found in wines. The hybrids generated herein were characterized by low ethanol production, high glycerol synthesis and the capacity to grow at low temperature and to produce malic acid with particular aroma profiles. These features make these hybrids useful for the new winemaking industry within the climate change era frame. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.
Population genetics inside a cell: Mutations and mitochondrial genome maintenance
NASA Astrophysics Data System (ADS)
Goyal, Sidhartha; Shraiman, Boris; Gottschling, Dan
2012-02-01
In realistic ecological and evolutionary systems natural selection acts on multiple levels, i.e. it acts on individuals as well as on collection of individuals. An understanding of evolutionary dynamics of such systems is limited in large part due to the lack of experimental systems that can challenge theoretical models. Mitochondrial genomes (mtDNA) are subjected to selection acting on cellular as well as organelle levels. It is well accepted that mtDNA in yeast Saccharomyces cerevisiae is unstable and can degrade over time scales comparable to yeast cell division time. We utilize a recent technology designed in Gottschling lab to extract DNA from populations of aged yeast cells and deep sequencing to characterize mtDNA variation in a population of young and old cells. In tandem, we developed a stochastic model that includes the essential features of mitochondrial biology that provides a null model for expected mtDNA variation. Overall, we find approximately 2% of the polymorphic loci that show significant increase in frequency as cells age providing direct evidence for organelle level selection. Such quantitative study of mtDNA dynamics is absolutely essential to understand the propagation of mtDNA mutations linked to a spectrum of age-related diseases in humans.
Evidence for multiple interspecific hybridization in Saccharomyces sensu stricto species.
de Barros Lopes, Miguel; Bellon, Jennifer R; Shirley, Neil J; Ganter, Philip F
2002-01-01
Fluorescent amplified fragment length polymorphism analysis demonstrates a high level of gene exchange between Saccharomyces sensu stricto species, with some strains having undergone multiple interspecific hybridization events with subsequent changes in genome complexity. Two lager strains were shown to be hybrids between Saccharomyces cerevisiae and the alloploid species Saccharomyces pastorianus. The genome structure of CBS 380(T), the type strain of Saccharomyces bayanus, is also consistent with S. pastorianus gene transfer. The results indicate that the cider yeast, CID1, possesses nuclear DNA from three separate species. Mating experiments show that there are no barriers to interspecific conjugation of haploid cells. Furthermore, the allopolyploid strains were able to undergo further hybridizations with other Saccharomyces sensu stricto yeasts. These results demonstrate that introgression between the Saccharomyces sensu stricto species is likely.
Gsell, Martina; Mascher, Gerald; Schuiki, Irmgard; Ploier, Birgit; Hrastnik, Claudia; Daum, Günther
2013-01-01
In the yeast, Saccharomyces cerevisiae, the synthesis of the essential phospholipid phosphatidylethanolamine (PE) is accomplished by a network of reactions which comprises four different pathways. The enzyme contributing most to PE formation is the mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) which catalyzes conversion of phosphatidylserine (PS) to PE. To study the genome wide effect of an unbalanced cellular and mitochondrial PE level and in particular the contribution of Psd1p to this depletion we performed a DNA microarray analysis with a ∆psd1 deletion mutant. This approach revealed that 54 yeast genes were significantly up-regulated in the absence of PSD1 compared to wild type. Surprisingly, marked down-regulation of genes was not observed. A number of different cellular processes in different subcellular compartments were affected in a ∆psd1 mutant. Deletion mutants bearing defects in all 54 candidate genes, respectively, were analyzed for their growth phenotype and their phospholipid profile. Only three mutants, namely ∆gpm2, ∆gph1 and ∆rsb1, were affected in one of these parameters. The possible link of these mutations to PE deficiency and PSD1 deletion is discussed.
Gey, Uta; Czupalla, Cornelia; Hoflack, Bernard; Krause, Udo; Rödel, Gerhard
2014-01-01
The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects. PMID:25117470
Cytotoxicity and gene induction by some essential oils in the yeast Saccharomyces cerevisiae.
Bakkali, F; Averbeck, S; Averbeck, D; Zhiri, A; Idaomar, M
2005-08-01
In order to get an insight into the possible genotoxicity of essential oils (EOs) used in traditional pharmacological applications we tested five different oils extracted from the medicinal plants Origanum compactum, Coriandrum sativum, Artemisia herba alba, Cinnamomum camphora (Ravintsara aromatica) and Helichrysum italicum (Calendula officinalis) for genotoxic effects using the yeast Saccharomyces cerevisiae. Clear cytotoxic effects were observed in the diploid yeast strain D7, with the cells being more sensitive to EOs in exponential than in stationary growth phase. The cytotoxicity decreased in the following order: Origanum compactum>Coriandrum sativum>Artemisia herba alba>Cinnamomum camphora>Helichrysum italicum. In the same order, all EOs, except that derived from Helichrysum italicum, clearly induced cytoplasmic petite mutations indicating damage to mitochondrial DNA. However, no nuclear genetic events such as point mutations or mitotic intragenic or intergenic recombination were induced. The capacity of EOs to induce nuclear DNA damage-responsive genes was tested using suitable Lac-Z fusion strains for RNR3 and RAD51, which are genes involved in DNA metabolism and DNA repair, respectively. At equitoxic doses, all EOs demonstrated significant gene induction, approximately the same as that caused by hydrogen peroxide, but much lower than that caused by methyl methanesulfonate (MMS). EOs affect mitochondrial structure and function and can stimulate the transcriptional expression of DNA damage-responsive genes. The induction of mitochondrial damage by EOs appears to be closely linked to overall cellular cytotoxicity and appears to mask the occurrence of nuclear genetic events. EO-induced cytotoxicity involves oxidative stress, as is evident from the protection observed in the presence of ROS inhibitors such as glutathione, catalase or the iron-chelating agent deferoxamine.
Molecular Characterization of Propolis-Induced Cell Death in Saccharomyces cerevisiae▿†
de Castro, Patrícia Alves; Savoldi, Marcela; Bonatto, Diego; Barros, Mário Henrique; Goldman, Maria Helena S.; Berretta, Andresa A.; Goldman, Gustavo Henrique
2011-01-01
Propolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in folk medicine for centuries. Here, we apply the power of Saccharomyces cerevisiae as a model organism for studies of genetics, cell biology, and genomics to determine how propolis affects fungi at the cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c but not endonuclease G (Nuc1p) is involved in propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death. To elucidate the gene functions that may be required for propolis sensitivity in eukaryotes, the full collection of about 4,800 haploid S. cerevisiae deletion strains was screened for propolis sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis sensitivity compared to the corresponding wild-type strains. Systems biology revealed enrichment for genes involved in the mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein targeting to vacuoles, and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. PMID:21193549
Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; Shirasaki, Dyna I.; Wang, Charles; Blaby-Haas, Crysten E.; Merchant, Sabeeha S.; Loo, Joseph A.; Clarke, Catherine F.
2015-01-01
Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11. PMID:25631044
Role of mitochondrial permeability transition pores in mitochondrial autophagy.
Rodriguez-Enriquez, Sara; He, Lihua; Lemasters, John J
2004-12-01
During autophagy, cells rid themselves of damaged and superfluous mitochondria, as well as other organelles. This activation of mitochondrial turnover could be the result of changes in the physiological state of mitochondria. Confocal microscopy and fluorescence techniques indicate that onset of mitochondrial permeability transition is one such change. The mitochondrial permeability transition is a reversible phenomenon whereby the mitochondrial inner membrane becomes freely permeable to solutes of less than 1500 Da. At onset of the mitochondrial permeability transition, mitochondria depolarize, uncouple, and undergo large amplitude swelling due to opening of permeability transition pores, which may form by aggregation of damaged, misfolded membrane proteins. When injurious cellular stresses occur, cells may protect themselves using autophagy to remove damaged mitochondria and mutated mitochondrial DNA. Ca(2+) overloading, reactive oxygen and nitrogen species, decreased mitochondrial membrane potential, and oxidation of pyridine nucleotides and glutathione all promote mitochondrial damage and onset of the mitochondrial permeability transition. The mitochondrial permeability transition is also associated with necrosis and apoptosis after a variety of stimuli. This review emphasizes the role of the mitochondrial permeability transition as a key event in mitochondrial autophagy.
Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation
Soloveychik, Maria; Xu, Mengshu; Zaslaver, Olga; Lee, Kwanyin; Narula, Ashrut; Jiang, River; Rosebrock, Adam P.; Caudy, Amy A.; Meneghini, Marc D.
2016-01-01
Histone demethylation by Jumonji-family proteins is coupled with the decarboxylation of α-ketoglutarate (αKG) to yield succinate, prompting hypotheses that their activities are responsive to levels of these metabolites in the cell. Consistent with this paradigm we show here that the Saccharomyces cerevisiae Jumonji demethylase Jhd2 opposes the accumulation of H3K4me3 in fermenting cells only when they are nutritionally manipulated to contain an elevated αKG/succinate ratio. We also find that Jhd2 opposes H3K4me3 in respiratory cells that do not exhibit such an elevated αKG/succinate ratio. While jhd2∆ caused only limited gene expression defects in fermenting cells, transcript profiling and physiological measurements show that JHD2 restricts mitochondrial respiratory capacity in cells grown in non-fermentable carbon in an H3K4me-dependent manner. In association with these phenotypes, we find that JHD2 limits yeast proliferative capacity under physiologically challenging conditions as measured by both replicative lifespan and colony growth on non-fermentable carbon. JHD2’s impact on nutrient response may reflect an ancestral role of its gene family in mediating mitochondrial regulation. PMID:27897198
Mitochondrial Dynamics: Coupling Mitochondrial Fitness with Healthy Aging.
Sebastián, David; Palacín, Manuel; Zorzano, Antonio
2017-03-01
Aging is associated with a decline in mitochondrial function and the accumulation of abnormal mitochondria. However, the precise mechanisms by which aging promotes these mitochondrial alterations and the role of the latter in aging are still not fully understood. Mitochondrial dynamics is a key process regulating mitochondrial function and quality. Altered expression of some mitochondrial dynamics proteins has been recently associated with aging and with age-related alterations in yeast, Caenorhabditis elegans, mice, and humans. Here, we review the link between alterations in mitochondrial dynamics, aging, and age-related impairment. We propose that the dysregulation of mitochondrial dynamics leads to age-induced accumulation of unhealthy mitochondria and contributes to alterations linked to aging, such as diabetes and neurodegeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.
Reenan, R A; Kolodner, R D
1992-12-01
The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (< 0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (< 0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus.
Georgiades, Kalliopi; Raoult, Didier
2011-10-20
Mitochondria are thought to have evolved from eubacteria-like endosymbionts; however, the origin of the mitochondrion remains a subject of debate. In this study, we investigated the phenomenon of chimerism in mitochondria to shed light on the origin of these organelles by determining which species played a role in their formation. We used the mitochondria of four distinct organisms, Reclinomonas americana, Homo sapiens, Saccharomyces cerevisiae and multichromosome Pediculus humanus, and attempted to identify the origin of each mitochondrial gene. Our results suggest that the origin of mitochondrial genes is not limited to the Rickettsiales and that the creation of these genes did not occur in a single event, but through multiple successive events. Some of these events are very old and were followed by events that are more recent and occurred through the addition of elements originating from current species. The points in time that the elements were added and the parental species of each gene in the mitochondrial genome are different to the individual species. These data constitute strong evidence that mitochondria do not have a single common ancestor but likely have numerous ancestors, including proto-Rickettsiales, proto-Rhizobiales and proto-Alphaproteobacteria, as well as current alphaproteobacterial species. The analysis of the multichromosome P. humanus mitochondrion supports this mechanism. The most plausible scenario of the origin of the mitochondrion is that ancestors of Rickettsiales and Rhizobiales merged in a proto-eukaryotic cell approximately one billion years ago. The fusion of the Rickettsiales and Rhizobiales cells was followed by gene loss, genomic rearrangements and the addition of alphaproteobacterial elements through ancient and more recent recombination events. Each gene of each of the four studied mitochondria has a different origin, while in some cases, multichromosomes may allow for enhanced gene exchange. Therefore, the tree of life is not
NASA Technical Reports Server (NTRS)
Satyanarayana, T.; Klein, Harold P.
1976-01-01
A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis. Sedimentation velocity runs revealed a single symmetric peak with an s(sub (20,w)) value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 +/- 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 +/- 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic Saccharomyces cerevisiae is composed of three subunits of identical or nearly identical size.
Kitagaki, Hiroshi; Cowart, L Ashley; Matmati, Nabil; Montefusco, David; Gandy, Jason; de Avalos, Silvia Vaena; Novgorodov, Sergei A; Zheng, Jim; Obeid, Lina M; Hannun, Yusuf A
2009-04-17
Growth of Saccharomyces cerevisiae following glucose depletion (the diauxic shift) depends on a profound metabolic adaptation accompanied by a global reprogramming of gene expression. In this study, we provide evidence for a heretofore unsuspected role for Isc1p in mediating this reprogramming. Initial studies revealed that yeast cells deleted in ISC1, the gene encoding inositol sphingolipid phospholipase C, which resides in mitochondria in the post-diauxic phase, showed defective aerobic respiration in the post-diauxic phase but retained normal intrinsic mitochondrial functions, including intact mitochondrial DNA, normal oxygen consumption, and normal mitochondrial polarization. Microarray analysis revealed that the Deltaisc1 strain failed to up-regulate genes required for nonfermentable carbon source metabolism during the diauxic shift, thus suggesting a mechanism for the defective supply of respiratory substrates into mitochondria in the post-diauxic phase. This defect in regulating nuclear gene induction in response to a defect in a mitochondrial enzyme raised the possibility that mitochondria may initiate diauxic shift-associated regulation of nucleus-encoded genes. This was established by demonstrating that in respiratory-deficient petite cells these genes failed to be up-regulated across the diauxic shift in a manner similar to the Deltaisc1 strain. Isc1p- and mitochondrial function-dependent genes significantly overlapped with Adr1p-, Snf1p-, and Cat8p-dependent genes, suggesting some functional link among these factors. However, the retrograde response was not activated in Deltaisc1, suggesting that the response of Deltaisc1 cannot be simply attributed to mitochondrial dysfunction. These results suggest a novel role for Isc1p in allowing the reprogramming of gene expression during the transition from anaerobic to aerobic metabolism.
Experience with Saccharomyces boulardii Probiotic in Oncohaematological Patients.
Sulik-Tyszka, Beata; Snarski, Emilian; Niedźwiedzka, Magda; Augustyniak, Małgorzata; Myhre, Thorvald Nilsen; Kacprzyk, Anna; Swoboda-Kopeć, Ewa; Roszkowska, Marta; Dwilewicz-Trojaczek, Jadwiga; Jędrzejczak, Wiesław Wiktor; Wróblewska, Marta
2018-06-01
Very few reports have been published to date on the bloodstream infections caused by Saccharomyces spp. in oncohaematological patients, and there are no guidelines on the use of this probiotic microorganism in this population. We describe the use of probiotic preparation containing Saccharomyces boulardii in a large group of oncohaematological patients. We retrospectively analysed the data from 32,000 patient hospitalisations at the haematological centre during 2011-2013 (including 196 haematopoietic stem cell transplant recipients) in a tertiary care university-affiliated hospital. During the study period, 2270 doses of Saccharomyces boulardii probiotic were administered to the oncohaematological patients. In total, 2816 mycological cultures were performed, out of which 772 (27.4%) were positive, with 52 indicating digestive tract colonisation by Saccharomyces spp., mainly in patients with acute myeloid leukaemia (AML), myelodysplastic syndrome (MDS) or multiple myeloma (MM). While colonised, they were hospitalised for 1683 days and 416 microbiological cultures of their clinical samples were performed. In the studied group of patients, there were six blood cultures positive for fungi; however, they comprised Candida species: two C. glabrata, one C. albicans, one C. krusei, one C. tropicalis and one C. parapsilosis. There was no blood culture positive for Saccharomyces spp. Our study indicates that despite colonisation of many oncohaematological patients with Saccharomyces spp., there were no cases of fungal sepsis caused by this species.
Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase.
Kuusk, Silja; Sedman, Tiina; Jõers, Priit; Sedman, Juhan
2005-07-01
Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.
Rodríguez, M Eugenia; Pérez-Través, Laura; Sangorrín, Marcela P; Barrio, Eladio; Lopes, Christian A
2014-09-01
Mudai is a traditional fermented beverage, made from the seeds of the Araucaria araucana tree by Mapuche communities. The main goal of the present study was to identify and characterize the yeast microbiota responsible of Mudai fermentation as well as from A. araucana seeds and bark from different locations in Northern Patagonia. Only Hanseniaspora uvarum and a commercial bakery strain of Saccharomyces cerevisiae were isolated from Mudai and all Saccharomyces isolates recovered from A. araucana seed and bark samples belonged to the cryotolerant species Saccharomyces eubayanus and Saccharomyces uvarum. These two species were already reported in Nothofagus trees from Patagonia; however, this is the first time that they were isolated from A. araucana, which extends their ecological distribution. The presence of these species in A. araucana seeds and bark samples, led us to postulate a potential role for them as the original yeasts responsible for the elaboration of Mudai before the introduction of commercial S. cerevisiae cultures. The molecular and genetic characterization of the S. uvarum and S. eubayanus isolates and their comparison with European S. uvarum strains and S. eubayanus hybrids (S. bayanus and S. pastorianus), allowed their ecology and evolution us to be examined. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Finsterer, Josef; Zarrouk-Mahjoub, Sinda
2016-01-01
Mitochondrial disorders (MIDs) are usually multisystem disorders (mitochondrial multiorgan disorder syndrome) either on from onset or starting at a point during the disease course. Most frequently affected tissues are those with a high oxygen demand such as the central nervous system, the muscle, endocrine glands, or the myocardium. Recently, it has been shown that rarely also the arteries may be affected (mitochondrial arteriopathy). This review focuses on the type, diagnosis, and treatment of mitochondrial vasculopathy in MID patients. A literature search using appropriate search terms was carried out. Mitochondrial vasculopathy manifests as either microangiopathy or macroangiopathy. Clinical manifestations of mitochondrial microangiopathy include leukoencephalopathy, migraine-like headache, stroke-like episodes, or peripheral retinopathy. Mitochondrial macroangiopathy manifests as atherosclerosis, ectasia of arteries, aneurysm formation, dissection, or spontaneous rupture of arteries. The diagnosis relies on the documentation and confirmation of the mitochondrial metabolic defect or the genetic cause after exclusion of non-MID causes. Treatment is not at variance compared to treatment of vasculopathy due to non-MID causes. Mitochondrial vasculopathy exists and manifests as micro- or macroangiopathy. Diagnosing mitochondrial vasculopathy is crucial since appropriate treatment may prevent from severe complications. PMID:27231520
Genomic Insights into the Saccharomyces sensu stricto Complex
Borneman, Anthony R.; Pretorius, Isak S.
2015-01-01
The Saccharomyces sensu stricto group encompasses species ranging from the industrially ubiquitous yeast Saccharomyces cerevisiae to those that are confined to geographically limited environmental niches. The wealth of genomic data that are now available for the Saccharomyces genus is providing unprecedented insights into the genomic processes that can drive speciation and evolution, both in the natural environment and in response to human-driven selective forces during the historical “domestication” of these yeasts for baking, brewing, and winemaking. PMID:25657346
Domizio, Paola; Romani, Cristina; Lencioni, Livio; Comitini, Francesca; Gobbi, Mirko; Mannazzu, Ilaria; Ciani, Maurizio
2011-06-30
The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol. Copyright © 2011 Elsevier B.V. All rights reserved.
Lapointe, Christopher P; Stefely, Jonathan A; Jochem, Adam; Hutchins, Paul D; Wilson, Gary M; Kwiecien, Nicholas W; Coon, Joshua J; Wickens, Marvin; Pagliarini, David J
2018-01-24
Coenzyme Q (CoQ) is a redox-active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a multi-omic strategy to identify mRNAs that not only bind Puf3p but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Puf3p target: Puf3p regulates the abundance of Coq5p and prevents its detrimental hyperaccumulation, thereby enabling efficient CoQ production. More broadly, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly (pathways essential to prime mitochondrial biogenesis). Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis, and they demonstrate the power of multi-omics for defining genuine targets of RBPs. Copyright © 2017 Elsevier Inc. All rights reserved.
Pallotta, Maria Luigia; Valenti, Daniela; Iacovino, Michelina; Passarella, Salvatore
2004-02-15
In this work we looked at whether and how mitochondria isolated from Saccharomyces cerevisiae (SCM) oxidize d-lactate. We found that: (1). externally added d-lactate causes oxygen uptake by SCM with P/O ratio equal to 1.5; in the presence of antimycin A (AA), P/O ratio was 1.8, differently in the presence of the non-penetrant alpha-cyanocinnamate (alpha-CCN-) no P/O ratio could be measured. Consistently, mitochondrial electrical membrane potential (deltapsi) generation was found, due to externally added d-lactate in the presence of antimycin A, but not of alpha-CCN-. (2). SCM oxidize d-lactate in two different manners: (i). via inner membrane d-lactate dehydrogenase which leads to d-lactate oxidation without driving deltapsi generation and ATP synthesis and (ii). via the matrix d-lactate dehydrogenase, which drives deltapsi generation and ATP synthesis by using taken up d-lactate. (3). Pyruvate newly synthesised in the mitochondrial matrix is exported via the novel d-lactate/pyruvate antiporter. d-Lactate/pyruvate antiport proved to regulate the rate of pyruvate efflux in vitro. (4). The existence of the d-lactate/H+ symporter is also proposed as shown by mitochondrial swelling. The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.
Guerrero-Castillo, Sergio; Araiza-Olivera, Daniela; Cabrera-Orefice, Alfredo; Espinasa-Jaramillo, Juan; Gutiérrez-Aguilar, Manuel; Luévano-Martínez, Luís A; Zepeda-Bastida, Armando; Uribe-Carvajal, Salvador
2011-06-01
Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O(2) consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O(2) consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.
Genomic insights into the Saccharomyces sensu stricto complex.
Borneman, Anthony R; Pretorius, Isak S
2015-02-01
The Saccharomyces sensu stricto group encompasses species ranging from the industrially ubiquitous yeast Saccharomyces cerevisiae to those that are confined to geographically limited environmental niches. The wealth of genomic data that are now available for the Saccharomyces genus is providing unprecedented insights into the genomic processes that can drive speciation and evolution, both in the natural environment and in response to human-driven selective forces during the historical "domestication" of these yeasts for baking, brewing, and winemaking. Copyright © 2015 by the Genetics Society of America.
CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.
Dumont, M E; Schlichter, J B; Cardillo, T S; Hayes, M K; Bethlendy, G; Sherman, F
1993-01-01
The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria. Images PMID:8413243
Nakai, Daisuke; Shimizu, Takahiko; Nojiri, Hidetoshi; Uchiyama, Satoshi; Koike, Hideo; Takahashi, Mayumi; Hirokawa, Katsuiku; Shirasawa, Takuji
2004-10-01
coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, and shows sluggish behaviours and an extended lifespan. In C. elegans and Saccharomyces cerevisiae, coq7/clk-1 is required for the biosynthesis of coenzyme Q (CoQ), an essential co-factor in mitochondrial respiration. The clk-1 mutant contains dietary CoQ(8) from Escherichia coli and demethoxyubiquinone 9 (DMQ9) instead of CoQ(9). In a previous study, we generated COQ7-deficient mice by targeted disruption of the coq7 gene and reported that mouse coq7/clk-1 is also essential for CoQ synthesis, maintenance of mitochondrial integrity and neurogenesis. In the present study, we rescued COQ7-deficient mice from embryonic lethality and established a mouse model with decreased CoQ level by transgene expression of COQ7/CLK-1. A biochemical analysis showed a concomitant decrease in CoQ(9), mitochondrial respiratory enzyme activity and the generation of reactive oxygen species (ROS) in the mitochondria of CoQ-insufficient mice. This implied that the depressed activity of respiratory enzymes and the depressed production of ROS may play a physiological role in the control of lifespan in mammalian species and of C. elegans.
The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy
Vincent, Amy E.; Ng, Yi Shiau; White, Kathryn; Davey, Tracey; Mannella, Carmen; Falkous, Gavin; Feeney, Catherine; Schaefer, Andrew M.; McFarland, Robert; Gorman, Grainne S.; Taylor, Robert W.; Turnbull, Doug M.; Picard, Martin
2016-01-01
Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease. PMID:27506553
Jeyaram, Kumaraswamy; Tamang, Jyoti Prakash; Capece, Angela; Romano, Patrizia
2011-11-01
Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.
DiMauro, Salvatore
2006-11-01
Our understanding of mitochondrial diseases (defined restrictively as defects of the mitochondrial respiratory chain) is expanding rapidly. In this review, I will give the latest information on disorders affecting predominantly or exclusively skeletal muscle. The most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency and mutations in genes controlling mitochondrial DNA abundance and structure, such as POLG, TK2, and MPV17. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with decreased amount and altered structure of cardiolipin, the main phospholipid of the inner mitochondrial membrane, but a secondary impairment of respiratory chain function is plausible. The role of mutations in protein-coding genes of mitochondrial DNA in causing isolated myopathies has been confirmed. Mutations in tRNA genes of mitochondrial DNA can also cause predominantly myopathic syndromes and--contrary to conventional wisdom--these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, cramps, recurrent myoglobinuria, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.
Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.
He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J
2012-07-01
Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.
Gomes, Fernando; Palma, Flávio Romero; Barros, Mario H; Tsuchida, Eduardo T; Turano, Helena G; Alegria, Thiago G P; Demasi, Marilene; Netto, Luis E S
2017-10-13
Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H 2 O 2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
van Rossum, Harmen M.; Kozak, Barbara U.; Niemeijer, Matthijs S.; Duine, Hendrik J.; Luttik, Marijke A. H.; Boer, Viktor M.; Kötter, Peter; Daran, Jean-Marc G.; van Maris, Antonius J. A.
2016-01-01
Pyruvate and acetyl-coenzyme A, located at the interface between glycolysis and TCA cycle, are important intermediates in yeast metabolism and key precursors for industrially relevant products. Rational engineering of their supply requires knowledge of compensatory reactions that replace predominant pathways when these are inactivated. This study investigates effects of individual and combined mutations that inactivate the mitochondrial pyruvate-dehydrogenase (PDH) complex, extramitochondrial citrate synthase (Cit2) and mitochondrial CoA-transferase (Ach1) in Saccharomyces cerevisiae. Additionally, strains with a constitutively expressed carnitine shuttle were constructed and analyzed. A predominant role of the PDH complex in linking glycolysis and TCA cycle in glucose-grown batch cultures could be functionally replaced by the combined activity of the cytosolic PDH bypass and Cit2. Strongly impaired growth and a high incidence of respiratory deficiency in pda1Δ ach1Δ strains showed that synthesis of intramitochondrial acetyl-CoA as a metabolic precursor requires activity of either the PDH complex or Ach1. Constitutive overexpression of AGP2, HNM1, YAT2, YAT1, CRC1 and CAT2 enabled the carnitine shuttle to efficiently link glycolysis and TCA cycle in l-carnitine-supplemented, glucose-grown batch cultures. Strains in which all known reactions at the glycolysis-TCA cycle interface were inactivated still grew slowly on glucose, indicating additional flexibility at this key metabolic junction. PMID:26895788
Allan, Christopher M.; Awad, Agape M.; Johnson, Jarrett S.; ...
2015-01-28
Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. In thismore » paper, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q 6 and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Finally, given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.« less
Špírek, Mário; Poláková, Silvia; Jatzová, Katarína; Sulo, Pavol
2015-01-01
Nucleo-mitochondrial interactions, particularly those determining the primary divergence of biological species, can be studied by means of xenomitochondrial cybrids, which are cells where the original mitochondria are substituted by their counterparts from related species. Saccharomyces cerevisiae cybrids are prepared simply by the mating of the ρ0 strain with impaired karyogamy and germinating spores from other Saccharomyces species and fall into three categories. Cybrids with compatible mitochondrial DNA (mtDNA) from Saccharomyces paradoxus CBS 432 and Saccharomyces cariocanus CBS 7994 are metabolically and genetically similar to cybrids containing mtDNA from various S. cerevisiae. Cybrids with mtDNA from other S. paradoxus strains, S. cariocanus, Saccharomyces kudriavzevii, and Saccharomyces mikatae require a period of adaptation to establish efficient oxidative phosphorylation. They exhibit a temperature-sensitive phenotype, slower growth rate on a non-fermentable carbon source and a long lag phase after the shift from glucose. Their decreased respiration capacity and reduced cytochrome aa3 content is associated with the inefficient splicing of cox1I3β, the intron found in all Saccharomyces species but not in S. cerevisiae. The splicing defect is compensated in cybrids by nuclear gain-of-function and can be alternatively suppressed by overexpression of MRP13 gene for mitochondrial ribosomal protein or the MRS2, MRS3, and MRS4 genes involved in intron splicing. S. cerevisiae with Saccharomyces bayanus mtDNA is unable to respire and the growth on ethanol–glycerol can be restored only after mating to some mit− strains. The nucleo-mitochondrial compatibility limit of S. cerevisiae and other Saccharomyces was set between S. kudriavzevii and S. bayanus at the divergence from S. cerevisiae about 15 MYA. The MRS1-cox1 S. cerevisiae/S. paradoxus cytonuclear Dobzhansky–Muller pair has a neglible impact on the separation of species since its imperfection is
Biotechnology of non-Saccharomyces yeasts--the ascomycetes.
Johnson, Eric A
2013-01-01
Saccharomyces cerevisiae and several other yeast species are among the most important groups of biotechnological organisms. S. cerevisiae and closely related ascomycetous yeasts are the major producer of biotechnology products worldwide, exceeding other groups of industrial microorganisms in productivity and economic revenues. Traditional industrial attributes of the S. cerevisiae group include their primary roles in food fermentations such as beers, cider, wines, sake, distilled spirits, bakery products, cheese, sausages, and other fermented foods. Other long-standing industrial processes involving S. cerevisae yeasts are production of fuel ethanol, single-cell protein (SCP), feeds and fodder, industrial enzymes, and small molecular weight metabolites. More recently, non-Saccharomyces yeasts (non-conventional yeasts) have been utilized as industrial organisms for a variety of biotechnological roles. Non-Saccharomyces yeasts are increasingly being used as hosts for expression of proteins, biocatalysts and multi-enzyme pathways for the synthesis of fine chemicals and small molecular weight compounds of medicinal and nutritional importance. Non-Saccharomyces yeasts also have important roles in agriculture as agents of biocontrol, bioremediation, and as indicators of environmental quality. Several of these products and processes have reached commercial utility, while others are in advanced development. The objective of this mini-review is to describe processes currently used by industry and those in developmental stages and close to commercialization primarily from non-Saccharomyces yeasts with an emphasis on new opportunities. The utility of S. cerevisiae in heterologous production of selected products is also described.
Schaefke, Bernhard; Wang, Tzi-Yuan; Wang, Chuen-Yi; Li, Wen-Hsiung
2015-01-01
Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains). PMID:26220934
Schiabor, Kelly M.; Quan, Allison S.; Eisen, Michael B.
2014-01-01
While screening a large collection of wild and laboratory yeast strains for their ability to attract Drosophila melanogaster adults, we noticed a large difference in fly preference for two nearly isogenic strains of Saccharomyces cerevisiae, BY4741 and BY4742. Using standard genetic analyses, we tracked the preference difference to the lack of mitochondria in the BY4742 strain used in the initial experiment. We used gas chromatography coupled with mass spectroscopy to examine the volatile compounds produced by BY4741 and the mitochondria-deficient BY4742, and found that they differed significantly. We observed that several ethyl esters are present at much higher levels in strains with mitochondria, even in fermentative conditions. We found that nitrogen levels in the substrate affect the production of these compounds, and that they are produced at the highest level by strains with mitochondria when fermenting natural fruit substrates. Collectively these observations demonstrate that core metabolic processes mediate the interaction between yeasts and insect vectors, and highlight the importance mitochondrial functions in yeast ecology. PMID:25462617
Di Fonzo, Alessio; Ronchi, Dario; Lodi, Tiziana; Fassone, Elisa; Tigano, Marco; Lamperti, Costanza; Corti, Stefania; Bordoni, Andreina; Fortunato, Francesco; Nizzardo, Monica; Napoli, Laura; Donadoni, Chiara; Salani, Sabrina; Saladino, Francesca; Moggio, Maurizio; Bresolin, Nereo; Ferrero, Iliana; Comi, Giacomo P
2009-05-01
A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.
Di Fonzo, Alessio; Ronchi, Dario; Lodi, Tiziana; Fassone, Elisa; Tigano, Marco; Lamperti, Costanza; Corti, Stefania; Bordoni, Andreina; Fortunato, Francesco; Nizzardo, Monica; Napoli, Laura; Donadoni, Chiara; Salani, Sabrina; Saladino, Francesca; Moggio, Maurizio; Bresolin, Nereo; Ferrero, Iliana; Comi, Giacomo P.
2009-01-01
A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1R182H mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease. PMID:19409522
Heude, M; Moustacchi, E
1979-09-01
Three main features regarding the loss of mitochondrial genetic markers among rho- mutants induced by ultraviolet irradiation are reported: (a) the frequency of loss of six loci examined increases with UV dose; (b) preferential loss of one region of the mitochondrial genome observed in spontaneous rho- mutants is enhanced by UV; and (c) the loss of each marker results from large deletions. Marker loss in rho- mutants was also investigated under conditions that modulate rho- induction. Liquid holding of irradiated exponential or stationary phase cells, as well as a split-dose regime applied to stationary phase cells, results in rho- mutants in which the loss of markers is correlated with rho- induction: the more sensitive the cells are to rho- induction, the more frequent are the marker losses among rho- clones derived from these cells. This correlation is not found in exponential-phase cells submitted to a split-dose treatment, suggesting that a different mechanism is involved in the latter case. It is known that UV-induced pyrimidine dimers are not excised in a controlled manner in mitochondrial DNA. However, our studies indicate that an accurate repair mechanism (of the recombinational type ?) can lead to the restoration of mitochondrial genetic information in growing cells.
Zhou, Wenchang; Leone, Vanessa; Krah, Alexander; Faraldo-Gómez, José D
2017-04-20
Recent years have witnessed a renewed interest in the ATP synthase as a drug target against human pathogens. Indeed, clinical, biochemical, and structural data indicate that hydrophobic inhibitors targeting the membrane-embedded proton-binding sites of the c-subunit ring could serve as last-resort antibiotics against multidrug resistant strains. However, because inhibition of the mitochondrial ATP synthase in humans is lethal, it is essential that these inhibitors be not only potent but also highly selective for the bacterial enzyme. To this end, a detailed understanding of the structure of this protein target is arguably instrumental. Here, we use computational methods to predict the atomic structures of the proton-binding sites in two prototypical c-rings: that of the ATP synthase from Saccharomyces cerevisiae, which is a model system for mitochondrial enzymes, and that from Escherichia coli, which can be pathogenic for humans. Our study reveals the structure of these binding sites loaded with protons and in the context of the membrane, that is, in the state that would mediate the recognition of a potential inhibitor. Both structures reflect a mode of proton coordination unlike those previously observed in other c-ring structures, whether experimental or modeled.
Guo, Zhenhua; Adomas, Aleksandra B; Jackson, Erin D; Qin, Hong; Townsend, Jeffrey P
2011-06-01
We investigated the mechanism underlying the natural variation in longevity within natural populations using the model budding yeast, Saccharomyces cerevisiae. We analyzed whole-genome gene expression in four progeny of a natural S. cerevisiae strain that display differential replicative aging. Genes with different expression levels in short- and long-lived strains were classified disproportionately into metabolism, transport, development, transcription or cell cycle, and organelle organization (mitochondrial, chromosomal, and cytoskeletal). With several independent validating experiments, we detected 15 genes with consistent differential expression levels between the long- and the short-lived progeny. Among those 15, SIR2, HSP30, and TIM17 were upregulated in long-lived strains, which is consistent with the known effects of gene silencing, stress response, and mitochondrial function on aging. The link between SIR2 and yeast natural life span variation offers some intriguing ties to the allelic association of the human homolog SIRT1 to visceral obesity and metabolic response to lifestyle intervention. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake.
Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M
2017-05-01
Mitochondrial calcium ([Ca 2+ ] m ) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca 2+ ] m uptake upon SK channel activation as detected by time lapse mitochondrial Ca 2+ measurements with the Ca 2+ -binding mitochondria-targeted aequorin and FRET-based [Ca 2+ ] m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca 2+ ] m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.
SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake
Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M
2017-01-01
Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death. PMID:28282037
Mitochondrial peptides modulate mitochondrial function during cellular senescence.
Kim, Su-Jeong; Mehta, Hemal H; Wan, Junxiang; Kuehnemann, Chisaka; Chen, Jingcheng; Hu, Ji-Fan; Hoffman, Andrew R; Cohen, Pinchas
2018-06-10
Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.
Schaefke, Bernhard; Wang, Tzi-Yuan; Wang, Chuen-Yi; Li, Wen-Hsiung
2015-07-27
Gene expression evolution occurs through changes in cis- or trans-regulatory elements or both. Interactions between transcription factors (TFs) and their binding sites (TFBSs) constitute one of the most important points where these two regulatory components intersect. In this study, we investigated the evolution of TFBSs in the promoter regions of different Saccharomyces strains and species. We divided the promoter of a gene into the proximal region and the distal region, which are defined, respectively, as the 200-bp region upstream of the transcription starting site and as the 200-bp region upstream of the proximal region. We found that the predicted TFBSs in the proximal promoter regions tend to be evolutionarily more conserved than those in the distal promoter regions. Additionally, Saccharomyces cerevisiae strains used in the fermentation of alcoholic drinks have experienced more TFBS losses than gains compared with strains from other environments (wild strains, laboratory strains, and clinical strains). We also showed that differences in TFBSs correlate with the cis component of gene expression evolution between species (comparing S. cerevisiae and its sister species Saccharomyces paradoxus) and within species (comparing two closely related S. cerevisiae strains). © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Origone, A C; Del Mónaco, S M; Ávila, J R; González Flores, M; Rodríguez, M E; Lopes, C A
2017-08-01
Evaluating the winemaking stress tolerance of a set of both Saccharomyces eubayanus and Saccharomyces uvarum strains from diverse Patagonian habitats. Yeast strains growth was analysed under increasing ethanol concentrations; all of them were able to grow until 8% v/v ethanol. The effect of different temperature and pH conditions as well as at SO 2 and hexose concentrations was evaluated by means of a central composite experimental design. Only two S. uvarum strains (NPCC 1289 and 1321) were able to grow in most stress conditions. Kinetic parameters analysed (μ max and λ) were statistically affected by temperature, pH and SO 2 , but not influenced by sugar concentration. The obtained growth model was used for predicting optimal growth conditions for both strains: 20°C, 0% w/v SO 2 and pH 4·5. Strains from human-associated environments (chichas) presented the highest diversity in the response to different stress factors. Two S. uvarum strains from chichas demonstrated to be the most tolerant to winemaking conditions. This work evidenced the potential use of two S. uvarum yeast strains as starter cultures in wines fermented at low temperatures. Saccharomyces eubayanus was significantly affected by winemaking stress conditions, limiting its use in this industry. © 2017 The Society for Applied Microbiology.
Tzagoloff, A; Foury, F; Akai, A
1976-11-24
1. Fourteen cytoplasmic mutants of Saccharomyces cerevisiae with a specific deficiency of cytochrome b have been studied. The mutations have been shown to occur in two separate genetic loci, COB 1 and COB 2. These loci can be distinguished by mit- X mit- crosses. Pairwise crosses of cytochrome b mutants belonging to different loci yield 4-6% wild type recombinants corresponding to recombinational frequencies of 8-12%. In intra-locus crosses, the recombinational frequencies range from 1% to less than 0.01%. The two loci can also be distinguished by mit- X rho- crosses. Twenty rho- testers have been isolated of which ten preferentially restore mutations in COB 1 and ten others in COB 2. 2. The COB 1 and COB 2 loci have been localized on mitochondrial DNA between the two antibiotic resistance loci OLI 1 and OLI 2 in the order OLI 2-COB 2-COB 1-OLI 1. The results of mit- X mit- and mit- X rho- crosses have also been used to map the cytochrome b mutations relative to each other. The maps obtained by the two independent methods are in good agreement. 3. Mutations in COB 1 have been found to be linked to the OLI1 locus in some but not in other strains of S. cervisiae. This evidence suggests that there may be a spacer region between the two loci whose length varies from strain to strain. 4. Two mutations in COB 2 have been found to cause a loss of a mitochondrial translation product corresponding to the cytochrome b apoprotein. Instead of the wild type protein the mutants have a new low-molecular weight product which is probably a fragment of cytochrome b. The fact that the mutations revert suggests that they are nonsense mutations in the structural gene of cytochrome b.
Dunn, Cory D
2011-10-01
Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.
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.
Atomic model for the dimeric FO region of mitochondrial ATP synthase.
Guo, Hui; Bueler, Stephanie A; Rubinstein, John L
2017-11-17
Mitochondrial adenosine triphosphate (ATP) synthase produces the majority of ATP in eukaryotic cells, and its dimerization is necessary to create the inner membrane folds, or cristae, characteristic of mitochondria. Proton translocation through the membrane-embedded F O region turns the rotor that drives ATP synthesis in the soluble F 1 region. Although crystal structures of the F 1 region have illustrated how this rotation leads to ATP synthesis, understanding how proton translocation produces the rotation has been impeded by the lack of an experimental atomic model for the F O region. Using cryo-electron microscopy, we determined the structure of the dimeric F O complex from Saccharomyces cerevisiae at a resolution of 3.6 angstroms. The structure clarifies how the protons travel through the complex, how the complex dimerizes, and how the dimers bend the membrane to produce cristae. Copyright © 2017, American Association for the Advancement of Science.
Gamero-Sandemetrio, Esther; Gómez-Pastor, Rocío; Matallana, Emilia
2013-05-01
Aerobic organisms have devised several enzymatic and non-enzymatic antioxidant defenses to deal with reactive oxygen species (ROS) produced by cellular metabolism. To combat such stress, cells induce ROS scavenging enzymes such as catalase, peroxidase, superoxide dismutase (SOD) and glutathione reductase. In the present research, we have used a double staining technique of SOD and catalase enzymes in the same polyacrylamide gel to analyze the different antioxidant enzymatic activities and protein isoforms present in Saccharomyces and non-Saccharomyces yeast species. Moreover, we used a technique to differentially detect Sod1p and Sod2p on gel by immersion in NaCN, which specifically inhibits the Sod1p isoform. We observed unique SOD and catalase zymogram profiles for all the analyzed yeasts and we propose this technique as a new approach for Saccharomyces and non-Saccharomyces yeast strains differentiation. In addition, we observed functional correlations between SOD and catalase enzyme activities, accumulation of essential metabolites, such as glutathione and trehalose, and the fermentative performance of different yeasts strains with industrial relevance.
Contamine, V; Picard, M
2000-06-01
Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho(+) mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho(0) petites) and/or lead to truncated forms (rho(-)) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho(+) genome depends on a centromere-like structure dispensable for the maintenance of rho(-) mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.
Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.
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.
Haploid deletion strains of Saccharomyces cerevisiae that determine survival during space flight
NASA Astrophysics Data System (ADS)
Johanson, Kelly; Allen, Patricia L.; Gonzalez-Villalobos, Romer A.; Nesbit, Jacqueline; Nickerson, Cheryl A.; Höner zu Bentrup, Kerstin; Wilson, James W.; Ramamurthy, Rajee; D'Elia, Riccardo; Muse, Kenneth E.; Hammond, Jeffrey; Freeman, Jake; Stodieck, Louis S.; Hammond, Timothy G.
2007-02-01
This study identifies genes that determine survival during a space flight, using the model eukaryotic organism, Saccharomyces cerevisiae. Select strains of a haploid yeast deletion series grew during storage in distilled water in space, but not in ground based static or clinorotation controls. The survival advantages in space in distilled water include a 133-fold advantage for the deletion of PEX19, a chaperone and import receptor for newly- synthesized class I peroxisomal membrane proteins, to 77-40 fold for deletion strains lacking elements of aerobic respiration, isocitrate metabolism, and mitochondrial electron transport. Following automated addition of rich growth media, the space flight was associated with a marked survival advantage of strains with deletions in catalytically active genes including hydrolases, oxidoreductases and transferases. When compared to static controls, space flight was associated with a marked survival disadvantage of deletion strains lacking transporter, antioxidant and catalytic activity. This study identifies yeast deletion strains with a survival advantage during storage in distilled water and space flight, and amplifies our understanding of the genes critical for survival in space.
Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics
Tan, Dun-Xian; Manchester, Lucien C.; Qin, Lilan; Reiter, Russel J.
2016-01-01
Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT) is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s). Melatonin protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting the mitochondrial permeability transition pore (MPTP), and activating uncoupling proteins (UCPs). Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria. PMID:27999288
Mitochondrial threshold effects.
Rossignol, Rodrigue; Faustin, Benjamin; Rocher, Christophe; Malgat, Monique; Mazat, Jean-Pierre; Letellier, Thierry
2003-01-01
The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases. PMID:12467494
Wang, H T; Rahaim, P; Robbins, P; Yocum, R R
1994-11-01
The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate. DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase [G3PDase; EC 1.1.1.8]) homologous to several other eukaryotic G3PDases. DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S. cerevisiae, a close relative of S. diastaticus. The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity. Disruption of DAR1 in a haploid S. cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ogawa, Tetsuhiro, E-mail: atetsu@mail.ecc.u-tokyo.ac.jp; Shimizu, Ayano; Takahashi, Kazutoshi
2014-08-15
Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrialmore » dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.« less
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
Saccharomyces boulardii CNCM I-745 in different clinical conditions.
Dinleyici, Ener Cagri; Kara, Ates; Ozen, Metehan; Vandenplas, Yvan
2014-11-01
Saccharomyces boulardii is a well-known probiotic worldwide, and there are numerous studies including experimental and clinical trials in children and adults by the use of S. boulardii. The objective of the present report is to provide an update on the evidence for the efficacy of S. boulardii CNCM I-745 in different clinical conditions. Saccharomyces boulardii is one of the best-studied probiotics in acute gastroenteritis (AGE) and is shown to be safe and to reduce the duration of diarrhea and hospitalization by about 1 day. Saccharomyces boulardii is one of the recommended probiotics for AGE in children by European Society of Paediatric Infectious Diseases and European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN). Saccharomyces boulardii is also a recommended probiotic for the prevention of antibiotic-associated diarrhea (AAD), and a recent study showed promising results for the treatment of AAD in children. There is insufficient evidence to recommend the long-term use of S. boulardii in patients with irritable bowel syndrome. Although some clinical studies showed positive effects of S. boulardii on inflammation, there is no clinical evidence that S. boulardii is useful in inflammatory bowel disease. Saccharomyces boulardii could be used in patients needing Helicobacter pylori eradication because the S. boulardii improves compliance, decreases the side effects and moderately increases the eradication rate. There are new promising results (improving feeding tolerance, shorten the course of hyperbilirubinemia), but we do still not recommend the routine use of S. boulardii in newborns. Saccharomyces boulardii CNCM I-745 is a good example for the statement that each probiotic needs to be taxonomically characterized and its efficacy and safety should be documented individually in different clinical settings.
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
Koch, Barbara; Tucey, Timothy M; Lo, Tricia L; Novakovic, Stevan; Boag, Peter; Traven, Ana
2017-01-01
The interactions of mitochondria with the endoplasmic reticulum (ER) are crucial for maintaining proper mitochondrial morphology, function and dynamics. This enables cells to utilize their mitochondria optimally for energy production and anabolism, and it further provides for metabolic control over developmental decisions. In fungi, a key mechanism by which ER and mitochondria interact is via a membrane tether, the protein complex ERMES (ER-Mitochondria Encounter Structure). In the model yeast Saccharomyces cerevisiae , the mitochondrial GTPase Gem1 interacts with ERMES, and it has been proposed to regulate its activity. Here we report on the first characterization of Gem1 in a human fungal pathogen. We show that in Candida albicans Gem1 has a dominant role in ensuring proper mitochondrial morphology, and our data is consistent with Gem1 working with ERMES in this role. Mitochondrial respiration and steady state cellular phospholipid homeostasis are not impacted by inactivation of GEM1 in C. albicans . There are two major virulence-related consequences of disrupting mitochondrial morphology by GEM1 inactivation: C. albicans becomes hypersusceptible to cell wall stress, and is unable to grow invasively. In the gem1 Δ / Δ mutant, it is specifically the invasive capacity of hyphae that is compromised, not the ability to transition from yeast to hyphal morphology, and this phenotype is shared with ERMES mutants. As a consequence of the hyphal invasion defect, the gem1 Δ / Δ mutant is drastically hypovirulent in the worm infection model. Activation of the mitogen activated protein (MAP) kinase Cek1 is reduced in the gem1 Δ / Δ mutant, and this function could explain both the susceptibility to cell wall stress and lack of invasive growth. This result establishes a new, respiration-independent mechanism of mitochondrial control over stress signaling and hyphal functions in C. albicans . We propose that ER-mitochondria interactions and the ER-Mitochondria Organizing
Koch, Barbara; Tucey, Timothy M.; Lo, Tricia L.; Novakovic, Stevan; Boag, Peter; Traven, Ana
2017-01-01
The interactions of mitochondria with the endoplasmic reticulum (ER) are crucial for maintaining proper mitochondrial morphology, function and dynamics. This enables cells to utilize their mitochondria optimally for energy production and anabolism, and it further provides for metabolic control over developmental decisions. In fungi, a key mechanism by which ER and mitochondria interact is via a membrane tether, the protein complex ERMES (ER-Mitochondria Encounter Structure). In the model yeast Saccharomyces cerevisiae, the mitochondrial GTPase Gem1 interacts with ERMES, and it has been proposed to regulate its activity. Here we report on the first characterization of Gem1 in a human fungal pathogen. We show that in Candida albicans Gem1 has a dominant role in ensuring proper mitochondrial morphology, and our data is consistent with Gem1 working with ERMES in this role. Mitochondrial respiration and steady state cellular phospholipid homeostasis are not impacted by inactivation of GEM1 in C. albicans. There are two major virulence-related consequences of disrupting mitochondrial morphology by GEM1 inactivation: C. albicans becomes hypersusceptible to cell wall stress, and is unable to grow invasively. In the gem1Δ/Δ mutant, it is specifically the invasive capacity of hyphae that is compromised, not the ability to transition from yeast to hyphal morphology, and this phenotype is shared with ERMES mutants. As a consequence of the hyphal invasion defect, the gem1Δ/Δ mutant is drastically hypovirulent in the worm infection model. Activation of the mitogen activated protein (MAP) kinase Cek1 is reduced in the gem1Δ/Δ mutant, and this function could explain both the susceptibility to cell wall stress and lack of invasive growth. This result establishes a new, respiration-independent mechanism of mitochondrial control over stress signaling and hyphal functions in C. albicans. We propose that ER-mitochondria interactions and the ER-Mitochondria Organizing Network
Constriction of the mitochondrial inner compartment is a priming event for mitochondrial division
Cho, Bongki; Cho, Hyo Min; Jo, Youhwa; Kim, Hee Dae; Song, Myungjae; Moon, Cheil; Kim, Hyongbum; Kim, Kyungjin; Sesaki, Hiromi; Rhyu, Im Joo; Kim, Hyun; Sun, Woong
2017-01-01
Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-membrane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2+ induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM–IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial division. PMID:28598422
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
Chen, Bin; Che, Tuanjie; Bai, Decheng; He, Xiangyi
2013-04-01
To evaluate the effects of non-Saccharomyces albicans metabolic products on the cell cycle distribution and proliferation of human umbilical vein endothelial cell ECV304 cells in vitro. The parallel dilution supernatant of Saccharomyces tropicalis, Saccharomyces krusei and Saccharomyces glabrata were prepared, and 1, 4, 16-fold(s) diluted concentration and control group were set up. The line of human umbilical vein endothelial cell ECV304 was cultured in vitro and treated by non-Saccharomyces albicans supernatant. The proliferous effect of ECV304 induced by non-Saccharomyces albicans supernatant after 24, 48, 72 h was detected by the methods of MTT, and the changes of cell density and cycle after 48 h were investigated by inverted microscope and flow cytometry. At the 24th hour, all of the higher concentration (1-fold) of non-Saccharomyces albicans supernatant and the 4-folds diluted Saccharomyces krusei could promote ECV304 proliferation(P < 0.05). After adding various non-Saccharomyces albicans supernatant at 48h and 72th hour, Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant significantly increased proliferation rate of ECV304, while Saccharomyces tropicalis supernatant group showed no significant change no matter which concentration was tested. At 48th hour after adding the non-Saccharomyces albicans supernatant, the ECV304 cells density treated by Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant were significantly higher under the inverted microscope. The G0/G1 population of ECV304 cells decreased while cell proliferation index (PI) increased after incubated with Saccharomyces krusei supernatant and Saccharomyces glabrata supernatant for 48 hours (P < 0.05). Saccharomyces tropicalis group showed no significant change (P > 0.05). The metabolic products of Sacharoymces krusei and Saccharomyces glabrata could induce proliferation of ECV304 cell, which suggests non-Saccharomyces albicans should be undergone more
Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando
2014-01-01
The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. PMID:25320081
Swerdlow, Russell H.
2012-01-01
Several sporadic neurodegenerative diseases display phenomena that directly or indirectly relate to mitochondrial function. Data suggesting altered mitochondrial function in these diseases could arise from mitochondrial DNA (mtDNA) are reviewed. Approaches for manipulating mitochondrial function and minimizing the downstream consequences of mitochondrial dysfunction are discussed. PMID:21902672
Oliver, N A; Wallace, D C
1982-01-01
Two mitochondrially synthesized marker polypeptides, MV-1 and MV-2, were found in human HeLa and HT1080 cells. These were assigned to the mitochondrial DNA in HeLa-HT1080 cybrids and hybrids by demonstrating their linkage to cytoplasmic genetic markers. These markers include mitochondrial DNA restriction site polymorphisms and resistance to chloramphenicol, an inhibitor of mitochondrial protein synthesis. In the absence of chloramphenicol, the expression of MV-1 and MV-2 in cybrids and hybrids was found to be directly proportional to the ratio of the parental mitochondrial DNAs. In the presence of chloramphenicol, the marker polypeptide linked to the chloramphenicol-sensitive mitochondrial DNA continued to be expressed. This demonstrated that resistant and sensitive mitochondrial DNAs can cooperate within a cell for gene expression and that the CAP-resistant allele was dominant or codominant to sensitive. Such cooperation suggests that mitochondrial DNAs can be exchanged between mitochondria. Images PMID:6955589
Saccharomyces cerevisiae possesses a stress-inducible glycyl-tRNA synthetase gene.
Chen, Shun-Jia; Wu, Yi-Hua; Huang, Hsiao-Yun; Wang, Chien-Chia
2012-01-01
Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydrogen peroxide addition, and high pH, while expression of GRS1 was somewhat repressed under those conditions. In addition, GlyRS2 (the enzyme encoded by GRS2) had a higher protein stability and a lower K(M) value for yeast tRNA(Gly) under heat shock conditions than under normal conditions. Moreover, GRS2 rescued the growth defect of a GRS1 knockout strain when highly expressed by a strong promoter at 37 °C, but not at the optimal temperature of 30 °C. These results suggest that GRS2 is actually an inducible gene that may function to rescue the activity of GRS1 under stress conditions.
The mitochondrial COB region in yeast codes for apocytochrome b and is mosaic.
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).
Clinical mitochondrial genetics
Chinnery, P.; Howell, N.; Andrews, R.; Turnbull, D.
1999-01-01
The last decade has been an age of enlightenment as far as mitochondrial pathology is concerned. Well established nuclear genetic diseases, such as Friedreich's ataxia,12 Wilson disease,3 and autosomal recessive hereditary spastic paraplegia,4 have been shown to have a mitochondrial basis, and we are just starting to unravel the complex nuclear genetic disorders which directly cause mitochondrial dysfunction (table 1). However, in addition to the 3 billion base pair nuclear genome, each human cell typically contains thousands of copies of a small, 16.5 kb circular molecule of double stranded DNA (fig 1). Mitochondrial DNA (mtDNA) accounts for only 1% of the total cellular nucleic acid content. It encodes for 13 polypeptides which are essential for aerobic metabolism and defects of the mitochondrial genome are an important cause of human disease.9293 Since the characterisation of the first pathogenic mtDNA defects in 1988,513 over 50 point mutations and well over 100 rearrangements of the mitochondrial genome have been associated with human disease9495 (http://www.gen.emory.edu/mitomap.html). These disorders form the focus of this article. Keywords: mitochondrial DNA; mitochondrial disease; heteroplasmy; genetic counselling PMID:10874629
Long, Aaron; Klimova, Nina; Kristian, Tibor
2017-10-01
NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.
Review of Saccharomyces boulardii as a treatment option in IBD.
Sivananthan, Kavitha; Petersen, Andreas Munk
2018-05-17
Review of the yeast Saccharomyces boulardii as a treatment option for the inflammatory bowel diseases (IBD) ulcerative colitis and Crohn's disease. IBD is caused by an inappropriate immune response to gut microbiota. Treatment options could therefore be prebiotics, probiotics, antibiotics and/or fecal transplant. In this review, we have looked at the evidence for the yeast S. boulardii as a treatment option. Searches in PubMed and the Cochrane Library with the MeSH words 'Saccharomyces boulardii AND IBD', 'Saccharomyces boulardii AND Inflammatory Bowel Disease', 'Saccharomyces boulardii AND ulcerative colitis' and 'Saccharomyces boulardii AND Crohn's disease' gave total a total of 80 articles. After exclusions because of irrelevance, articles in other languages and some articles that were not available, 16 articles were included in this review. Three of the clinical trials showed a positive effect of S. boulardii in IBD patients (two Crohn's disease, one ulcerative colitis), while there was one trial that didn't prove any effect (Crohn's disease). Included Animal trials and cell assays describes different anti-inflammatory mechanisms of S. boulardii supporting a possible effect when treating IBD patients. The number of studies of S. boulardii as treatment for IBD is limited. Furthermore, the existing trials have small populations and short duration. We do not have enough evidence to prove the effect of S. boulardii in IBD. Saccharomyces boulardii is, however, a plausible treatment option in the future, but more placebo-controlled clinical studies on both patients with ulcerative colitis and Crohn's disease are needed.
Kucharczyk, Roza; Salin, Bénédicte; di Rago, J-P
2009-08-01
The Leigh syndrome is a severe neurological disorder that has been associated with mutations affecting the mitochondrial energy transducing system. One of these mutations, T9176G, has been localized in the mitochondrial ATP6 gene encoding the Atp6p (or a) subunit of the ATP synthase. This mutation converts a highly conserved leucine residue into arginine within a presumed trans-membrane alpha-helical segment, at position 217 of Atp6p. The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation. However, the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase, we have created and analyzed the properties of a yeast strain bearing an equivalent of this mutation. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on previous partial biochemical characterization of human T9176G cells, it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been suggested. Interestingly, the T9176G yeast exhibits important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176G may not be limited to a bioenergetic deficiency.
Reversible infantile mitochondrial diseases.
Boczonadi, Veronika; Bansagi, Boglarka; Horvath, Rita
2015-05-01
Mitochondrial diseases are usually severe and progressive conditions; however, there are rare forms that show remarkable spontaneous recoveries. Two homoplasmic mitochondrial tRNA mutations (m.14674T>C/G in mt-tRNA(Glu)) have been reported to cause severe infantile mitochondrial myopathy in the first months of life. If these patients survive the first year of life by extensive life-sustaining measures they usually recover and develop normally. Another mitochondrial disease due to deficiency of the 5-methylaminomethyl-2-thiouridylate methyltransferase (TRMU) causes severe liver failure in infancy, but similar to the reversible mitochondrial myopathy, within the first year of life these infants may also recover completely. Partial recovery has been noted in some other rare forms of mitochondrial disease due to deficiency of mitochondrial tRNA synthetases and mitochondrial tRNA modifying enzymes. Here we summarize the clinical presentation of these unique reversible mitochondrial diseases and discuss potential molecular mechanisms behind the reversibility. Understanding these mechanisms may provide the key to treatments of potential broader relevance in mitochondrial disease, where for the majority of the patients no effective treatment is currently available.
miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor.
Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung
2014-11-28
Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.
Mehrotra, Arpit; Kanwal, Abhinav; Banerjee, Sanjay Kumar; Sandhir, Rajat
2015-06-01
Huntington's disease (HD) is a chronic neurodegenerative condition involving impaired mitochondrial functions. The present study evaluates the therapeutic potential of combined administration of mitochondrial modulators: alpha-lipoic acid and acetyl-l-carnitine on mitochondrial dysfunctions in 3-NP-induced HD. Our results reveal 3-NP administration resulted in compromise of mitochondrial functions in terms of: (1) impaired activity of mitochondrial respiratory chain enzymes, altered cytochrome levels, reduced histochemical staining of complex-II and IV, reduced in-gel activity of complex-I to V, and reduced mRNA expression of respiratory chain complexes; (2) enhanced mitochondrial oxidative stress indicated by increased malondialdehyde, protein carbonyls, reactive oxygen species and nitrite levels, along with decreased Mn-superoxide dismutase and catalase activity; (3) mitochondrial structural changes measured by mitochondrial swelling, reduced mitochondrial membrane potential and ultra-structure changes; (4) increased cytosolic cytochrome c levels, caspase-3 and -9 activity along with altered expression of apoptotic proteins (AIF, Bim, Bad, and Bax); and (5) impaired cognitive functions assessed using Morris water maze and Y-maze. Combination of mitochondrial modulators (alpha-lipoic acid + acetyl-l-carnitine) on the other hand ameliorated 3-NP-induced mitochondrial dysfunctions, oxidative stress, histologic alterations, and behavioral deficits, suggesting their therapeutic efficacy in the management of HD. Copyright © 2015 Elsevier Inc. All rights reserved.
Organization of the SUC gene family in Saccharomyces.
Carlson, M; Botstein, D
1983-01-01
The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains. Images PMID:6843548
Distribution patterns of Saccharomyces species in cultural landscapes of Germany.
Brysch-Herzberg, Michael; Seidel, Martin
2017-08-01
The distribution patterns of the three Saccharomyces species, Saccharomyces paradoxus, S. uvarum and S. cerevisiae, were investigated by a culture-dependent approach in order to understand better how these species propagate in the cultural landscape of Germany. Saccharomyces paradoxus, the closest relative of S. cerevisiae, is shown to be a true woodland species. It was frequently found in the soil under conifers indicating that S. paradoxus is an autochthonous member of the microbial community in this habitat. Physiological characteristics of the species like the Crabtree effect and high tolerance against ethanol suggest that the species is adapted to regular supply with considerable amounts of sugars. Additionally, a high proportion of the S. paradoxus strains isolated in this study are shown to have the rare ability to ferment melezitose. For these reasons, it is hypothesized that S. paradoxus may be closely associated with the honeydew system in forests. Saccharomyces cerevisiae was rare in most habitats and only exceeded the frequency of S. paradoxus in habitats characterized by modern agricultural mass production of fruit. Both the landscape structure and the agricultural system heavily influence the frequencies of Saccharomyces species. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Mitochondrial medicine for neurodegenerative diseases.
Du, Heng; Yan, Shirley ShiDu
2010-05-01
Mitochondrial dysfunction has been reported in a wide array of neurological disorders ranging from neuromuscular to neurodegenerative diseases. Recent studies on neurodegenerative diseases have revealed that mitochondrial pathology is generally found in inherited or sporadic neurodegenerative diseases and is believed to be involved in the pathophysiological process of these diseases. Commonly seen types of mitochondrial dysfunction in neurodegenerative diseases include excessive free radical generation, lowered ATP production, mitochondrial permeability transition, mitochondrial DNA lesions, perturbed mitochondrial dynamics and apoptosis. Mitochondrial medicine as an emerging therapeutic strategy targeted to mitochondrial dysfunction in neurodegenerative diseases has been proven to be of value, though this area of research is still at in its early stage. In this article, we report on recent progress in the development of several mitochondrial therapies including antioxidants, blockade of mitochondrial permeability transition, and mitochondrial gene therapy as evidence that mitochondrial medicine has promise in the treatment of neurodegenerative diseases. 2010 Elsevier Ltd. All rights reserved.
Nagi, Minoru; Tanabe, Koichi; Nakayama, Hironobu; Ueno, Keigo; Yamagoe, Satoshi; Umeyama, Takashi; Ohno, Hideaki; Miyazaki, Yoshitsugu
2016-01-01
ABSTRACT Candida glabrata, a haploid budding yeast, is the cause of severe systemic infections in immune-compromised hosts. The amount of free iron supplied to C. glabrata cells during systemic infections is severely limited by iron-chelating proteins such as transferrin. Thus, the iron-deficiency response in C. glabrata cells is thought to play important roles in their survival inside the host's body. In this study, we found that mitophagy was induced under iron-depleted conditions, and that the disruption of a gene homologous to ATG32, which is responsible for mitophagy in Saccharomyces cerevisiae, blocked mitophagy in C. glabrata. The mitophagic activity in C. glabrata cells was not detected on short-period exposure to nitrogen-starved conditions, which is a mitophagy-inducing condition used in S. cerevisiae. The mitophagy-deficient atg32Δ mutant of C. glabrata also exhibited decreased longevity under iron-deficient conditions. The mitochondrial membrane potential in Cgatg32Δ cells was significantly lower than that in wild-type cells under iron-depleted conditions. In a mouse model of disseminated infection, the Cgatg32Δ strain resulted in significantly decreased kidney and spleen fungal burdens compared with the wild-type strain. These results indicate that mitophagy in C. glabrata occurs in an iron-poor host tissue environment, and it may contribute to the longevity of cells, mitochondrial quality control, and pathogenesis. PMID:27347716
Wang, H T; Rahaim, P; Robbins, P; Yocum, R R
1994-01-01
The Saccharomyces diastaticus DAR1 gene was cloned by complementation in an Escherichia coli strain auxogrophic for glycerol-3-phosphate. DAR1 encodes an NADH-dependent dihydroxyacetone phosphate reductase (sn-glycerol-3-phosphate dehydrogenase [G3PDase; EC 1.1.1.8]) homologous to several other eukaryotic G3PDases. DAR1 is distinct from GUT2, which encodes a glucose-repressed mitochondrial G3PDase, but is identical to GPD1 from S. cerevisiae, a close relative of S. diastaticus. The level of DAR1-encoded G3PDase was increased about threefold in a medium of high osmolarity. Disruption of DAR1 in a haploid S. cerevisiae was not lethal but led to a decrease in cytoplasmic NADH-dependent G3PDase activity, an increase in osmotic sensitivity, and a 25% reduction in glycerol secretion from cells grown anaerobically on glucose. PMID:7961476
Bellon, Jennifer R; Schmid, Frank; Capone, Dimitra L; Dunn, Barbara L; Chambers, Paul J
2013-01-01
Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment.
miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor
Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung
2014-01-01
Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3′-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF. PMID:25431021
Evidence for Divergent Evolution of Growth Temperature Preference in Sympatric Saccharomyces Species
Gonçalves, Paula; Valério, Elisabete; Correia, Cláudia; de Almeida, João M. G. C. F.; Sampaio, José Paulo
2011-01-01
The genus Saccharomyces currently includes eight species in addition to the model yeast Saccharomyces cerevisiae, most of which can be consistently isolated from tree bark and soil. We recently found sympatric pairs of Saccharomyces species, composed of one cryotolerant and one thermotolerant species in oak bark samples of various geographic origins. In order to contribute to explain the occurrence in sympatry of Saccharomyces species, we screened Saccharomyces genomic data for protein divergence that might be correlated to distinct growth temperature preferences of the species, using the dN/dS ratio as a measure of protein evolution rates and pair-wise species comparisons. In addition to proteins previously implicated in growth at suboptimal temperatures, we found that glycolytic enzymes were among the proteins exhibiting higher than expected divergence when one cryotolerant and one thermotolerant species are compared. By measuring glycolytic fluxes and glycolytic enzymatic activities in different species and at different temperatures, we subsequently show that the unusual divergence of glycolytic genes may be related to divergent evolution of the glycolytic pathway aligning its performance to the growth temperature profiles of the different species. In general, our results support the view that growth temperature preference is a trait that may have undergone divergent selection in the course of ecological speciation in Saccharomyces. PMID:21674061
Gurvitz, Aner
2009-01-01
Our recognition of the mitochondria as being important sites of fatty acid biosynthesis is continuously unfolding, especially in light of new data becoming available on compromised fatty acid synthase type 2 (FASII) in mammals. For example, perturbed regulation of murine 17β-HSD8 encoding a component of the mitochondrial FASII enzyme 3-oxoacyl-thioester reductase is implicated in polycystic kidney disease. In addition, over-expression in mice of the Mecr gene coding for 2-trans-enoyl-thioester reductase, also of mitochondrial FASII, leads to impaired heart function. However, mouse knockouts for mitochondrial FASII have hitherto not been reported and, hence, there is a need to develop alternate metazoan models such as nematodes or fruit flies. Here, the identification of Caenorhabditis elegans W09H1.5/MECR-1 as a 2-trans-enoyl-thioester reductase of mitochondrial FASII is reported. To identify MECR-1, Saccharomyces cerevisiae etr1Δ mutant cells were employed that are devoid of mitochondrial 2-trans-enoyl-thioester reductase Etr1p. These yeast mutants fail to synthesize sufficient levels of lipoic acid or form cytochrome complexes, and cannot respire or grow on non-fermentable carbon sources. A mutant yeast strain ectopically expressing nematode mecr-1 was shown to contain reductase activity and resemble the self-complemented mutant strain for these phenotype characteristics. Since MECR-1 was not intentionally targeted for compartmentalization using a yeast mitochondrial leader sequence, this inferred that the protein represented a physiologically functional mitochondrial 2-trans-enoyl-thioester reductase. In accordance with published findings, RNAi-mediated knockdown of mecr-1 in C. elegans resulted in life span extension, presumably due to mitochondrial dysfunction. Moreover, old mecr-1(RNAi) worms had better internal organ appearance and were more mobile than control worms, indicating a reduced physiological age. This is the first report on RNAi work dedicated
Shahi, Puja; Gulshan, Kailash; Näär, Anders M; Moye-Rowley, W Scott
2010-07-15
The multiprotein transcriptional Mediator complex provides a key link between RNA polymerase II and upstream transcriptional activator proteins. Previous work has established that the multidrug resistance transcription factors Pdr1 and Pdr3 interact with the Mediator component Med15/Gal11 to drive normal levels of expression of the ATP-binding cassette transporter-encoding gene PDR5 in Saccharomyces cerevisiae. PDR5 transcription is induced upon loss of the mitochondrial genome (rho(0) cells) and here we provide evidence that this rho(0) induction is Med15 independent. A search through other known Mediator components determined that Med12/Srb8, a member of the CDK8 Mediator submodule, is required for rho(0) activation of PDR5 transcription. The CDK8 submodule contains the cyclin C homologue (CycC/Srb11), cyclin-dependent kinase Cdk8/Srb10, and the large Med13/Srb9 protein. Loss of these other proteins did not lead to the same block in PDR5 induction. Chromatin immunoprecipitation analyses demonstrated that Med15 is associated with the PDR5 promoter in both rho(+) and rho(0), whereas Med12 recruitment to this target promoter is highly responsive to loss of the mitochondrial genome. Coimmunoprecipitation experiments revealed that association of Pdr3 with Med12 can only be detected in rho(0) cells. These experiments uncover the unique importance of Med12 in activated transcription of PDR5 seen in rho(0) cells.
... disorder, something goes wrong with this process. Mitochondrial diseases are a group of metabolic disorders. Mitochondria are ... cells and cause damage. The symptoms of mitochondrial disease can vary. It depends on how many mitochondria ...
A novel mitochondrial carrier protein Mme1 acts as a yeast mitochondrial magnesium exporter.
Cui, Yixian; Zhao, Shanke; Wang, Juan; Wang, Xudong; Gao, Bingquan; Fan, Qiangwang; Sun, Fei; Zhou, Bing
2015-03-01
The homeostasis of magnesium (Mg2+), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. Previously, two mitochondrial Mg2+ importers, Mrs2 and Lpe10, were characterized for mitochondrial Mg2+ uptake. We now show that mitochondrial Mg2+ homeostasis is accurately controlled through the combined effects of previously known importers and a novel exporter, Mme1 (mitochondrial magnesium exporter 1). Mme1 belongs to the mitochondrial carrier family and was isolated for its mutation that is able to suppress the mrs2Δ respiration defect. Deletion of MME1 significantly increased steady-state mitochondrial Mg2+ concentration, while overexpression decreased it. Measurements of Mg2+ exit from proteoliposomes reconstituted with purified Mme1 provided definite evidence for Mme1 as an Mg2+ exporter. Our studies identified, for the first time, a mitochondrial Mg2+ exporter that works together with mitochondrial importers to ensure the precise control of mitochondrial Mg2+ homeostasis. Copyright © 2015 Elsevier B.V. All rights reserved.
Bachmann, Rosilla F; Wang, Yun; Yuan, Peixiong; Zhou, Rulun; Li, Xiaoxia; Alesci, Salvatore; Du, Jing; Manji, Husseini K
2009-07-01
Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.
Mitochondrial Nucleoid: Shield and Switch of the Mitochondrial Genome
2017-01-01
Mitochondria preserve very complex and distinctively unique machinery to maintain and express the content of mitochondrial DNA (mtDNA). Similar to chromosomes, mtDNA is packaged into discrete mtDNA-protein complexes referred to as a nucleoid. In addition to its role as a mtDNA shield, over 50 nucleoid-associated proteins play roles in mtDNA maintenance and gene expression through either temporary or permanent association with mtDNA or other nucleoid-associated proteins. The number of mtDNA(s) contained within a single nucleoid is a fundamental question but remains a somewhat controversial issue. Disturbance in nucleoid components and mutations in mtDNA were identified as significant in various diseases, including carcinogenesis. Significant interest in the nucleoid structure and its regulation has been stimulated in relation to mitochondrial diseases, which encompass diseases in multicellular organisms and are associated with accumulation of numerous mutations in mtDNA. In this review, mitochondrial nucleoid structure, nucleoid-associated proteins, and their regulatory roles in mitochondrial metabolism are briefly addressed to provide an overview of the emerging research field involving mitochondrial biology. PMID:28680532
Atamna, Hani; Mackey, Jeanette; Dhahbi, Joseph M
2012-01-01
Mitochondrial dysfunction (primary or secondary) is detrimental to intermediary metabolism. Therapeutic strategies to treat/prevent mitochondrial dysfunction could be valuable for managing metabolic and age-related disorders. Here, we review strategies proposed to treat mitochondrial impairment. We then concentrate on redox-active agents, with mild-redox potential, who shuttle electrons among specific cytosolic or mitochondrial redox-centers. We propose that specific redox agents with mild redox potential (-0.1 V; 0.1 V) improve mitochondrial function because they can readily donate or accept electrons in biological systems, thus they enhance metabolic activity and prevent reactive oxygen species (ROS) production. These agents are likely to lack toxic effects because they lack the risk of inhibiting electron transfer in redox centers. This is different from redox agents with strong negative (-0.4 V; -0.2 V) or positive (0.2 V; 0.4 V) redox potentials who alter the redox status of redox-centers (i.e., become permanently reduced or oxidized). This view has been demonstrated by testing the effect of several redox active agents on cellular senescence. Methylene blue (MB, redox potential ≅10 mV) appears to readily cycle between the oxidized and reduced forms using specific mitochondrial and cytosolic redox centers. MB is most effective in delaying cell senescence and enhancing mitochondrial function in vivo and in vitro. Mild-redox agents can alter the biochemical activity of specific mitochondrial components, which then in response alters the expression of nuclear and mitochondrial genes. We present the concept of mitochondrial electron-carrier bypass as a potential result of mild-redox agents, a method to prevent ROS production, improve mitochondrial function, and delay cellular aging. Thus, mild-redox agents may prevent/delay mitochondria-driven disorders. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.
Thermal resistance of Saccharomyces yeast ascospores in beers.
Milani, Elham A; Gardner, Richard C; Silva, Filipa V M
2015-08-03
The industrial production of beer ends with a process of thermal pasteurization. Saccharomyces cerevisiae and Saccharomyces pastorianus are yeasts used to produce top and bottom fermenting beers, respectively. In this research, first the sporulation rate of 12 Saccharomyces strains was studied. Then, the thermal resistance of ascospores of three S. cerevisiae strains (DSMZ 1848, DSMZ 70487, Ethanol Red(®)) and one strain of S. pastorianus (ATCC 9080) was determined in 4% (v/v) ethanol lager beer. D60 °C-values of 11.2, 7.5, 4.6, and 6.0 min and z-values of 11.7, 14.3, 12.4, and 12.7 °C were determined for DSMZ 1848, DSMZ 70487, ATCC 9080, and Ethanol Red(®), respectively. Lastly, experiments with 0 and 7% (v/v) beers were carried out to investigate the effect of ethanol content on the thermal resistance of S. cerevisiae (DSMZ 1848). D55 °C-values of 34.2 and 15.3 min were obtained for 0 and 7% beers, respectively, indicating lower thermal resistance in the more alcoholic beer. These results demonstrate similar spore thermal resistance for different Saccharomyces strains and will assist in the design of appropriate thermal pasteurization conditions for preserving beers with different alcohol contents. Copyright © 2015 Elsevier B.V. All rights reserved.
Characterization of Alcohol-induced Filamentous Growth in Saccharomyces cerevisiae
Lorenz, Michael C.; Cutler, N. Shane; Heitman, Joseph
2000-01-01
Diploid cells of the budding yeast Saccharomyces cerevisiae starved for nitrogen differentiate into a filamentous growth form. Poor carbon sources such as starches can also stimulate filamentation, whereas haploid cells undergo a similar invasive growth response in rich medium. Previous work has demonstrated a role for various alcohols, by-products of amino acid metabolism, in altering cellular morphology. We found that several alcohols, notably isoamyl alcohol and 1-butanol, stimulate filamentous growth in haploid cells in which this differentiation is normally repressed. Butanol also induces cell elongation and changes in budding pattern, leading to a pseudohyphal morphology, even in liquid medium. The filamentous colony morphology and cell elongation require elements of the pheromone-responsive MAPK cascade and TEC1, whereas components of the nutrient-sensing machinery, such as MEP2, GPA2, and GPR1, do not affect this phenomenon. A screen for 1-butanol–insensitive mutants identified additional proteins that regulate polarized growth (BUD8, BEM1, BEM4, and FIG1), mitochondrial function (MSM1, MRP21, and HMI1), and a transcriptional regulator (CHD1). Furthermore, we have also found that ethanol stimulates hyperfilamentation in diploid cells, again in a MAPK-dependent manner. Together, these results suggest that yeast may sense a combination of nutrient limitation and metabolic by-products to regulate differentiation. PMID:10637301
Nikulin, Jarkko; Krogerus, Kristoffer
2017-01-01
Abstract The lager yeast hybrid (Saccharomyces cerevisiae × Saccharomyces eubayanus) possesses two key characteristics that are essential for lager brewing: efficient sugar utilization and cold tolerance. Here we explore the possibility that the lager yeast phenotype can be recreated by hybridizing S. cerevisiae ale yeast with a number of cold‐tolerant Saccharomyces species including Saccharomyces arboricola, Saccharomyces eubayanus, Saccharomyces mikatae and Saccharomyces uvarum. Interspecies hybrids performed better than parental strains in lager brewing conditions (12°C and 12°P wort), with the S. mikatae hybrid performing as well as the S. eubayanus hybrid. Where the S. cerevisiae parent was capable of utilizing maltotriose, this trait was inherited by the hybrids. A greater production of higher alcohols and esters by the hybrids resulted in the production of more aromatic beers relative to the parents. Strong fermentation performance relative to the parents was dependent on ploidy, with polyploid hybrids (3n, 4n) performing better than diploid hybrids. All hybrids produced 4‐vinyl guaiacol, a smoke/clove aroma generally considered an off flavour in lager beer. This characteristic could however be eliminated by isolating spore clones from a fertile hybrid of S. cerevisiae and S. mikatae. The results suggest that S. eubayanus is dispensable when constructing yeast hybrids that express the typical lager yeast phenotype. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:28755430
Mitochondrial fatty acid synthesis, fatty acids and mitochondrial physiology.
Kastaniotis, Alexander J; Autio, Kaija J; Kerätär, Juha M; Monteuuis, Geoffray; Mäkelä, Anne M; Nair, Remya R; Pietikäinen, Laura P; Shvetsova, Antonina; Chen, Zhijun; Hiltunen, J Kalervo
2017-01-01
Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the "classic" cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both β-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum. Copyright © 2016 Elsevier B.V. All rights reserved.
De Nicola, Raffaele; Hazelwood, Lucie A.; De Hulster, Erik A. F.; Walsh, Michael C.; Knijnenburg, Theo A.; Reinders, Marcel J. T.; Walker, Graeme M.; Pronk, Jack T.; Daran, Jean-Marc; Daran-Lapujade, Pascale
2007-01-01
Transcriptional responses of the yeast Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. To investigate the context dependency of this transcriptional response and eliminate growth rate-dependent variations in transcription, yeast was grown under several chemostat regimens, resulting in various carbon (glucose), nitrogen (ammonium), zinc, and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified, and the set enabled the definition of the Zn-specific Zap1p regulon, comprised of 26 genes and characterized by a broader zinc-responsive element consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large number of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified. PMID:17933919
Vandenbosch, Davy; De Canck, Evelien; Dhondt, Inne; Rigole, Petra; Nelis, Hans J; Coenye, Tom
2013-12-01
Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Matsushika, Akinori; Hoshino, Tamotsu
2015-12-01
The Saccharomyces cerevisiae HAP4 gene encodes a transcription activator that plays a key role in controlling the expression of genes involved in mitochondrial respiration and reductive pathways. This work examines the effect of knockout of the HAP4 gene on aerobic ethanol production in a xylose-utilizing S. cerevisiae strain. A hap4-deleted recombinant yeast strain (B42-DHAP4) showed increased maximum concentration, production rate, and yield of ethanol compared with the reference strain MA-B42, irrespective of cultivation medium (glucose, xylose, or glucose/xylose mixtures). Notably, B42-DHAP4 was capable of producing ethanol from xylose as the sole carbon source under aerobic conditions, whereas no ethanol was produced by MA-B42. Moreover, the rate of ethanol production and ethanol yield (0.44 g/g) from the detoxified hydrolysate of wood chips was markedly improved in B42-DHAP4 compared to MA-B42. Thus, the results of this study support the view that deleting HAP4 in xylose-utilizing S. cerevisiae strains represents a useful strategy in ethanol production processes.
Saccharomyces cerevisiae as a model for the study of extranuclear functions of mammalian telomerase.
Simonicova, Lucia; Dudekova, Henrieta; Ferenc, Jaroslav; Prochazkova, Katarina; Nebohacova, Martina; Dusinsky, Roman; Nosek, Jozef; Tomaska, Lubomir
2015-11-01
The experimental evidence from the last decade made telomerase a prominent member of a family of moonlighting proteins performing different functions at various cellular loci. However, the study of extratelomeric functions of the catalytic subunit of mammalian telomerase (TERT) is often complicated by the fact that it is sometimes difficult to distinguish them from its role(s) at the chromosomal ends. Here, we present an experimental model for studying the extranuclear function(s) of mammalian telomerase in the yeast Saccharomyces cerevisiae. We demonstrate that the catalytic subunit of mammalian telomerase protects the yeast cells against oxidative stress and affects the stability of the mitochondrial genome. The advantage of using S. cerevisiae to study of mammalian telomerase is that (1) mammalian TERT does not interfere with its yeast counterpart in the maintenance of telomeres, (2) yeast telomerase is not localized in mitochondria and (3) it does not seem to be involved in the protection of cells against oxidative stress and stabilization of mtDNA. Thus, yeast cells can be used as a 'test tube' for reconstitution of mammalian TERT extranuclear function(s).
Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando
2014-11-28
The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Bellon, Jennifer R.; Schmid, Frank; Capone, Dimitra L.; Dunn, Barbara L.; Chambers, Paul J.
2013-01-01
Interspecific hybrids are commonplace in agriculture and horticulture; bread wheat and grapefruit are but two examples. The benefits derived from interspecific hybridisation include the potential of generating advantageous transgressive phenotypes. This paper describes the generation of a new breed of wine yeast by interspecific hybridisation between a commercial Saccharomyces cerevisiae wine yeast strain and Saccharomyces mikatae, a species hitherto not associated with industrial fermentation environs. While commercially available wine yeast strains provide consistent and reliable fermentations, wines produced using single inocula are thought to lack the sensory complexity and rounded palate structure obtained from spontaneous fermentations. In contrast, interspecific yeast hybrids have the potential to deliver increased complexity to wine sensory properties and alternative wine styles through the formation of novel, and wider ranging, yeast volatile fermentation metabolite profiles, whilst maintaining the robustness of the wine yeast parent. Screening of newly generated hybrids from a cross between a S. cerevisiae wine yeast and S. mikatae (closely-related but ecologically distant members of the Saccharomyces sensu stricto clade), has identified progeny with robust fermentation properties and winemaking potential. Chemical analysis showed that, relative to the S. cerevisiae wine yeast parent, hybrids produced wines with different concentrations of volatile metabolites that are known to contribute to wine flavour and aroma, including flavour compounds associated with non-Saccharomyces species. The new S. cerevisiae x S. mikatae hybrids have the potential to produce complex wines akin to products of spontaneous fermentation while giving winemakers the safeguard of an inoculated ferment. PMID:23614011
Ohshima, Yohei; Takata, Natsuhiko; Suzuki-Karasaki, Miki; Yoshida, Yukihiro; Tokuhashi, Yasuaki; Suzuki-Karasaki, Yoshihiro
2017-10-01
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent with high tumor-selective cytotoxicity. The congenital and acquired resistance of some cancer types including malignant melanoma and osteosarcoma impede the current TRAIL therapy of these cancers. Since fine tuning of the intracellular Ca2+ level is essential for cell function and survival, Ca2+ dynamics could be a promising target for cancer treatment. Recently, we demonstrated that mitochondrial Ca2+ removal increased TRAIL efficacy toward malignant melanoma and osteosarcoma cells. Here we report that mitochondrial Ca2+ overload leads to tumor-selective sensitization to TRAIL cytotoxicity. Treatment with the mitochondrial Na+/Ca2+ exchanger inhibitor CGP-37157 and oxidative phosphorylation inhibitor antimycin A and FCCP resulted in a rapid and persistent mitochondrial Ca2+ rise. These agents also increased TRAIL sensitivity in a tumor-selective manner with a switching from apoptosis to a nonapoptotic cell death. Moreover, we found that mitochondrial Ca2+ overload led to increased mitochondrial fragmentation, while mitochondrial Ca2+ removal resulted in mitochondrial hyperfusion. Regardless of their reciprocal actions on the mitochondrial dynamics, both interventions commonly exacerbated TRAIL-induced mitochondrial network abnormalities. These results expand our previous study and suggest that an appropriate level of mitochondrial Ca2+ is essential for maintaining the mitochondrial dynamics and the survival of these cells. Thus, disturbing mitochondrial Ca2+ homeostasis may serve as a promising approach to overcome the TRAIL resistance of these cancers with minimally compromising the tumor-selectivity.
United Mitochondrial Disease Foundation
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The DNLZ/HEP zinc-binding subdomain is critical for regulation of the mitochondrial chaperone HSPA9
Vu, Michael T; Zhai, Peng; Lee, Juhye; Guerra, Cecilia; Liu, Shirley; Gustin, Michael C; Silberg, Jonathan J
2012-01-01
Human mitochondrial DNLZ/HEP regulates the catalytic activity and solubility of the mitochondrial hsp70 chaperone HSPA9. Here, we investigate the role that the DNLZ zinc-binding and C-terminal subdomains play in regulating HSPA9. We show that truncations lacking portions of the zinc-binding subdomain (ZBS) do not affect the solubility of HSPA9 or its ATPase domain, whereas those containing the ZBS and at least 10 residues following this subdomain enhance chaperone solubility. Binding measurements further show that DNLZ requires its ZBS to form a stable complex with the HSPA9 ATPase domain, and ATP hydrolysis measurements reveal that the ZBS is critical for full stimulation of HSPA9 catalytic activity. We also examined if DNLZ is active in vivo. We found that DNLZ partially complements the growth of Δzim17Saccharomyces cerevisiae, and we discovered that a Zim17 truncation lacking a majority of the C-terminal subdomain strongly complements growth like full-length Zim17. These findings provide direct evidence that human DNLZ is a functional ortholog of Zim17. In addition, they implicate the pair of antiparallel β-strands that coordinate zinc in Zim17/DNLZ-type proteins as critical for binding and regulating hsp70 chaperones. PMID:22162012
The DNLZ/HEP zinc-binding subdomain is critical for regulation of the mitochondrial chaperone HSPA9.
Vu, Michael T; Zhai, Peng; Lee, Juhye; Guerra, Cecilia; Liu, Shirley; Gustin, Michael C; Silberg, Jonathan J
2012-02-01
Human mitochondrial DNLZ/HEP regulates the catalytic activity and solubility of the mitochondrial hsp70 chaperone HSPA9. Here, we investigate the role that the DNLZ zinc-binding and C-terminal subdomains play in regulating HSPA9. We show that truncations lacking portions of the zinc-binding subdomain (ZBS) do not affect the solubility of HSPA9 or its ATPase domain, whereas those containing the ZBS and at least 10 residues following this subdomain enhance chaperone solubility. Binding measurements further show that DNLZ requires its ZBS to form a stable complex with the HSPA9 ATPase domain, and ATP hydrolysis measurements reveal that the ZBS is critical for full stimulation of HSPA9 catalytic activity. We also examined if DNLZ is active in vivo. We found that DNLZ partially complements the growth of Δzim17 Saccharomyces cerevisiae, and we discovered that a Zim17 truncation lacking a majority of the C-terminal subdomain strongly complements growth like full-length Zim17. These findings provide direct evidence that human DNLZ is a functional ortholog of Zim17. In addition, they implicate the pair of antiparallel β-strands that coordinate zinc in Zim17/DNLZ-type proteins as critical for binding and regulating hsp70 chaperones. Copyright © 2011 The Protein Society.
de Moura, Michelle Barbi; Uppala, Radha; Zhang, Yuxun; Van Houten, Bennett; Goetzman, Eric S
2014-01-01
SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.
Cho, J H; Lee, Y K; Chae, C B
2001-12-30
The mitochondrial histone, Abf2p, of Saccharomyces cerevisiae is essential for the maintenance of mitochondrial DNA (mtDNA) and appears to play an important role in the recombination and copy number determination of mtDNA. Abf2p, encoded by a nuclear gene, is a member of HMG1 DNA-binding protein family and has two HMG1-Box domains, HMG1-Box A and B. To investigate the role of Abf2p in the control of mtDNA copy number, we asked if the in vivo functions of Abf2p are regulated by the possible modification such as phosphorylation. We found that the N-terminal extended segment (KRPT(21)S(22)) of HMG1-Box A is rapidly and specifically phosphorylated by cAMP-dependent protein kinase (PKA) in vitro. The phosphorylation in this region inhibits the binding of Abf2p to all kinds of DNA including four-way junction DNA and the supercoiling activity of Abf2p itself. The abf2 mutant cells with an abf2(T21A/S22A) allele defective in the phosphorylation site have a severe defect in the regulation of mtDNA content during glucose repression in vivo. These observations suggest that the phosphorylation via PKA, that is activated during glucose repression, may regulate the in vivo functions of Abf2p for the control of mtDNA content during shift from gluconeogenic to fermentative growth.
Magyar, Ildikó; Nyitrai-Sárdy, Diána; Leskó, Annamária; Pomázi, Andrea; Kállay, Miklós
2014-05-16
Organic acid production under oxygen-limited conditions has been thoroughly studied in the Saccharomyces species, but practically never investigated in Candida zemplinina, which seems to be an acidogenic species under oxidative laboratory conditions. In this study, several strains of C. zemplinina were tested for organic acid metabolism, in comparison with Saccharomyces cerevisiae, Saccharomyces uvarum and Candida stellata, under fermentative conditions. Only C. stellata produced significantly higher acidity in simple minimal media (SM) with low sugar content and two different nitrogen sources (ammonia or glutamic acid) at low level. However, the acid profile differed largely between the Saccharomyces and Candida species and showed inverse types of N-dependence in some cases. Succinic acid production was strongly enhanced on glutamic acid in Saccharomyces species, but not in Candida species. 2-oxoglutarate production was strongly supported on ammonium nitrogen in Candida species, but remained low in Saccharomyces. Candida species, C. stellata in particular, produced more pyruvic acid regardless of N-sources. From the results, we concluded that the anaerobic organic acid metabolisms of C. zemplinina and C. stellata are different from each other and also from that of the Saccharomyces species. In the formation of succinic acid, the oxidative pathway from glutamic acid seems to play little or no role in C. zemplinina. The reductive branch of the TCA cycle, however, produces acidic intermediates (malic, fumaric, and succinic acid) in a level comparable with the production of the Saccharomyces species. An unidentified organic acid, which was produced on glutamic acid only by the Candida species, needs further investigation. Copyright © 2014 Elsevier B.V. All rights reserved.
Human Mitochondrial Protein Database
National Institute of Standards and Technology Data Gateway
SRD 131 Human Mitochondrial Protein Database (Web, free access) The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.
Mitochondrial biogenesis: pharmacological approaches.
Valero, Teresa
2014-01-01
Organelle biogenesis is concomitant to organelle inheritance during cell division. It is necessary that organelles double their size and divide to give rise to two identical daughter cells. Mitochondrial biogenesis occurs by growth and division of pre-existing organelles and is temporally coordinated with cell cycle events [1]. However, mitochondrial biogenesis is not only produced in association with cell division. It can be produced in response to an oxidative stimulus, to an increase in the energy requirements of the cells, to exercise training, to electrical stimulation, to hormones, during development, in certain mitochondrial diseases, etc. [2]. Mitochondrial biogenesis is therefore defined as the process via which cells increase their individual mitochondrial mass [3]. Recent discoveries have raised attention to mitochondrial biogenesis as a potential target to treat diseases which up to date do not have an efficient cure. Mitochondria, as the major ROS producer and the major antioxidant producer exert a crucial role within the cell mediating processes such as apoptosis, detoxification, Ca2+ buffering, etc. This pivotal role makes mitochondria a potential target to treat a great variety of diseases. Mitochondrial biogenesis can be pharmacologically manipulated. This issue tries to cover a number of approaches to treat several diseases through triggering mitochondrial biogenesis. It contains recent discoveries in this novel field, focusing on advanced mitochondrial therapies to chronic and degenerative diseases, mitochondrial diseases, lifespan extension, mitohormesis, intracellular signaling, new pharmacological targets and natural therapies. It contributes to the field by covering and gathering the scarcely reported pharmacological approaches in the novel and promising field of mitochondrial biogenesis. There are several diseases that have a mitochondrial origin such as chronic progressive external ophthalmoplegia (CPEO) and the Kearns- Sayre syndrome (KSS
Kato, Michiko; Lin, Su-Ju
2014-01-01
Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD+ is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD+ homeostasis is essential for proper cellular function and aberrant NAD+ metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD+ metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD+ metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD+ metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD+ metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD+ metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD+-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD+ intermediates, and their potential roles in NAD+ homeostasis. To date, it remains unclear how NAD+ and NAD+ intermediates shuttle between different
Escobar-Henriques, Mafalda; Langer, Thomas
2006-01-01
A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.
Tak, Hyosun; Eun, Jung Woo; Kim, Jihye; Park, So Jung; Kim, Chongtae; Ji, Eunbyul; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Lee, Kyungbun; Kim, Wook; Nam, Suk Woo; Lee, Eun Kyung
2017-01-01
Mitochondrial morphology is dynamically regulated by the formation of small fragmented units or interconnected mitochondrial networks, and this dynamic morphological change is a pivotal process in normal mitochondrial function. In the present study, we identified a novel regulator responsible for the regulation of mitochondrial dynamics. An assay using CHANG liver cells stably expressing mitochondrial-targeted yellow fluorescent protein (mtYFP) and a group of siRNAs revealed that T-cell intracellular antigen protein-1 (TIA-1) affects mitochondrial morphology by enhancing mitochondrial fission. The function of TIA-1 in mitochondrial dynamics was investigated through various biological approaches and expression analysis in human specimen. Downregulation of TIA-1-enhanced mitochondrial elongation, whereas ectopic expression of TIA-1 resulted in mitochondria fragmentation. In addition, TIA-1 increased mitochondrial activity, including the rate of ATP synthesis and oxygen consumption. Further, we identified mitochondrial fission factor (MFF) as a direct target of TIA-1, and showed that TIA-1 promotes mitochondrial fragmentation by enhancing MFF translation. TIA-1 null cells had a decreased level of MFF and less mitochondrial Drp1, a critical factor for mitochondrial fragmentation, thereby enhancing mitochondrial elongation. Taken together, our results indicate that TIA-1 is a novel factor that facilitates mitochondrial dynamics by enhancing MFF expression and contributes to mitochondrial dysfunction. PMID:27612012
Foster, Andrew W; Dainty, Samantha J; Patterson, Carl J; Pohl, Ehmke; Blackburn, Hannah; Wilson, Clare; Hess, Corinna R; Rutherford, Julian C; Quaranta, Laura; Corran, Andy; Robinson, Nigel J
2014-07-01
The extreme resistance of Saccharomyces cerevisiae to copper is overcome by 2-(6-benzyl-2-pyridyl)quinazoline (BPQ), providing a chemical-biology tool which has been exploited in two lines of discovery. First, BPQ is shown to form a red (BPQ)2 Cu(I) complex and promote Ctr1-independent copper-accumulation in whole cells and in mitochondria isolated from treated cells. Multiple phenotypes, including loss of aconitase activity, are consistent with copper-BPQ mediated damage to mitochondrial iron-sulphur clusters. Thus, a biochemical basis of copper-toxicity in S. cerevisiae is analogous to other organisms. Second, iron regulons controlled by Aft1/2, Cth2 and Yap5 that respond to mitochondrial iron-sulphur cluster status are modulated by copper-BPQ causing iron hyper-accumulation via upregulated iron-import. Comparison of copper-BPQ treated, untreated and copper-only treated wild-type and fra2Δ by RNA-seq has uncovered a new candidate Aft1 target-gene (LSO1) and paralogous non-target (LSO2), plus nine putative Cth2 target-transcripts. Two lines of evidence confirm that Fra2 dominates basal repression of the Aft1/2 regulons in iron-replete cultures. Fra2-independent control of these regulons is also observed but CTH2 itself appears to be atypically Fra2-dependent. However, control of Cth2-target transcripts which is independent of CTH2 transcript abundance or of Fra2, is also quantified. Use of copper-BPQ supports a substantial contribution of metabolite repression to iron-regulation. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.
Foster, Andrew W; Dainty, Samantha J; Patterson, Carl J; Pohl, Ehmke; Blackburn, Hannah; Wilson, Clare; Hess, Corinna R; Rutherford, Julian C; Quaranta, Laura; Corran, Andy; Robinson, Nigel J
2014-01-01
The extreme resistance of Saccharomyces cerevisiae to copper is overcome by 2-(6-benzyl-2-pyridyl)quinazoline (BPQ), providing a chemical-biology tool which has been exploited in two lines of discovery. First, BPQ is shown to form a red (BPQ)2Cu(I) complex and promote Ctr1-independent copper-accumulation in whole cells and in mitochondria isolated from treated cells. Multiple phenotypes, including loss of aconitase activity, are consistent with copper-BPQ mediated damage to mitochondrial iron–sulphur clusters. Thus, a biochemical basis of copper-toxicity in S. cerevisiae is analogous to other organisms. Second, iron regulons controlled by Aft1/2, Cth2 and Yap5 that respond to mitochondrial iron–sulphur cluster status are modulated by copper-BPQ causing iron hyper-accumulation via upregulated iron-import. Comparison of copper-BPQ treated, untreated and copper-only treated wild-type and fra2Δ by RNA-seq has uncovered a new candidate Aft1 target-gene (LSO1) and paralogous non-target (LSO2), plus nine putative Cth2 target-transcripts. Two lines of evidence confirm that Fra2 dominates basal repression of the Aft1/2 regulons in iron-replete cultures. Fra2-independent control of these regulons is also observed but CTH2 itself appears to be atypically Fra2-dependent. However, control of Cth2-target transcripts which is independent of CTH2 transcript abundance or of Fra2, is also quantified. Use of copper-BPQ supports a substantial contribution of metabolite repression to iron-regulation. PMID:24895027
Robertson, Aaron; Schaltz, Kyle; Neimanis, Karina; Staples, James F; McDonald, Allison E
2016-10-01
Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.
Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering
Shiba-Fukushima, Kahori; Arano, Taku; Matsumoto, Gen; Inoshita, Tsuyoshi; Yoshida, Shigeharu; Ishihama, Yasushi; Ryu, Kwon-Yul; Nukina, Nobuyuki; Hattori, Nobutaka; Imai, Yuzuru
2014-01-01
The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin's ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved. PMID:25474007
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mkaouar-Rebai, Emna, E-mail: emna.mkaouar@gmail.com; Felhi, Rahma; Tabebi, Mouna
Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes ofmore » complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder. - Highlights: • We reported 2 patients with mitochondrial neuromuscular disorders. • The heteroplasmic MT-ATP6 9157G>A variation was reported. • A triplication of 9 bp in the mitochondrial NC7 region was detected. • The m.14924T>C transition (S60P) in the MT-CYB gene was found.« less
Papanicolaou, Kyriakos N.; Phillippo, Matthew M.
2012-01-01
Mitofusins (Mfn-1 and Mfn-2) are transmembrane proteins that bind and hydrolyze guanosine 5′-triphosphate to bring about the merging of adjacent mitochondrial membranes. This event is necessary for mitochondrial fusion, a biological process that is critical for organelle function. The broad effects of mitochondrial fusion on cell bioenergetics have been extensively studied, whereas the local effects of mitofusin activity on the structure and integrity of the fusing mitochondrial membranes have received relatively little attention. From the study of fusogenic proteins, theoretical models, and simulations, it has been noted that the fusion of biological membranes is associated with local perturbations on the integrity of the membrane that present in the form of lipidic holes which open on the opposing bilayers. These lipidic holes represent obligate intermediates that make the fusion process thermodynamically more favorable and at the same time induce leakage to the fusing membranes. In this perspectives article we present the relevant evidence selected from a spectrum of membrane fusion/leakage models and attempt to couple this information with observations conducted with cardiac myocytes or mitochondria deficient in Mfn-1 and Mfn-2. More specifically, we argue in favor of a situation whereby mitochondrial fusion in cardiac myocytes is coupled with outer mitochondrial membrane destabilization that is opportunistically employed during the process of mitochondrial permeability transition. We hope that these insights will initiate research on this new hypothesis of mitochondrial permeability transition regulation, a poorly understood mitochondrial function with significant consequences on myocyte survival. PMID:22636681
Nicotinamide Riboside and Mitochondrial Biogenesis
2018-03-15
Mitochondrial Diseases; Mitochondrial Myopathies; Progressive External Ophthalmoplegia; Progressive Ophthalmoplegia; Progressive; Ophthalmoplegia, External; Mitochondria DNA Deletion; MELAS; Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-Like Episodes; Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-Like Episodes (MELAS Syndrome)
Sun, Ren; Eriksson, Staffan
2014-01-01
Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of deoxynucleosides in the synthesis of the DNA precursors required for mitochondrial DNA (mtDNA) replication and are essential for mitochondrial function. Antiviral nucleosides are known to cause toxic mitochondrial side effects. Here, we examined the effects of 3′-azido-2′,3′-dideoxythymidine (AZT) (zidovudine) on mitochondrial TK2 and dGK levels and found that AZT treatment led to downregulation of mitochondrial TK2 and dGK in U2OS cells, whereas cytosolic deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) levels were not affected. The AZT effects on mitochondrial TK2 and dGK were similar to those of oxidants (e.g., hydrogen peroxide); therefore, we examined the oxidative effects of AZT. We found a modest increase in cellular reactive oxygen species (ROS) levels in the AZT-treated cells. The addition of uridine to AZT-treated cells reduced ROS levels and protein oxidation and prevented the degradation of mitochondrial TK2 and dGK. In organello studies indicated that the degradation of mitochondrial TK2 and dGK is a mitochondrial event. These results suggest that downregulation of mitochondrial TK2 and dGK may lead to decreased mitochondrial DNA precursor pools and eventually mtDNA depletion, which has significant implications for the regulation of mitochondrial nucleotide biosynthesis and for antiviral therapy using nucleoside analogs. PMID:25182642
The power of yeast to model diseases of the powerhouse of the cell
Baile, Matthew G.; Claypool, Steven M
2013-01-01
Mitochondria participate in a variety of cellular functions. As such, mitochondrial diseases exhibit numerous clinical phenotypes. Because mitochondrial functions are highly conserved between humans and Saccharomyces cerevisiae, yeast are an excellent model to study mitochondrial disease, providing insight into both physiological and pathophysiological processes. PMID:23276920
Schuller, Dorit; Pereira, Leonor; Alves, Hugo; Cambon, Brigitte; Dequin, Sylvie; Casal, Margarida
2007-08-01
One hundred isolates of the commercial Saccharomyces cerevisiae strain Zymaflore VL1 were recovered from spontaneous fermentations carried out with grapes collected from vineyards located close to wineries in the Vinho Verde wine region of Portugal. Isolates were differentiated based on their mitochondrial DNA restriction patterns and the evaluation of genetic polymorphisms was carried out by microsatellite analysis, interdelta sequence typing and pulsed-field gel electrophoresis (PFGE). Genetic patterns were compared to those obtained for 30 isolates of the original commercialized Zymaflore VL1 strain. Among the 100 recovered isolates we found a high percentage of chromosomal size variations, most evident for the smaller chromosomes III and VI. Complete loss of heterozygosity was observed for two isolates that had also lost chromosomal heteromorphism; their growth and fermentative capacity in a synthetic must medium was also affected. A considerably higher number of variant patterns for interdelta sequence amplifications was obtained for grape-derived strains compared to the original VL1 isolates. Our data show that the long-term presence of strain VL1 in natural grapevine environments induced genetic changes that can be detected using different fingerprinting methods. The observed genetic changes may reflect adaptive mechanisms to changed environmental conditions that yeast cells encounter during their existence in nature. (c) 2007 John Wiley & Sons, Ltd.
Sulfate Assimilation Mediates Tellurite Reduction and Toxicity in Saccharomyces cerevisiae▿†
Ottosson, Lars-Göran; Logg, Katarina; Ibstedt, Sebastian; Sunnerhagen, Per; Käll, Mikael; Blomberg, Anders; Warringer, Jonas
2010-01-01
Despite a century of research and increasing environmental and human health concerns, the mechanistic basis of the toxicity of derivatives of the metalloid tellurium, Te, in particular the oxyanion tellurite, Te(IV), remains unsolved. Here, we provide an unbiased view of the mechanisms of tellurium metabolism in the yeast Saccharomyces cerevisiae by measuring deviations in Te-related traits of a complete collection of gene knockout mutants. Reduction of Te(IV) and intracellular accumulation as metallic tellurium strongly correlated with loss of cellular fitness, suggesting that Te(IV) reduction and toxicity are causally linked. The sulfate assimilation pathway upstream of Met17, in particular, the sulfite reductase and its cofactor siroheme, was shown to be central to tellurite toxicity and its reduction to elemental tellurium. Gene knockout mutants with altered Te(IV) tolerance also showed a similar deviation in tolerance to both selenite and, interestingly, selenomethionine, suggesting that the toxicity of these agents stems from a common mechanism. We also show that Te(IV) reduction and toxicity in yeast is partially mediated via a mitochondrial respiratory mechanism that does not encompass the generation of substantial oxidative stress. The results reported here represent a robust base from which to attack the mechanistic details of Te(IV) toxicity and reduction in a eukaryotic organism. PMID:20675578
DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhao, Lantao; Li, Shuhong; Wang, Shilei, E-mail: wshlei@aliyun.com
The mitochondrial calcium uniporter (MCU) transports free Ca{sup 2+} into the mitochondrial matrix, maintaining Ca{sup 2+} homeostasis, thus regulates the mitochondrial morphology. Previous studies have indicated that there was closely crosstalk between MCU and mitochondrial fission during the process of ischemia/reperfusion injury. This study constructed a hypoxia reoxygenation model using primary hippocampus neurons to mimic the cerebral ischemia/reperfusion injury and aims to explore the exactly effect of MCU on the mitochondrial fission during the process of ischemia/reperfusion injury and so as the mechanisms. Our results found that the inhibitor of the MCU, Ru360, decreased mitochondrial Ca{sup 2+} concentration, suppressed themore » expression of mitochondrial fission protein Drp1, MIEF1 and Fis1, and thus improved mitochondrial morphology significantly. Whereas spermine, the agonist of the MCU, had no significant impact compared to the I/R group. This study demonstrated that the MCU regulates the process of mitochondrial fission by controlling the Ca{sup 2+} transport, directly upregulating mitochondrial fission proteins Drp1, Fis1 and indirectly reversing the MIEF1-induced mitochondrial fusion. It also provides new targets for brain protection during ischemia/reperfusion injury. - Highlights: • We study MCU with primary neuron culture. • MCU induces mitochondrial fission. • MCU reverses MIEF1 effect.« less
The clinical maze of mitochondrial neurology
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
Asmat, Shakila; Shaukat, Fouzia; Asmat, Raheela; Bakhat, Hafiz Faiq Siddique Gul; Asmat, Tauseef M
2018-03-01
To compare the efficacy of Saccharomyces boulardii and lactic acid producing probiotics in addition to usual treatment regimen to cure diarrhea among children (6 months to 5 years of age). Randomized controlled trial. Department of Pediatrics, Sheikh Zayed Hospital, Lahore, from February to July 2015. Children suffering from acute diarrhea were orally administered Saccharomyces boulardii and lactic acid producing probiotics for 5 days. The efficacy of administered probiotics was monitored. Patients were given Saccharomyces boulardii and lactic acid producing probiotics randomly to remove the bias. Two hundred patients randomly selected for trials; out of which, 100 were treated with Saccharomyces boulardii while the other 100 were supplemented with lactic acid concomitantly along with conventional diarrhea treatment. Results indicated that Saccharomyces boulardii treatment group has significantly higher efficacy rate (45%) compared to lactic acid producing probiotics (26%). This study concluded that Saccharomyces boulardii has a better efficacy compared to lactic acid and may be adopted as a probiotic of choice.
The Saccharomyces Genome Database Variant Viewer
Sheppard, Travis K.; Hitz, Benjamin C.; Engel, Stacia R.; Song, Giltae; Balakrishnan, Rama; Binkley, Gail; Costanzo, Maria C.; Dalusag, Kyla S.; Demeter, Janos; Hellerstedt, Sage T.; Karra, Kalpana; Nash, Robert S.; Paskov, Kelley M.; Skrzypek, Marek S.; Weng, Shuai; Wong, Edith D.; Cherry, J. Michael
2016-01-01
The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer. PMID:26578556
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.
Ozer, Hatice K; Dlouhy, Adrienne C; Thornton, Jeremy D; Hu, Jingjing; Liu, Yilin; Barycki, Joseph J; Balk, Janneke; Outten, Caryn E
2015-11-13
The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. Although all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, whereas ruling out significant roles for Erv1 or Mia40 in these pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
Peris, D; Pérez-Través, L; Belloch, C; Querol, A
2016-02-01
Wine fermentation and innovation have focused mostly on Saccharomyces cerevisiae strains. However, recent studies have shown that other Saccharomyces species can also be involved in wine fermentation or are useful for wine bouquet, such as Saccharomyces uvarum and Saccharomyces paradoxus. Many interspecies hybrids have also been isolated from wine fermentation, such as S. cerevisiae × Saccharomyces kudriavzevii hybrids. In this study, we explored the genetic diversity and fermentation performance of Spanish S. kudriavzevii strains, which we compared to other S. kudriavzevii strains. Fermentations of red and white grape musts were performed, and the phenotypic differences between Spanish S. kudriavzevii strains under different temperature conditions were examined. An ANOVA analysis suggested striking similarity between strains for glycerol and ethanol production, although a high diversity of aromatic profiles among fermentations was found. The sources of these phenotypic differences are not well understood and require further investigation. Although the Spanish S. kudriavzevii strains showed desirable properties, particularly must fermentations, the quality of their wines was no better than those produced with a commercial S. cerevisiae. We suggest hybridization or directed evolution as methods to improve and innovate wine. Copyright © 2015 Elsevier Ltd. All rights reserved.
Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu
2012-07-15
Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that
Bartz, Raquel R.; Fu, Ping; Suliman, Hagir B.; Crowley, Stephen D.; MacGarvey, Nancy Chou; Welty-Wolf, Karen; Piantadosi, Claude A.
2014-01-01
Acute kidney injury (AKI) contributes to the high morbidity and mortality of multi-system organ failure in sepsis. However, recovery of renal function after sepsis-induced AKI suggests active repair of energy-producing pathways. Here, we tested the hypothesis in mice that Staphyloccocus aureus sepsis damages mitochondrial DNA (mtDNA) in the kidney and activates mtDNA repair and mitochondrial biogenesis. Sepsis was induced in wild-type C57Bl/6J and Cox-8 Gfp-tagged mitochondrial-reporter mice via intraperitoneal fibrin clots embedded with S. aureus. Kidneys from surviving mice were harvested at time zero (control), 24, or 48 hours after infection and evaluated for renal inflammation, oxidative stress markers, mtDNA content, and mitochondrial biogenesis markers, and OGG1 and UDG mitochondrial DNA repair enzymes. We examined the kidneys of the mitochondrial reporter mice for changes in staining density and distribution. S. aureus sepsis induced sharp amplification of renal Tnf, Il-10, and Ngal mRNAs with decreased renal mtDNA content and increased tubular and glomerular cell death and accumulation of protein carbonyls and 8-OHdG. Subsequently, mtDNA repair and mitochondrial biogenesis was evidenced by elevated OGG1 levels and significant increases in NRF-1, NRF-2, and mtTFA expression. Overall, renal mitochondrial mass, tracked by citrate synthase mRNA and protein, increased in parallel with changes in mitochondrial GFP-fluorescence especially in proximal tubules in the renal cortex and medulla. Sub-lethal S. aureus sepsis thus induces widespread renal mitochondrial damage that triggers the induction of the renal mtDNA repair protein, OGG1, and mitochondrial biogenesis as a conspicuous resolution mechanism after systemic bacterial infection. PMID:24988481
Mitochondrial lipids in neurodegeneration.
Aufschnaiter, Andreas; Kohler, Verena; Diessl, Jutta; Peselj, Carlotta; Carmona-Gutierrez, Didac; Keller, Walter; Büttner, Sabrina
2017-01-01
Mitochondrial dysfunction is a common feature of many neurodegenerative diseases, including proteinopathies such as Alzheimer's or Parkinson's disease, which are characterized by the deposition of aggregated proteins in the form of insoluble fibrils or plaques. The distinct molecular processes that eventually result in mitochondrial dysfunction during neurodegeneration are well studied but still not fully understood. However, defects in mitochondrial fission and fusion, mitophagy, oxidative phosphorylation and mitochondrial bioenergetics have been linked to cellular demise. These processes are influenced by the lipid environment within mitochondrial membranes as, besides membrane structure and curvature, recruitment and activity of different proteins also largely depend on the respective lipid composition. Hence, the interaction of neurotoxic proteins with certain lipids and the modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration. Here, we discuss the relevance of mitochondrial lipids in the pathological alterations that result in neuronal demise, focussing on proteinopathies.
Mitochondrial DNA Damage and its Consequences for Mitochondrial Gene Expression
Cline, Susan D.
2012-01-01
How mitochondria process DNA damage and whether a change in the steady-state level of mitochondrial DNA damage (mtDNA) contributes to mitochondrial dysfunction are questions that fuel burgeoning areas of research into aging and disease pathogenesis. Over the past decade, researchers have identified and measured various forms of endogenous and environmental mtDNA damage and have elucidated mtDNA repair pathways. Interestingly, mitochondria do not appear to contain the full range of DNA repair mechanisms that operate in the nucleus, although mtDNA contains types of damage that are targets of each nuclear DNA repair pathway. The reduced repair capacity may, in part, explain the high mutation frequency of the mitochondrial chromosome. Since mtDNA replication is dependent on transcription, mtDNA damage may alter mitochondrial gene expression at three levels: by causing DNA polymerase γ nucleotide incorporation errors leading to mutations, by interfering with the priming of mtDNA replication by the mitochondrial RNA polymerase, or by inducing transcriptional mutagenesis or premature transcript termination. This review summarizes our current knowledge of mtDNA damage, its repair, and its effects on mtDNA integrity and gene expression. PMID:22728831
Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance
Fazakerley, Daniel J; Chaudhuri, Rima; Yang, Pengyi; Maghzal, Ghassan J; Thomas, Kristen C; Krycer, James R; Humphrey, Sean J; Parker, Benjamin L; Fisher-Wellman, Kelsey H; Meoli, Christopher C; Hoffman, Nolan J; Diskin, Ciana; Burchfield, James G; Cowley, Mark J; Kaplan, Warren; Modrusan, Zora; Kolumam, Ganesh; Yang, Jean YH; Chen, Daniel L; Samocha-Bonet, Dorit; Greenfield, Jerry R; Hoehn, Kyle L
2018-01-01
Insulin resistance in muscle, adipocytes and liver is a gateway to a number of metabolic diseases. Here, we show a selective deficiency in mitochondrial coenzyme Q (CoQ) in insulin-resistant adipose and muscle tissue. This defect was observed in a range of in vitro insulin resistance models and adipose tissue from insulin-resistant humans and was concomitant with lower expression of mevalonate/CoQ biosynthesis pathway proteins in most models. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered mitochondrial oxidants and insulin resistance while CoQ supplementation in either insulin-resistant cell models or mice restored normal insulin sensitivity. Specifically, lowering of mitochondrial CoQ caused insulin resistance in adipocytes as a result of increased superoxide/hydrogen peroxide production via complex II. These data suggest that mitochondrial CoQ is a proximal driver of mitochondrial oxidants and insulin resistance, and that mechanisms that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance. PMID:29402381
Tigchelaar, Wardit; Yu, Hongjuan; de Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Silljé, Herman H W
2015-01-15
Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy. Copyright © 2015 the American Physiological Society.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Barbier, Vincent; Lang, Diane; Valois, Sierra
Mitochondria are highly dynamic organelles that undergo continuous cycles of fission and fusion to maintain essential cellular functions. An imbalance between these two processes can result in many pathophysiological outcomes. Dengue virus (DENV) interacts with cellular organelles, including mitochondria, to successfully replicate in cells. This study used live-cell imaging and found an increase in mitochondrial length and respiration during DENV infection. The level of mitochondrial fission protein, Dynamin-related protein 1 (Drp1), was decreased on mitochondria during DENV infection, as well as Drp1 phosphorylated on serine 616, which is important for mitochondrial fission. DENV proteins NS4b and NS3 were also associatedmore » with subcellular fractions of mitochondria. Induction of fission through uncoupling of mitochondria or overexpression of Drp1 wild-type and Drp1 with a phosphomimetic mutation (S616D) significantly reduced viral replication. These results demonstrate that DENV infection causes an imbalance in mitochondrial dynamics by inhibiting Drp1-triggered mitochondrial fission, which promotes viral replication. - Highlights: •Mitochondrial length and respiration are increased during DENV infection. •DENV inhibits Drp1-triggered mitochondrial fission. •DENV titers are reduced by mitochondrial fragmentation, Drp1 WT and S616D expression. •Viral proteins NS4b and NS3 are associated with subcellular fractions of mitochondria.« less
Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina
2014-11-01
Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.
Gobert, Antoine; Tourdot-Maréchal, Raphaëlle; Morge, Christophe; Sparrow, Céline; Liu, Youzhong; Quintanilla-Casas, Beatriz; Vichi, Stefania; Alexandre, Hervé
2017-01-01
Nitrogen sources in the must are important for yeast metabolism, growth, and performance, and wine volatile compounds profile. Yeast assimilable nitrogen (YAN) deficiencies in grape must are one of the main causes of stuck and sluggish fermentation. The nitrogen requirement of Saccharomyces cerevisiae metabolism has been described in detail. However, the YAN preferences of non-Saccharomyces yeasts remain unknown despite their increasingly widespread use in winemaking. Furthermore, the impact of nitrogen consumption by non-Saccharomyces yeasts on YAN availability, alcoholic performance and volatile compounds production by S. cerevisiae in sequential fermentation has been little studied. With a view to improving the use of non-Saccharomyces yeasts in winemaking, we studied the use of amino acids and ammonium by three strains of non-Saccharomyces yeasts (Starmerella bacillaris, Metschnikowia pulcherrima, and Pichia membranifaciens) in grape juice. We first determined which nitrogen sources were preferentially used by these yeasts in pure cultures at 28 and 20°C (because few data are available). We then carried out sequential fermentations at 20°C with S. cerevisiae, to assess the impact of the non-Saccharomyces yeasts on the availability of assimilable nitrogen for S. cerevisiae. Finally, 22 volatile compounds were quantified in sequential fermentation and their levels compared with those in pure cultures of S. cerevisiae. We report here, for the first time, that non-Saccharomyces yeasts have specific amino-acid consumption profiles. Histidine, methionine, threonine, and tyrosine were not consumed by S. bacillaris, aspartic acid was assimilated very slowly by M. pulcherrima, and glutamine was not assimilated by P. membranifaciens. By contrast, cysteine appeared to be a preferred nitrogen source for all non-Saccharomyces yeasts. In sequential fermentation, these specific profiles of amino-acid consumption by non-Saccharomyces yeasts may account for some of the
Gobert, Antoine; Tourdot-Maréchal, Raphaëlle; Morge, Christophe; Sparrow, Céline; Liu, Youzhong; Quintanilla-Casas, Beatriz; Vichi, Stefania; Alexandre, Hervé
2017-01-01
Nitrogen sources in the must are important for yeast metabolism, growth, and performance, and wine volatile compounds profile. Yeast assimilable nitrogen (YAN) deficiencies in grape must are one of the main causes of stuck and sluggish fermentation. The nitrogen requirement of Saccharomyces cerevisiae metabolism has been described in detail. However, the YAN preferences of non- Saccharomyces yeasts remain unknown despite their increasingly widespread use in winemaking. Furthermore, the impact of nitrogen consumption by non- Saccharomyces yeasts on YAN availability, alcoholic performance and volatile compounds production by S. cerevisiae in sequential fermentation has been little studied. With a view to improving the use of non- Saccharomyces yeasts in winemaking, we studied the use of amino acids and ammonium by three strains of non- Saccharomyces yeasts ( Starmerella bacillaris, Metschnikowia pulcherrima , and Pichia membranifaciens ) in grape juice. We first determined which nitrogen sources were preferentially used by these yeasts in pure cultures at 28 and 20°C (because few data are available). We then carried out sequential fermentations at 20°C with S. cerevisiae , to assess the impact of the non- Saccharomyces yeasts on the availability of assimilable nitrogen for S. cerevisiae . Finally, 22 volatile compounds were quantified in sequential fermentation and their levels compared with those in pure cultures of S. cerevisiae . We report here, for the first time, that non- Saccharomyces yeasts have specific amino-acid consumption profiles. Histidine, methionine, threonine, and tyrosine were not consumed by S. bacillaris , aspartic acid was assimilated very slowly by M. pulcherrima , and glutamine was not assimilated by P. membranifaciens . By contrast, cysteine appeared to be a preferred nitrogen source for all non- Saccharomyces yeasts. In sequential fermentation, these specific profiles of amino-acid consumption by non- Saccharomyces yeasts may account for
Mitochondrial Dynamics in Diabetes
Galloway, Chad A.; Jhun, Bong Sook; Yu, Tianzheng
2011-01-01
Abstract Mitochondria are at the center of cellular energy metabolism and regulate cell life and death. The cell biological aspect of mitochondria, especially mitochondrial dynamics, has drawn much attention through implications in human pathology, including neurological disorders and metabolic diseases. Mitochondrial fission and fusion are the main processes governing the morphological plasticity and are controlled by multiple factors, including mechanochemical enzymes and accessory proteins. Emerging evidence suggests that mitochondrial dynamics plays an important role in metabolism–secretion coupling in pancreatic β-cells as well as complications of diabetes. This review describes an overview of mechanistic and functional aspects of mitochondrial fission and fusion, and comments on the recent advances connecting mitochondrial dynamics with diabetes and diabetic complications. Antioxid. Redox Signal. 14, 439–457. PMID:20518704
Jiang, Dan; Gao, Fei; Zhang, Yuelin; Wong, David Sai Hung; Li, Qing; Tse, Hung-Fat; Xu, Goufeng; Yu, Zhendong; Lian, Qizhou
2016-11-10
Recent studies have demonstrated that mesenchymal stem cells (MSCs) can donate mitochondria to airway epithelial cells and rescue mitochondrial damage in lung injury. We sought to determine whether MSCs could donate mitochondria and protect against oxidative stress-induced mitochondrial dysfunction in the cornea. Co-culturing of MSCs and corneal epithelial cells (CECs) indicated that the efficiency of mitochondrial transfer from MSCs to CECs was enhanced by Rotenone (Rot)-induced oxidative stress. The efficient mitochondrial transfer was associated with increased formation of tunneling nanotubes (TNTs) between MSCs and CECs, tubular connections that allowed direct intercellular communication. Separation of MSCs and CECs by a transwell culture system revealed no mitochiondrial transfer from MSCs to CECs and mitochondrial function was impaired when CECs were exposed to Rot challenge. CECs with or without mitochondrial transfer from MSCs displayed a distinct survival capacity and mitochondrial oxygen consumption rate. Mechanistically, increased filopodia outgrowth in CECs for TNT formation was associated with oxidative inflammation-activated NFκB/TNFαip2 signaling pathways that could be attenuated by reactive oxygen species scavenger N-acetylcysteine (NAC) treatment. Furthermore, MSCs grown on a decellularized porcine corneal scaffold were transplanted onto an alkali-injured eye in a rabbit model. Enhanced corneal wound healing was evident following healthy MSC scaffold transplantation. And transferred mitochondria was detected in corneal epithelium. In conclusion, mitochondrial transfer from MSCs provides novel protection for the cornea against oxidative stress-induced mitochondrial damage. This therapeutic strategy may prove relevant for a broad range of mitochondrial diseases.
Ridge, Perry G; Maxwell, Taylor J; Foutz, Spencer J; Bailey, Matthew H; Corcoran, Christopher D; Tschanz, JoAnn T; Norton, Maria C; Munger, Ronald G; O'Brien, Elizabeth; Kerber, Richard A; Cawthon, Richard M; Kauwe, John S K
2014-01-01
The mitochondria are essential organelles and are the location of cellular respiration, which is responsible for the majority of ATP production. Each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. The regulation of mitochondrial copy number by nuclear genes has been studied extensively. While mitochondrial variation has been associated with longevity and some of the diseases known to have reduced mitochondrial copy number, the role that the mitochondrial genome itself has in regulating mitochondrial copy number remains poorly understood. We analyzed the complete mitochondrial genomes from 1007 individuals randomly selected from the Cache County Study on Memory Health and Aging utilizing the inferred evolutionary history of the mitochondrial haplotypes present in our dataset to identify sequence variation and mitochondrial haplotypes associated with changes in mitochondrial copy number. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. We identified three variants associated with higher mitochondrial copy number and suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that causes changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes.
Lin, Pei-Hsuan; Lin, Hsien-Yi; Kuo, Cheng-Chin; Yang, Liang-Tung
2015-06-24
The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3's action remain unanswered. We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants. Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on
Powering Up Mitochondrial Functions to Treat Mitochondrial Disease
2017-10-01
derived hormone whose serum level correlates positively with the severity of mitochondrial cardiomyopathy (recently published with DOD grant support...o Pei lab has recently discovered that GDF15 is a heart-derived hormone that regulates body growth. Circulating GDF15 level correlates positively...Circulating GDF15 level correlates positively with the severity of mitochondrial cardiomyopathy and can be used as a serum biomarker for our 5
Genetic and phenotypic characterization of Saccharomyces spp. strains isolated in distillery plants.
Úbeda, Juan F; Chacón-Ocaña, Maria; Díaz-Hellín, Patricia; Ramírez-Pérez, Hector; Briones, Ana
2016-06-01
In this study, the biodiversity and some interesting phenotypic properties of Saccharomyces wild yeasts isolated in distilleries, at least 100 years old, located in La Mancha (Spain), were determined. Strains were genetically characterized by RFLP-mtDNA, which confirmed a great genetic biodiversity with 73% of strains with different mtDNA profiles, highlighting the large variability found in sweet and fermented piquette substrata. The predominant species identified was S. cerevisiae, followed by S. paradoxus and S. bayanus Due to the residual sugar-alcohol extraction process using warm water, a great number of thermophilic Saccharomyces strains with a great cell vitality were found to have potential use as starters in distillery plants. Interesting technological properties such as cell vitality and growth rate at different temperatures were studied. The thermal washing process for the extraction of alcohol and reducing sugars of some raw materials contributes to the presence of Saccharomyces strains with technologically interesting properties, especially in terms of vitality and resistance to high temperatures. Due to the fact that fermentation is spontaneous, the yeast biota of these environments, Saccharomyces and non-Saccharomyces, is very varied so these ecological niches are microbial reserves of undoubted biotechnological interest. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Pharmacological approaches to restore mitochondrial function
Andreux, Pénélope A.; Houtkooper, Riekelt H.; Auwerx, Johan
2014-01-01
Mitochondrial dysfunction is not only a hallmark of rare inherited mitochondrial disorders, but is also implicated in age-related diseases, including those that affect the metabolic and nervous system, such as type 2 diabetes and Parkinson’s disease. Numerous pathways maintain and/or restore proper mitochondrial function, including mitochondrial biogenesis, mitochondrial dynamics, mitophagy, and the mitochondrial unfolded protein response. New and powerful phenotypic assays in cell-based models, as well as multicellular organisms, have been developed to explore these different aspects of mitochondrial function. Modulating mitochondrial function has therefore emerged as an attractive therapeutic strategy for a range of diseases, which has spurred active drug discovery efforts in this area. PMID:23666487
Lukša, Juliana; Ravoitytė, Bazilė; Konovalovas, Aleksandras; Aitmanaitė, Lina; Butenko, Anzhelika; Serva, Saulius; Servienė, Elena
2017-01-01
Competitive and naturally occurring yeast killer phenotype is governed by coinfection with dsRNA viruses. Long-term relationship between the host cell and viruses appear to be beneficial and co-adaptive; however, the impact of viral dsRNA on the host gene expression has barely been investigated. Here, we determined the transcriptomic profiles of the host Saccharomyces cerevisiae upon the loss of the M-2 dsRNA alone and the M-2 along with the L-A-lus dsRNAs. We provide a comprehensive study based on the high-throughput RNA-Seq data, Gene Ontology and the analysis of the interaction networks. We identified 486 genes differentially expressed after curing yeast cells of the M-2 dsRNA and 715 genes affected by the elimination of both M-2 and L-A-lus dsRNAs. We report that most of the transcriptional responses induced by viral dsRNAs are moderate. Differently expressed genes are related to ribosome biogenesis, mitochondrial functions, stress response, biosynthesis of lipids and amino acids. Our study also provided insight into the virus–host and virus–virus interplays. PMID:28757599
Lukša, Juliana; Ravoitytė, Bazilė; Konovalovas, Aleksandras; Aitmanaitė, Lina; Butenko, Anzhelika; Yurchenko, Vyacheslav; Serva, Saulius; Servienė, Elena
2017-07-25
Competitive and naturally occurring yeast killer phenotype is governed by coinfection with dsRNA viruses. Long-term relationship between the host cell and viruses appear to be beneficial and co-adaptive; however, the impact of viral dsRNA on the host gene expression has barely been investigated. Here, we determined the transcriptomic profiles of the host Saccharomyces cerevisiae upon the loss of the M-2 dsRNA alone and the M-2 along with the L-A-lus dsRNAs. We provide a comprehensive study based on the high-throughput RNA-Seq data, Gene Ontology and the analysis of the interaction networks. We identified 486 genes differentially expressed after curing yeast cells of the M-2 dsRNA and 715 genes affected by the elimination of both M-2 and L-A-lus dsRNAs. We report that most of the transcriptional responses induced by viral dsRNAs are moderate. Differently expressed genes are related to ribosome biogenesis, mitochondrial functions, stress response, biosynthesis of lipids and amino acids. Our study also provided insight into the virus-host and virus-virus interplays.
Fouret, Gilles; Tolika, Evanthia; Lecomte, Jérôme; Bonafos, Béatrice; Aoun, Manar; Murphy, Michael P; Ferreri, Carla; Chatgilialoglu, Chryssostomos; Dubreucq, Eric; Coudray, Charles; Feillet-Coudray, Christine
2015-10-01
Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats. To do this, twenty-four 6week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF+MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production. These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats. Copyright © 2015 Elsevier B.V. All rights reserved.
Elastocapillary Instability in Mitochondrial Fission
NASA Astrophysics Data System (ADS)
Gonzalez-Rodriguez, David; Sart, Sébastien; Babataheri, Avin; Tareste, David; Barakat, Abdul I.; Clanet, Christophe; Husson, Julien
2015-08-01
Mitochondria are dynamic cell organelles that constantly undergo fission and fusion events. These dynamical processes, which tightly regulate mitochondrial morphology, are essential for cell physiology. Here we propose an elastocapillary mechanical instability as a mechanism for mitochondrial fission. We experimentally induce mitochondrial fission by rupturing the cell's plasma membrane. We present a stability analysis that successfully explains the observed fission wavelength and the role of mitochondrial morphology in the occurrence of fission events. Our results show that the laws of fluid mechanics can describe mitochondrial morphology and dynamics.
Naseeb, Samina; James, Stephen A; Alsammar, Haya; Michaels, Christopher J; Gini, Beatrice; Nueno-Palop, Carmen; Bond, Christopher J; McGhie, Henry; Roberts, Ian N; Delneri, Daniela
2017-06-01
Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain.
D'Elia, Riccardo; Allen, Patricia L; Johanson, Kelly; Nickerson, Cheryl A; Hammond, Timothy G
2005-06-01
This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.
NASA Technical Reports Server (NTRS)
D'Elia, Riccardo; Allen, Patricia L.; Johanson, Kelly; Nickerson, Cheryl A.; Hammond, Timothy G.
2005-01-01
This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which > 70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.
Itooka, Koki; Takahashi, Kazuo; Izawa, Shingo
2016-11-01
Cold atmospheric pressure plasma (CAP) has potential to be utilized as an alternative method for sterilization in food industries without thermal damage or toxic residues. In contrast to the bactericidal effects of CAP, information regarding the efficacy of CAP against eukaryotic microorganisms is very limited. Therefore, herein we investigated the effects of CAP on the budding yeast Saccharomyces cerevisiae, with a focus on the cellular response to CAP. The CAP treatment caused oxidative stress responses including the nuclear accumulation of the oxidative stress responsive transcription factor Yap1, mitochondrial fragmentation, and enhanced intracellular oxidation. Yeast cells also induced the expression of heat shock protein (HSP) genes and formation of Hsp104 aggregates when treated with CAP, suggesting that CAP denatures proteins. As phenomena unique to eukaryotic cells, the formation of cytoplasmic mRNP granules such as processing bodies and stress granules and changes in the intracellular localization of Ire1 were caused by the treatment with CAP, indicating that translational repression and endoplasmic reticulum (ER) stress were induced by the CAP treatment. These results suggest that the fungicidal effects of CAP are attributed to the multiple severe stresses.
Saccharomyces cerevisiae as a model for the study of extranuclear functions of mammalian telomerase
Simonicova, Lucia; Dudekova, Henrieta; Ferenc, Jaroslav; Prochazkova, Katarina; Nebohacova, Martina; Dusinsky, Roman; Nosek, Jozef; Tomaska, Lubomir
2015-01-01
The experimental evidence from the last decade made telomerase a prominent member of a family of moonlighting proteins performing different functions at various cellular loci. However, the study of extratelomeric function(s) of the catalytic subunit of mammalian telomerase (TERT) is often complicated by the fact that it is sometimes difficult to distinguish them from its role(s) at chromosomal ends. Here we describe an experimental model for studying extranuclear function(s) of mammalian telomerase in the yeast Saccharomyces cerevisiae. We demonstrate that the catalytic subunit of mammalian telomerase protects the yeast cells against oxidative stress and affect the stability of mitochondrial genome. The advantage of using S. cerevisiae for the study of mammalian telomerase is that (i) mammalian TERT does not interfere with its yeast counterpart in the maintenance of telomeres, (ii) yeast telomerase is not localized in mitochondria and (iii) it does not seem to be involved in the protection of the cells against oxidative stress and in the stabilization of mtDNA. Thus yeast cells can be used as a ‘test tube’ for reconstitution of mammalian TERT extranuclear function(s). PMID:25567623
Sayas, Enric; García-López, Federico; Serrano, Ramón
2015-09-01
We have compared the toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three DNA-intercalating fluorescent dyes widely used to stain DNA in gels. Safety data about ethidium bromide (EtBr) are contradictory, and two compounds of undisclosed structure (Redsafe and Gelred) have been proposed as safe alternatives. Our results indicate that all three compounds inhibit yeast growth, with Gelred being the most inhibitory and also the only one causing cell death. EtBr and Gelred, but not Redsafe, induce massive formation of petite (non-respiratory) mutants, but only EtBr induces massive loss of mitochondrial DNA. All three compounds increase reversion of a chromosomal point mutation (lys2-801(amber) ), with Gelred being the most mutagenic and Redsafe the least. These dyes are all cationic and are probably taken by cells through non-selective cation channels. We could measure the glucose-energized transport of EtBr and Gelred inside the cells, while uptake of Redsafe was below our detection limit. We conclude that although all three compounds are toxic and mutagenic in the yeast system, Redsafe is the safest for yeast, probably because of very limited uptake by these cells. Copyright © 2015 John Wiley & Sons, Ltd.
Higuchi-Sanabria, Ryo; Vevea, Jason D; Charalel, Joseph K; Sapar, Maria L; Pon, Liza A
2016-01-18
Increasing the stability or dynamics of the actin cytoskeleton can extend lifespan in C. elegans and S. cerevisiae . Actin cables of budding yeast, bundles of actin filaments that mediate cargo transport, affect lifespan control through effects on mitochondrial quality control. Sir2p, the founding member of the Sirtuin family of lifespan regulators, also affects actin cable dynamics, assembly, and function in mitochondrial quality control. Here, we obtained evidence for novel interactions between Sir2p and Sum1p, a transcriptional repressor that was originally identified through mutations that genetically suppress sir2 ∆ phenotypes unrelated to lifespan. We find that deletion of SUM1 in wild-type cells results in increased mitochondrial function and actin cable abundance. Furthermore, deletion of SUM1 suppresses defects in actin cables and mitochondria of sir2 ∆ yeast, and extends the replicative lifespan and cellular health span of sir2 ∆ cells. Thus, Sum1p suppresses Sir2p function in control of specific aging determinants and lifespan in budding yeast.
Shirakabe, Akihiro; Zhai, Peiyong; Ikeda, Yoshiyuki; Saito, Toshiro; Maejima, Yasuhiro; Hsu, Chiao-Po; Nomura, Masatoshi; Egashira, Kensuke; Levine, Beth; Sadoshima, Junichi
2016-03-29
Mitochondrial autophagy is an important mediator of mitochondrial quality control in cardiomyocytes. The occurrence of mitochondrial autophagy and its significance during cardiac hypertrophy are not well understood. Mice were subjected to transverse aortic constriction (TAC) and observed at multiple time points up to 30 days. Cardiac hypertrophy developed after 5 days, the ejection fraction was reduced after 14 days, and heart failure was observed 30 days after TAC. General autophagy was upregulated between 1 and 12 hours after TAC but was downregulated below physiological levels 5 days after TAC. Mitochondrial autophagy, evaluated by electron microscopy, mitochondrial content, and Keima with mitochondrial localization signal, was transiently activated at ≈3 to 7 days post-TAC, coinciding with mitochondrial translocation of Drp1. However, it was downregulated thereafter, followed by mitochondrial dysfunction. Haploinsufficiency of Drp1 abolished mitochondrial autophagy and exacerbated the development of both mitochondrial dysfunction and heart failure after TAC. Injection of Tat-Beclin 1, a potent inducer of autophagy, but not control peptide, on day 7 after TAC, partially rescued mitochondrial autophagy and attenuated mitochondrial dysfunction and heart failure induced by overload. Haploinsufficiency of either drp1 or beclin 1 prevented the rescue by Tat-Beclin 1, suggesting that its effect is mediated in part through autophagy, including mitochondrial autophagy. Mitochondrial autophagy is transiently activated and then downregulated in the mouse heart in response to pressure overload. Downregulation of mitochondrial autophagy plays an important role in mediating the development of mitochondrial dysfunction and heart failure, whereas restoration of mitochondrial autophagy attenuates dysfunction in the heart during pressure overload. © 2016 American Heart Association, Inc.
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.
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
The Saccharomyces Genome Database Variant Viewer.
Sheppard, Travis K; Hitz, Benjamin C; Engel, Stacia R; Song, Giltae; Balakrishnan, Rama; Binkley, Gail; Costanzo, Maria C; Dalusag, Kyla S; Demeter, Janos; Hellerstedt, Sage T; Karra, Kalpana; Nash, Robert S; Paskov, Kelley M; Skrzypek, Marek S; Weng, Shuai; Wong, Edith D; Cherry, J Michael
2016-01-04
The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the authoritative community resource for the Saccharomyces cerevisiae reference genome sequence and its annotation. In recent years, we have moved toward increased representation of sequence variation and allelic differences within S. cerevisiae. The publication of numerous additional genomes has motivated the creation of new tools for their annotation and analysis. Here we present the Variant Viewer: a dynamic open-source web application for the visualization of genomic and proteomic differences. Multiple sequence alignments have been constructed across high quality genome sequences from 11 different S. cerevisiae strains and stored in the SGD. The alignments and summaries are encoded in JSON and used to create a two-tiered dynamic view of the budding yeast pan-genome, available at http://www.yeastgenome.org/variant-viewer. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Nakashima-Kamimura, Naomi; Asoh, Sadamitsu; Ishibashi, Yoshitomo; Mukai, Yuri; Shidara, Yujiro; Oda, Hideaki; Munakata, Kae; Goto, Yu-Ichi; Ohta, Shigeo
2005-11-15
To investigate the regulatory system in mitochondrial biogenesis involving crosstalk between the mitochondria and nucleus, we found a factor named MIDAS (mitochondrial DNA absence sensitive factor) whose expression was enhanced by the absence of mitochondrial DNA (mtDNA). In patients with mitochondrial diseases, MIDAS expression was increased only in dysfunctional muscle fibers. A majority of MIDAS localized to mitochondria with a small fraction in the Golgi apparatus in HeLa cells. To investigate the function of MIDAS, we stably transfected HeLa cells with an expression vector carrying MIDAS cDNA or siRNA. Cells expressing the MIDAS protein and the siRNA constitutively showed an increase and decrease in the total mass of mitochondria, respectively, accompanying the regulation of a mitochondria-specific phospholipid, cardiolipin. In contrast, amounts of the mitochondrial DNA, RNA and proteins did not depend upon MIDAS. Thus, MIDAS is involved in the regulation of mitochondrial lipids, leading to increases of total mitochondrial mass in response to mitochondrial dysfunction.
Wang, Jiu-Qiang; Chen, Qian; Wang, Xianhua; Wang, Qiao-Chu; Wang, Yun; Cheng, He-Ping; Guo, Caixia; Sun, Qinmiao; Chen, Quan; Tang, Tie-Shan
2013-02-01
Huntington disease (HD) is an inherited, fatal neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Indications of oxidative stress are apparent in brain tissues from both HD patients and HD mouse models; however, the origin of this oxidant stress remains a mystery. Here, we used a yeast artificial chromosome transgenic mouse model of HD (YAC128) to investigate the potential connections between dysregulation of cytosolic Ca(2+) signaling and mitochondrial oxidative damage in HD cells. We found that YAC128 mouse embryonic fibroblasts exhibit a strikingly higher level of mitochondrial matrix Ca(2+) loading and elevated superoxide generation compared with WT cells, indicating that both mitochondrial Ca(2+) signaling and superoxide generation are dysregulated in HD cells. The excessive mitochondrial oxidant stress is critically dependent on mitochondrial Ca(2+) loading in HD cells, because blocking mitochondrial Ca(2+) uptake abolished elevated superoxide generation. Similar results were obtained using neurons from HD model mice and fibroblast cells from HD patients. More importantly, mitochondrial Ca(2+) loading in HD cells caused a 2-fold higher level of mitochondrial genomic DNA (mtDNA) damage due to the excessive oxidant generation. This study provides strong evidence to support a new causal link between dysregulated mitochondrial Ca(2+) signaling, elevated mitochondrial oxidant stress, and mtDNA damage in HD. Our results also indicate that reducing mitochondrial Ca(2+) uptake could be a therapeutic strategy for HD.
Coutinho, Eduarda; Batista, Cátia; Sousa, Fani; Queiroz, João; Costa, Diana
2017-03-06
Mitochondrial gene therapy seems to be a valuable and promising strategy to treat mitochondrial disorders. The use of a therapeutic vector based on mitochondrial DNA, along with its affinity to the site of mitochondria, can be considered a powerful tool in the reestablishment of normal mitochondrial function. In line with this and for the first time, we successfully cloned the mitochondrial gene ND1 that was stably maintained in multicopy pCAG-GFP plasmid, which is used to transform E. coli. This mitochondrial-gene-based plasmid was encapsulated into nanoparticles. Furthermore, the functionalization of nanoparticles with polymers, such as cellulose or gelatin, enhances their overall properties and performance for gene therapy. The fluorescence arising from rhodamine nanoparticles in mitochondria and a fluorescence microscopy study show pCAG-GFP-ND1-based nanoparticles' cell internalization and mitochondria targeting. The quantification of GFP expression strongly supports this finding. This work highlights the viability of gene therapy based on mitochondrial DNA instigating further in vitro research and clinical translation.
Mitochondrial functionality in female reproduction.
Gąsior, Łukasz; Daszkiewicz, Regina; Ogórek, Mateusz; Polański, Zbigniew
2017-01-04
In most animal species female germ cells are the source of mitochondrial genome for the whole body of individuals. As a source of mitochondrial DNA for future generations the mitochondria in the female germ line undergo dynamic quantitative and qualitative changes. In addition to maintaining the intact template of mitochondrial genome from one generation to another, mitochondrial role in oocytes is much more complex and pleiotropic. The quality of mitochondria determines the ability of meiotic divisions, fertilization ability, and activation after fertilization or sustaining development of a new embryo. The presence of normal number of functional mitochondria is also crucial for proper implantation and pregnancy maintaining. This article addresses issues of mitochondrial role and function in mammalian oocyte and presents new approaches in studies of mitochondrial function in female germ cells.
Gramegna, L L; Pisano, A; Testa, C; Manners, D N; D'Angelo, R; Boschetti, E; Giancola, F; Pironi, L; Caporali, L; Capristo, M; Valentino, M L; Plazzi, G; Casali, C; Dotti, M T; Cenacchi, G; Hirano, M; Giordano, C; Parchi, P; Rinaldi, R; De Giorgio, R; Lodi, R; Carelli, V; Tonon, C
2018-01-18
Mitochondrial neurogastrointestinal encephalopathy is a rare disorder due to recessive mutations in the thymidine phosphorylase gene, encoding thymidine phosphorylase protein required for mitochondrial DNA replication. Clinical manifestations include gastrointestinal dysmotility and diffuse asymptomatic leukoencephalopathy. This study aimed to elucidate the mechanisms underlying brain leukoencephalopathy in patients with mitochondrial neurogastrointestinal encephalopathy by correlating multimodal neuroradiologic features to postmortem pathology. Seven patients underwent brain MR imaging, including single-voxel proton MR spectroscopy and diffusion imaging. Absolute concentrations of metabolites calculated by acquiring unsuppressed water spectra at multiple TEs, along with diffusion metrics based on the tensor model, were compared with those of healthy controls using unpaired t tests in multiple white matters regions. Brain postmortem histologic, immunohistochemical, and molecular analyses were performed in 1 patient. All patients showed bilateral and nearly symmetric cerebral white matter hyperintensities on T2-weighted images, extending to the cerebellar white matter and brain stem in 4. White matter, N -acetylaspartate, creatine, and choline concentrations were significantly reduced compared with those in controls, with a prominent increase in the radial water diffusivity component. At postmortem examination, severe fibrosis of brain vessel smooth muscle was evident, along with mitochondrial DNA replication depletion in brain and vascular smooth-muscle and endothelial cells, without neuronal loss, myelin damage, or gliosis. Prominent periependymal cytochrome C oxidase deficiency was also observed. Vascular functional and histologic alterations account for leukoencephalopathy in mitochondrial neurogastrointestinal encephalopathy. Thymidine toxicity and mitochondrial DNA replication depletion may induce microangiopathy and blood-brain-barrier dysfunction, leading to
Alsammar, Haya; Michaels, Christopher J.; Gini, Beatrice; Nueno-Palop, Carmen; Bond, Christopher J.; McGhie, Henry; Roberts, Ian N.
2017-01-01
Two strains, D5088T and D5095, representing a novel yeast species belonging to the genus Saccharomyces were isolated from oak tree bark and surrounding soil located at an altitude of 1000 m above sea level in Saint Auban, France. Sequence analyses of the internal transcribed spacer (ITS) region and 26S rRNA D1/D2 domains indicated that the two strains were most closely related to Saccharomyces mikatae and Saccharomyces paradoxus. Genetic hybridization analyses showed that both strains are reproductively isolated from all other Saccharomyces species and, therefore, represent a distinct biological species. The species name Saccharomyces jurei sp. nov. is proposed to accommodate these two strains, with D5088T (=CBS 14759T=NCYC 3947T) designated as the type strain. PMID:28639933
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.
Parallel evolution of the make–accumulate–consume strategy in Saccharomyces and Dekkera yeasts
Rozpędowska, Elżbieta; Hellborg, Linda; Ishchuk, Olena P.; Orhan, Furkan; Galafassi, Silvia; Merico, Annamaria; Woolfit, Megan; Compagno, Concetta; Piškur, Jure
2011-01-01
Saccharomyces yeasts degrade sugars to two-carbon components, in particular ethanol, even in the presence of excess oxygen. This characteristic is called the Crabtree effect and is the background for the 'make–accumulate–consume' life strategy, which in natural habitats helps Saccharomyces yeasts to out-compete other microorganisms. A global promoter rewiring in the Saccharomyces cerevisiae lineage, which occurred around 100 mya, was one of the main molecular events providing the background for evolution of this strategy. Here we show that the Dekkera bruxellensis lineage, which separated from the Saccharomyces yeasts more than 200 mya, also efficiently makes, accumulates and consumes ethanol and acetic acid. Analysis of promoter sequences indicates that both lineages independently underwent a massive loss of a specific cis-regulatory element from dozens of genes associated with respiration, and we show that also in D. bruxellensis this promoter rewiring contributes to the observed Crabtree effect. PMID:21556056
Characterization of mitochondrial ferritin in Drosophila.
Missirlis, Fanis; Holmberg, Sara; Georgieva, Teodora; Dunkov, Boris C; Rouault, Tracey A; Law, John H
2006-04-11
Mitochondrial function depends on iron-containing enzymes and proteins, whose maturation requires available iron for biosynthesis of iron-sulfur clusters and heme. Little is known about how mitochondrial iron homeostasis is maintained, although the recent discovery of a mitochondrial ferritin in mammals and plants has uncovered a potential key player in the process. Here, we show that Drosophila melanogaster expresses mitochondrial ferritin from an intron-containing gene. It has high similarity to the mouse and human mitochondrial ferritin sequences and, as in mammals, is expressed mainly in testis. This ferritin contains a putative mitochondrial targeting sequence and an epitope-tagged version localizes to mitochondria in transfected cells. Overexpression of mitochondrial ferritin fails to alter both total-body iron levels and iron that is bound to secretory ferritins. However, the viability of iron-deficient flies is compromised by overexpression of mitochondrial ferritin, suggesting that it may sequester iron at the expense of other important cellular functions. The conservation of mitochondrial ferritin in an insect species underscores the importance of this iron-storage molecule.
Brandão, Rogelio L.; Castro, Ieso M.; Bambirra, Eduardo A.; Amaral, Sheila C.; Fietto, Luciano G.; Tropia, Maria José M.; Neves, Maria José; Dos Santos, Raquel G.; Gomes, Newton C. M.; Nicoli, Jacques R.
1998-01-01
As is the case for Saccharomyces boulardii, Saccharomyces cerevisiae W303 protects Fisher rats against cholera toxin (CT). The addition of glucose or dinitrophenol to cells of S. boulardii grown on a nonfermentable carbon source activated trehalase in a manner similar to that observed for S. cerevisiae. The addition of CT to the same cells also resulted in trehalase activation. Experiments performed separately on the A and B subunits of CT showed that both are necessary for activation. Similarly, the addition of CT but not of its separate subunits led to a cyclic AMP (cAMP) signal in both S. boulardii and S. cerevisiae. These data suggest that trehalase stimulation by CT probably occurred through the cAMP-mediated protein phosphorylation cascade. The requirement of CT subunit B for both the cAMP signal and trehalase activation indicates the presence of a specific receptor on the yeasts able to bind to the toxin, a situation similar to that observed for mammalian cells. This hypothesis was reinforced by experiments with 125I-labeled CT showing specific binding of the toxin to yeast cells. The adhesion of CT to a receptor on the yeast surface through the B subunit and internalization of the A subunit (necessary for the cAMP signal and trehalase activation) could be one more mechanism explaining protection against the toxin observed for rats treated with yeasts. PMID:9464394
Mitochondrial Ubiquitin Ligase in Cardiovascular Disorders.
Yu, Tao; Zhang, Yinfeng; Li, Pei-Feng
2017-01-01
Mitochondrial dynamics play a critical role in cellular responses and physiological process. However, their dysregulation leads to a functional degradation, which results in a diverse array of common disorders, including cardiovascular disease. In this background, the mitochondrial ubiquitin ligase has been attracting substantial research interest in recent years. Mitochondrial ubiquitin ligase is localized in the mitochondrial outer membrane, where it plays an essential role in the regulation of mitochondrial dynamics and apoptosis. In this chapter, we provide a comprehensive overview of the functions of mitochondrial ubiquitin ligases identified hitherto, with a special focus on cardiovascular disorders.
Bryan, Anthony C; Rodeheffer, Matthew S; Wearn, Christopher M; Shadel, Gerald S
2002-01-01
Mitochondrial translation is largely membrane-associated in S. cerevisiae. Recently, we discovered that the matrix protein Nam1p binds the amino-terminal domain of yeast mtRNA polymerase to couple translation and/or RNA-processing events to transcription. To gain additional insight into these transcription-coupled processes, we performed a genetic screen for genes that suppress the petite phenotype of a point mutation in mtRNA polymerase (rpo41-R129D) when overexpressed. One suppressor identified in this screen was SLS1, which encodes a mitochondrial membrane protein required for assembly of respiratory-chain enzyme complexes III and IV. The mtRNA-processing defects associated with the rpo41-R129D mutation were corrected in the suppressed strain, linking Sls1p to a pathway that includes mtRNA polymerase and Nam1p. This was supported by the observation that SLS1 overexpression rescued the petite phenotype of a NAM1 null mutation. In contrast, overexpression of Nam1p did not rescue the petite phenotype of a SLS1 null mutation, indicating that Nam1p and Sls1p are not functionally redundant but rather exist in an ordered pathway. On the basis of these data, a model in which Nam1p coordinates the delivery of newly synthesized transcripts to the membrane, where Sls1p directs or regulates their subsequent handling by membrane-bound factors involved in translation, is proposed. PMID:11805046
Agrawal, Sonal; Fox, Julia; Thyagarajan, Baskaran; Fox, Jonathan H
2018-05-20
Mitochondrial bioenergetic dysfunction is involved in neurodegeneration in Huntington's disease (HD). Iron is critical for normal mitochondrial bioenergetics but can also contribute to pathogenic oxidation. The accumulation of iron in the brain occurs in mouse models and in human HD. Yet the role of mitochondria-related iron dysregulation as a contributor to bioenergetic pathophysiology in HD is unclear. We demonstrate here that human HD and mouse model HD (12-week R6/2 and 12-month YAC128) brains accumulated mitochondrial iron and showed increased expression of iron uptake protein mitoferrin 2 and decreased iron-sulfur cluster synthesis protein frataxin. Mitochondria-enriched fractions from mouse HD brains had deficits in membrane potential and oxygen uptake and increased lipid peroxidation. In addition, the membrane-permeable iron-selective chelator deferiprone (1 μM) rescued these effects ex-vivo, whereas hydrophilic iron and copper chelators did not. A 10-day oral deferiprone treatment in 9-week R6/2 HD mice indicated that deferiprone removed mitochondrial iron, restored mitochondrial potentials, decreased lipid peroxidation, and improved motor endurance. Neonatal iron supplementation potentiates neurodegeneration in mouse models of HD by unknown mechanisms. We found that neonatal iron supplementation increased brain mitochondrial iron accumulation and potentiated markers of mitochondrial dysfunction in HD mice. Therefore, bi-directional manipulation of mitochondrial iron can potentiate and protect against markers of mouse HD. Our findings thus demonstrate the significance of iron as a mediator of mitochondrial dysfunction and injury in mouse models of human HD and suggest that targeting the iron-mitochondrial pathway may be protective. Copyright © 2018 Elsevier Inc. All rights reserved.
Saccharomyces genome database informs human biology
Skrzypek, Marek S; Nash, Robert S; Wong, Edith D; MacPherson, Kevin A; Karra, Kalpana; Binkley, Gail; Simison, Matt; Miyasato, Stuart R
2018-01-01
Abstract The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is an expertly curated database of literature-derived functional information for the model organism budding yeast, Saccharomyces cerevisiae. SGD constantly strives to synergize new types of experimental data and bioinformatics predictions with existing data, and to organize them into a comprehensive and up-to-date information resource. The primary mission of SGD is to facilitate research into the biology of yeast and to provide this wealth of information to advance, in many ways, research on other organisms, even those as evolutionarily distant as humans. To build such a bridge between biological kingdoms, SGD is curating data regarding yeast-human complementation, in which a human gene can successfully replace the function of a yeast gene, and/or vice versa. These data are manually curated from published literature, made available for download, and incorporated into a variety of analysis tools provided by SGD. PMID:29140510
Saccharomyces genome database informs human biology.
Skrzypek, Marek S; Nash, Robert S; Wong, Edith D; MacPherson, Kevin A; Hellerstedt, Sage T; Engel, Stacia R; Karra, Kalpana; Weng, Shuai; Sheppard, Travis K; Binkley, Gail; Simison, Matt; Miyasato, Stuart R; Cherry, J Michael
2018-01-04
The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is an expertly curated database of literature-derived functional information for the model organism budding yeast, Saccharomyces cerevisiae. SGD constantly strives to synergize new types of experimental data and bioinformatics predictions with existing data, and to organize them into a comprehensive and up-to-date information resource. The primary mission of SGD is to facilitate research into the biology of yeast and to provide this wealth of information to advance, in many ways, research on other organisms, even those as evolutionarily distant as humans. To build such a bridge between biological kingdoms, SGD is curating data regarding yeast-human complementation, in which a human gene can successfully replace the function of a yeast gene, and/or vice versa. These data are manually curated from published literature, made available for download, and incorporated into a variety of analysis tools provided by SGD. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio
2014-02-01
trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. © 2014 FEBS.
Padilla, Beatriz; Gil, José V.; Manzanares, Paloma
2016-01-01
It is well established that non-Saccharomyces wine yeasts, considered in the past as undesired or spoilage yeasts, can enhance the analytical composition, and aroma profile of the wine. The contribution of non-Saccharomyces yeasts, including the ability to secret enzymes and produce secondary metabolites, glycerol and ethanol, release of mannoproteins or contributions to color stability, is species- and strain-specific, pointing out the key importance of a clever strain selection. The use of mixed starters of selected non-Saccharomyces yeasts with strains of Saccharomyces cerevisiae represents an alternative to both spontaneous and inoculated wine fermentations, taking advantage of the potential positive role that non-Saccharomyces wine yeast species play in the organoleptic characteristics of wine. In this context mixed starters can meet the growing demand for new and improved wine yeast strains adapted to different types and styles of wine. With the aim of presenting old and new evidences on the potential of non-Saccharomyces yeasts to address this market trend, we mainly review the studies focused on non-Saccharomyces strain selection and design of mixed starters directed to improve primary and secondary aroma of wines. The ability of non-Saccharomyces wine yeasts to produce enzymes and metabolites of oenological relevance is also discussed. PMID:27065975
Padilla, Beatriz; Gil, José V; Manzanares, Paloma
2016-01-01
It is well established that non-Saccharomyces wine yeasts, considered in the past as undesired or spoilage yeasts, can enhance the analytical composition, and aroma profile of the wine. The contribution of non-Saccharomyces yeasts, including the ability to secret enzymes and produce secondary metabolites, glycerol and ethanol, release of mannoproteins or contributions to color stability, is species- and strain-specific, pointing out the key importance of a clever strain selection. The use of mixed starters of selected non-Saccharomyces yeasts with strains of Saccharomyces cerevisiae represents an alternative to both spontaneous and inoculated wine fermentations, taking advantage of the potential positive role that non-Saccharomyces wine yeast species play in the organoleptic characteristics of wine. In this context mixed starters can meet the growing demand for new and improved wine yeast strains adapted to different types and styles of wine. With the aim of presenting old and new evidences on the potential of non-Saccharomyces yeasts to address this market trend, we mainly review the studies focused on non-Saccharomyces strain selection and design of mixed starters directed to improve primary and secondary aroma of wines. The ability of non-Saccharomyces wine yeasts to produce enzymes and metabolites of oenological relevance is also discussed.
Exploring Protein Function Using the Saccharomyces Genome Database.
Wong, Edith D
2017-01-01
Elucidating the function of individual proteins will help to create a comprehensive picture of cell biology, as well as shed light on human disease mechanisms, possible treatments, and cures. Due to its compact genome, and extensive history of experimentation and annotation, the budding yeast Saccharomyces cerevisiae is an ideal model organism in which to determine protein function. This information can then be leveraged to infer functions of human homologs. Despite the large amount of research and biological data about S. cerevisiae, many proteins' functions remain unknown. Here, we explore ways to use the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org ) to predict the function of proteins and gain insight into their roles in various cellular processes.
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.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Jofuku, Akihiro; Ishihara, Naotada; Mihara, Katsuyoshi
2005-07-29
In yeast, mitochondrial-fission is regulated by the cytosolic dynamin-like GTPase (Dnm1p) in conjunction with a peripheral protein, Mdv1p, and a C-tail-anchored outer membrane protein, Fis1p. In mammals, a dynamin-related protein (Drp1) and Fis1 are involved in the mitochondrial-fission reaction as Dnm1 and Fis1 orthologues, respectively. The involvement of other component(s), such as the Mdv1 homologue, and the mechanisms regulating mitochondrial-fission remain unclear. Here, we identified rat Fis1 (rFis1) and analyzed its structure-function relationship. Blue-native-polyacrylamide gel electrophoresis revealed that rFis1 formed a {approx}200-kDa complex in the outer mitochondrial membrane. Its expression in HeLa cells promoted extensive mitochondrial fragmentation, and gene knock-downmore » by RNAi induced extension of the mitochondrial networks. Taking advantage of these properties, we analyzed functional domains of rFis1. These experiments revealed that the N-terminal and C-terminal segments are both essential for oligomeric rFis1 interaction, and the middle TPR-like domains regulate proper oligomer assembly. Any mutations that disturb the proper oligomeric assembly compromise mitochondrial division-stimulating activity of rFis1.« less
Mitochondrial Dynamics in Diabetic Cardiomyopathy
Galloway, Chad A.
2015-01-01
Abstract Significance: Cardiac function is energetically demanding, reliant on efficient well-coupled mitochondria to generate adenosine triphosphate and fulfill the cardiac demand. Predictably then, mitochondrial dysfunction is associated with cardiac pathologies, often related to metabolic disease, most commonly diabetes. Diabetic cardiomyopathy (DCM), characterized by decreased left ventricular function, arises independently of coronary artery disease and atherosclerosis. Dysregulation of Ca2+ handling, metabolic changes, and oxidative stress are observed in DCM, abnormalities reflected in alterations in mitochondrial energetics. Cardiac tissue from DCM patients also presents with altered mitochondrial morphology, suggesting a possible role of mitochondrial dynamics in its pathological progression. Recent Advances: Abnormal mitochondrial morphology is associated with pathologies across diverse tissues, suggesting that this highly regulated process is essential for proper cell maintenance and physiological homeostasis. Highly structured cardiac myofibers were hypothesized to limit alterations in mitochondrial morphology; however, recent work has identified morphological changes in cardiac tissue, specifically in DCM. Critical Issues: Mitochondrial dysfunction has been reported independently from observations of altered mitochondrial morphology in DCM. The temporal relationship and causative nature between functional and morphological changes of mitochondria in the establishment/progression of DCM is unclear. Future Directions: Altered mitochondrial energetics and morphology are not only causal for but also consequential to reactive oxygen species production, hence exacerbating oxidative damage through reciprocal amplification, which is integral to the progression of DCM. Therefore, targeting mitochondria for DCM will require better mechanistic characterization of morphological distortion and bioenergetic dysfunction. Antioxid. Redox Signal. 22, 1545–1562. PMID
Ayer, Anita; Sanwald, Julia; Pillay, Bethany A.; Meyer, Andreas J.; Perrone, Gabriel G.; Dawes, Ian W.
2013-01-01
Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. E GSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (−340 to −350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H+/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H+/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions. PMID:23762325
How did Saccharomyces evolve to become a good brewer?
Piskur, Jure; Rozpedowska, Elzbieta; Polakova, Silvia; Merico, Annamaria; Compagno, Concetta
2006-04-01
Brewing and wine production are among the oldest technologies and their products are almost indispensable in our lives. The central biological agents of beer and wine fermentation are yeasts belonging to the genus Saccharomyces, which can accumulate ethanol. Recent advances in comparative genomics and bioinformatics have made it possible to elucidate when and why yeasts produce ethanol in high concentrations, and how this remarkable trait originated and developed during their evolutionary history. Two research groups have shed light on the origin of the genes encoding alcohol dehydrogenase and the process of ethanol accumulation in Saccharomyces cerevisiae.
Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts
Gorter de Vries, Arthur R.; Pronk, Jack T.
2017-01-01
ABSTRACT Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyces strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. PMID:28341679
Kato, Michiko; Lin, Su-Ju
2014-11-01
Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates
Gray, Michael W.
2012-01-01
Viewed through the lens of the genome it contains, the mitochondrion is of unquestioned bacterial ancestry, originating from within the bacterial phylum α-Proteobacteria (Alphaproteobacteria). Accordingly, the endosymbiont hypothesis—the idea that the mitochondrion evolved from a bacterial progenitor via symbiosis within an essentially eukaryotic host cell—has assumed the status of a theory. Yet mitochondrial genome evolution has taken radically different pathways in diverse eukaryotic lineages, and the organelle itself is increasingly viewed as a genetic and functional mosaic, with the bulk of the mitochondrial proteome having an evolutionary origin outside Alphaproteobacteria. New data continue to reshape our views regarding mitochondrial evolution, particularly raising the question of whether the mitochondrion originated after the eukaryotic cell arose, as assumed in the classical endosymbiont hypothesis, or whether this organelle had its beginning at the same time as the cell containing it. PMID:22952398
Structural dynamics of the mitochondrial compartment.
Thorsness, P E
1992-09-01
The metabolic activities of mitochondria have been extensively characterized. However, there is much less known about the morphogenic changes of the mitochondrial compartment during growth, development and aging of the cell and the consequences of those structural changes on cellular metabolism. There is a growing body of evidence for interactions of mitochondria with cytoskeletal components and changes of mitochondrial structure during development and in response to changing environmental conditions. Segregation and recombination of mitochondrial genomes are also processes dependent upon the dynamic nature of the mitochondrial compartment. These regulatory and structural aspects of mitochondrial compartment dynamics will play an important role in the analysis of mitochondrial function and pathology.
Mitochondrial dynamics in Parkinson's disease
Van Laar, Victor S.; Berman, Sarah B.
2009-01-01
The unique energy demands of neurons require well-orchestrated distribution and maintenance of mitochondria. Thus, dynamic properties of mitochondria, including fission, fusion, trafficking, biogenesis, and degradation, are critical to all cells, but may be particularly important in neurons. Dysfunction in mitochondrial dynamics has been linked to neuropathies and is increasingly being linked to several neurodegenerative diseases, but the evidence is particularly strong, and continuously accumulating, in Parkinson's disease (PD). The unique characteristics of neurons that degenerate in PD may predispose those neuronal populations to susceptibility to alterations in mitochondrial dynamics. In addition, evidence from PD-related toxins supports that mitochondrial fission, fusion, and transport may be involved in pathogenesis. Furthermore, rapidly increasing evidence suggests that two proteins linked to familial forms of the disease, parkin and PINK1, interact in a common pathway to regulate mitochondrial fission/fusion. Parkin may also play a role in maintaining mitochondrial homeostasis through targeting damaged mitochondria for mitophagy. Taken together, the current data suggests that mitochondrial dynamics may play a role in PD pathogenesis, and a better understanding of mitochondrial dynamics within the neuron may lead to future therapeutic treatments for PD, potentially aimed at some of the earliest pathogenic events. PMID:19332061
DOE Office of Scientific and Technical Information (OSTI.GOV)
Watanabe, Tomoyuki; Saotome, Masao, E-mail: msaotome@hama-med.ac.jp; Nobuhara, Mamoru
Purpose: Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance. Methods and Results: DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨ{sub m}) depolarization, exhibited attenuated insulin signaling and 2-deoxy-D-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H{sub 2}O{sub 2}),more » they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨ{sub m} depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H{sub 2}O{sub 2}-induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨ{sub m} depolarization and impaired 2-DG uptake, however they improved insulin signaling. Conclusions: A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance. - Highlights: • DRP1 promotes mitochondrial fragmentation and insulin-resistance. • A mutual enhancement between DRP1 and ROS ipromotes insulin-resistance. • Palmitate increases DRP1 expression and induces insulin
Dunn, Barbara; Richter, Chandra; Kvitek, Daniel J; Pugh, Tom; Sherlock, Gavin
2012-05-01
Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species--i.e., its "pan-genome"--has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes-plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae.
Dunn, Barbara; Richter, Chandra; Kvitek, Daniel J.; Pugh, Tom; Sherlock, Gavin
2012-01-01
Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species—i.e., its “pan-genome”—has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes–plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae. PMID:22369888
Daenen, L; Saison, D; Sterckx, F; Delvaux, F R; Verachtert, H; Derdelinckx, G
2008-02-01
The aim of this study was to select and examine Saccharomyces and Brettanomyces brewing yeasts for hydrolase activity towards glycosidically bound volatile compounds. A screening for glucoside hydrolase activity of 58 brewing yeasts belonging to the genera Saccharomyces and Brettanomyces was performed. The studied Saccharomyces brewing yeasts did not show 1,4-beta-glucosidase activity, but a strain dependent beta-glucanase activity was observed. Some Brettanomyces species did show 1,4-beta-glucosidase activity. The highest constitutive activity was found in Brettanomyces custersii. For the most interesting strains the substrate specificity was studied and their activity was evaluated in fermentation experiments with added hop glycosides. Fermentations with Br. custersii led to the highest release of aglycones. Pronounced exo-beta-glucanase activity in Saccharomyces brewing yeasts leads to a higher release of certain aglycones. Certain Brettanomyces brewing yeasts, however, are more interesting for hydrolysis of glycosidically bound volatiles of hops. The release of flavour active compounds from hop glycosides opens perspectives for the bioflavouring and product diversification of beverages like beer. The release can be enhanced by using Saccharomyces strains with high exo-beta-glucanase activity. Higher activities can be found in Brettanomyces species with beta-glucosidase activity.
Saccharomyces boulardii improves humoral immune response to DNA vaccines against leptospirosis.
Silveira, Marcelle Moura; Conceição, Fabricio Rochedo; Mendonça, Marcelo; Moreira, Gustavo Marçal Schmidt Garcia; Da Cunha, Carlos Eduardo Pouey; Conrad, Neida Lucia; Oliveira, Patrícia Diaz de; Hartwig, Daiane Drawanz; De Leon, Priscila Marques Moura; Moreira, Ângela Nunes
2017-02-01
Saccharomyces boulardii may improve the immune response by enhancing the production of anti-inflammatory cytokines, T-cell proliferation and dendritic cell activation. The immunomodulator effect of this probiotic has never been tested with DNA vaccines, which frequently induce low antibody titers. This study evaluated the capacity of Saccharomyces boulardii to improve the humoral and cellular immune responses using DNA vaccines coding for the leptospiral protein fragments LigAni and LigBrep. BALB/c mice were fed with rodent-specific feed containing 108 c.f.u. of Saccharomycesboulardii per gram. Animals were immunized three times intramuscularly with 100 µg of pTARGET plasmids containing the coding sequences for the above mentioned proteins. Antibody titers were measured by indirect ELISA. Expression levels of IL-4, IL-10, IL-12, IL-17, IFN-γ and TGF-β were determined by quantitative real-time PCR from RNA extracted from whole blood, after an intraperitoneal boost with 50 µg of the recombinant proteins.Results/Key findings. Antibody titers increased significantly after the second and third application when pTARGET/ligAni and pTARGET/ligBrep were used to vaccinate the animals in comparison with the control group (P<0.05). In addition, there was a significant increase in the expression of the IL-10 in mice immunized with pTARGET/ligBrep and fed with Saccharomyces boulardii. The results suggested that Saccharomyces boulardii has an immunomodulator effect in DNA vaccines, mainly by stimulating the humoral response, which is often limited in this kind of vaccine. Therefore, the use of Saccharomyces boulardii as immunomodulator represents a new alternative strategy for more efficient DNA vaccination.
Resveratrol induces mitochondrial biogenesis in endothelial cells.
Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan
2009-07-01
Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.
Resveratrol induces mitochondrial biogenesis in endothelial cells
Csiszar, Anna; Labinskyy, Nazar; Pinto, John T.; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan
2009-01-01
Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1α, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases. PMID:19429820
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sheng Y.; Cabelli D.; Stich, T.A.
Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O{sub 2}{sup -}). This behavior limits the amount of H{sub 2}O{sub 2} produced at high [O{sub 2}{sup -}]; its desirability can be explained by the multiple roles of H{sub 2}O{sub 2} in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy, and redox chemistry, and they both rest predominantlymore » in the reduced state (unlike most other MnSODs). At high [O{sub 2}{sup -}], the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn{sup 3+} species in yeast Mn{sup 3+}SODs, including the well-characterized 5-coordinate Mn{sup 3+} species and a 6-coordinate L-Mn{sup 3+} species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O{sub 2}{sup -}].« less
Sheng, Yuewei; Stich, Troy A.; Barnese, Kevin; Gralla, Edith B.; Cascio, Duilio; Britt, R. David; Cabelli, Diane E.; Valentine, Joan Selverstone
2011-01-01
Human MnSOD is significantly more product-inhibited than bacterial MnSODs at high concentrations of superoxide (O2−). This behavior limits the amount of H2O2 produced at high [O2−]; its desirability can be explained by the multiple roles of H2O2 in mammalian cells, particularly its role in signaling. To investigate the mechanism of product inhibition in MnSOD, two yeast MnSODs, one from Saccharomyces cerevisiae mitochondria (ScMnSOD) and the other from Candida albicans cytosol (CaMnSODc), were isolated and characterized. ScMnSOD and CaMnSODc are similar in catalytic kinetics, spectroscopy and redox chemistry, and they both rest predominantly in the reduced state (unlike most other MnSODs). At high [O2−] the dismutation efficiencies of the yeast MnSODs surpass those of human and bacterial MnSODs, due to very low level of product inhibition. Optical and parallel-mode electron paramagnetic resonance (EPR) spectra suggest the presence of two Mn3+ species in yeast Mn3+SODs, including the well-characterized 5-coordinate Mn3+ species and a 6-coordinate L-Mn3+ species with hydroxide as the putative sixth ligand (L). The first and second coordination spheres of ScMnSOD are more similar to bacterial than to human MnSOD. Gln154, an H-bond donor to the Mn-coordinated solvent molecule, is slightly further away from Mn in yeast MnSODs, which may result in their unusual resting state. Mechanistically, the high efficiency of yeast MnSODs could be ascribed to putative translocation of an outer-sphere solvent molecule, which could destabilize the inhibited complex and enhance proton transfer from protein to peroxide. Our studies on yeast MnSODs indicate the unique nature of human MnSOD in that it predominantly undergoes the inhibited pathway at high [O2−]. PMID:22077216
Cancer: Mitochondrial Origins.
Stefano, George B; Kream, Richard M
2015-12-01
The primacy of glucose derived from photosynthesis as an existential source of chemical energy across plant and animal phyla is universally accepted as a core principle in the biological sciences. In mammalian cells, initial processing of glucose to triose phosphate intermediates takes place within the cytosolic glycolytic pathway and terminates with temporal transport of reducing equivalents derived from pyruvate metabolism by membrane-associated respiratory complexes in the mitochondrial matrix. The intra-mitochondrial availability of molecular oxygen as the ultimate electron acceptor drives the evolutionary fashioned chemiosmotic production of ATP as a high-efficiency biological process. The mechanistic bases of carcinogenesis have demonstrated profound alteration of normative mitochondrial function, notably dysregulated respiratory processes. Accordingly, the classic Warburg effect functionally links aerobic glycolysis, aberrant production and release of lactate, and metabolic down-regulation of mitochondrial oxidative processes with the carcinogenetic phenotype. We surmise, however, that aerobic fermentation by cancer cells may also represent a developmental re-emergence of an evolutionarily conserved early phenotype, which was "sidelined" with the emergence of mitochondrial oxidative phosphorylation as a primary mechanism for ATP production in normal cells. Regardless of state-dependent physiological status in mixed populations of cancer cells, it has been established that mitochondria are functionally linked to the initiation of cancer and its progression. Biochemical, molecular, and physiological differences in cancer cell mitochondria, notably mtDNA heteroplasmy and allele-specific expression of selected nuclear genes, may represent major focal points for novel targeting and elimination of cancer cells in metastatic disease afflicting human populations. To date, and despite considerable research efforts, the practical realization of advanced mitochondrial
Dai, Dao-Fu; Hsieh, Edward J.; Liu, Yonggang; Chen, Tony; Beyer, Richard P.; Chin, Michael T.; MacCoss, Michael J.; Rabinovitch, Peter S.
2012-01-01
Aims We investigate the role of mitochondrial oxidative stress in mitochondrial proteome remodelling using mouse models of heart failure induced by pressure overload. Methods and results We demonstrate that mice overexpressing catalase targeted to mitochondria (mCAT) attenuate pressure overload-induced heart failure. An improved method of label-free unbiased analysis of the mitochondrial proteome was applied to the mouse model of heart failure induced by transverse aortic constriction (TAC). A total of 425 mitochondrial proteins were compared between wild-type and mCAT mice receiving TAC or sham surgery. The changes in the mitochondrial proteome in heart failure included decreased abundance of proteins involved in fatty acid metabolism, an increased abundance of proteins in glycolysis, apoptosis, mitochondrial unfolded protein response and proteolysis, transcription and translational control, and developmental processes as well as responses to stimuli. Overexpression of mCAT better preserved proteins involved in fatty acid metabolism and attenuated the increases in apoptotic and proteolytic enzymes. Interestingly, gene ontology analysis also showed that monosaccharide metabolic processes and protein folding/proteolysis were only overrepresented in mCAT but not in wild-type mice in response to TAC. Conclusion This is the first study to demonstrate that scavenging mitochondrial reactive oxygen species (ROS) by mCAT not only attenuates most of the mitochondrial proteome changes in heart failure, but also induces a subset of unique alterations. These changes represent processes that are adaptive to the increased work and metabolic requirements of pressure overload, but which are normally inhibited by overproduction of mitochondrial ROS. PMID:22012956
Mitochondrial Function in Allergic Disease.
Iyer, Divyaanka; Mishra, Navya; Agrawal, Anurag
2017-05-01
The connections between allergy, asthma and metabolic syndrome are becoming increasingly clear. Recent research suggests a unifying mitochondrial link between the diverse phenotypes of these interlinked morbidities. The scope of this review is to highlight cellular mechanisms, epidemiology and environmental allergens influencing mitochondrial function and its importance in allergy and asthma. We briefly also consider the potential of mitochondria-targeted therapies in prevention and cure. Recent research has shown allergy, asthma and metabolic syndrome to be linked to mitochondrial dysfunction. Environmental pollutants and allergens are observed to cause mitochondrial dysfunction, primarily by inducing oxidative stress and ROS production. Malfunctioning mitochondria change the bioenergetics of the cell and its metabolic profile to favour systemic inflammation, which drives all three types of morbidities. Given the existing experimental evidence, approaches targeting mitochondria (e.g. antioxidant therapy and mitochondrial replacement) are being conducted in relevant disease models-with some progressing towards clinical trials, making mitochondrial function the focus of translational therapy research in asthma, allergy and linked metabolic syndrome.
Cousseau, F E M; Alves, S L; Trichez, D; Stambuk, B U
2013-01-01
The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose-H(+) symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort. © 2012 The Society for Applied Microbiology.
Mitochondrial respiration is sensitive to cytoarchitectural breakdown.
Kandel, Judith; Angelin, Alessia A; Wallace, Douglas C; Eckmann, David M
2016-11-07
An abundance of research suggests that cellular mitochondrial and cytoskeletal disruption are related, but few studies have directly investigated causative connections between the two. We previously demonstrated that inhibiting microtubule and microfilament polymerization affects mitochondrial motility on the whole-cell level in fibroblasts. Since mitochondrial motility can be indicative of mitochondrial function, we now further characterize the effects of these cytoskeletal inhibitors on mitochondrial potential, morphology and respiration. We found that although they did not reduce mitochondrial inner membrane potential, cytoskeletal toxins induced significant decreases in basal mitochondrial respiration. In some cases, basal respiration was only affected after cells were pretreated with the calcium ionophore A23187 in order to stress mitochondrial function. In most cases, mitochondrial morphology remained unaffected, but extreme microfilament depolymerization or combined intermediate doses of microtubule and microfilament toxins resulted in decreased mitochondrial lengths. Interestingly, these two particular exposures did not affect mitochondrial respiration in cells not sensitized with A23187, indicating an interplay between mitochondrial morphology and respiration. In all cases, inducing maximal respiration diminished differences between control and experimental groups, suggesting that reduced basal respiration originates as a largely elective rather than pathological symptom of cytoskeletal impairment. However, viability experiments suggest that even this type of respiration decrease may be associated with cell death.
Alberio, Tiziana; Pieroni, Luisa; Ronci, Maurizio; Banfi, Cristina; Bongarzone, Italia; Bottoni, Patrizia; Brioschi, Maura; Caterino, Marianna; Chinello, Clizia; Cormio, Antonella; Cozzolino, Flora; Cunsolo, Vincenzo; Fontana, Simona; Garavaglia, Barbara; Giusti, Laura; Greco, Viviana; Lucacchini, Antonio; Maffioli, Elisa; Magni, Fulvio; Monteleone, Francesca; Monti, Maria; Monti, Valentina; Musicco, Clara; Petrosillo, Giuseppe; Porcelli, Vito; Saletti, Rosaria; Scatena, Roberto; Soggiu, Alessio; Tedeschi, Gabriella; Zilocchi, Mara; Roncada, Paola; Urbani, Andrea; Fasano, Mauro
2017-12-01
The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.
Pharmacologic Effects on Mitochondrial Function
ERIC Educational Resources Information Center
Cohen, Bruce H.
2010-01-01
The vast majority of energy necessary for cellular function is produced in mitochondria. Free-radical production and apoptosis are other critical mitochondrial functions. The complex structure, electrochemical properties of the inner mitochondrial membrane (IMM), and genetic control from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) are…
Habitual physical activity in mitochondrial disease.
Apabhai, Shehnaz; Gorman, Grainne S; Sutton, Laura; Elson, Joanna L; Plötz, Thomas; Turnbull, Douglass M; Trenell, Michael I
2011-01-01
Mitochondrial disease is the most common neuromuscular disease and has a profound impact upon daily life, disease and longevity. Exercise therapy has been shown to improve mitochondrial function in patients with mitochondrial disease. However, no information exists about the level of habitual physical activity of people with mitochondrial disease and its relationship with clinical phenotype. Habitual physical activity, genotype and clinical presentations were assessed in 100 patients with mitochondrial disease. Comparisons were made with a control group individually matched by age, gender and BMI. Patients with mitochondrial disease had significantly lower levels of physical activity in comparison to matched people without mitochondrial disease (steps/day; 6883±3944 vs. 9924±4088, p = 0.001). 78% of the mitochondrial disease cohort did not achieve 10,000 steps per day and 48% were classified as overweight or obese. Mitochondrial disease was associated with less breaks in sedentary activity (Sedentary to Active Transitions, % per day; 13±0.03 vs. 14±0.03, p = 0.001) and an increase in sedentary bout duration (bout lengths/fraction of total sedentary time; 0.206±0.044 vs. 0.187±0.026, p = 0.001). After adjusting for covariates, higher physical activity was moderately associated with lower clinical disease burden (steps/day; r(s) = -0.49; 95% CI -0.33, -0.63, P<0.01). There were no systematic differences in physical activity between different genotypes mitochondrial disease. These results demonstrate for the first time that low levels of physical activity are prominent in mitochondrial disease. Combined with a high prevalence of obesity, physical activity may constitute a significant and potentially modifiable risk factor in mitochondrial disease.
Mitochondria inheritance is a key factor for tolerance to dehydration in wine yeast production.
Picazo, C; Gamero-Sandemetrio, E; Orozco, H; Albertin, W; Marullo, P; Matallana, E; Aranda, A
2015-03-01
Mitochondria are the cell's powerhouse when organisms are grown in the presence of oxygen. They are also the source of reactive oxygen species that cause damage to the biochemical components of the cell and lead to cellular ageing and death. Under winemaking conditions, Saccharomyces yeasts exclusively have a fermentative metabolism due to the high sugar content of grape must. However, their production as an active dry yeast (ADY) form required aerobic propagation and a dehydration process. In these industrial steps, oxidative stress is particularly harmful for the cell. In this work, we analysed the impact of the mitochondrial genome on oxidative stress response, longevity and dehydration tolerance using the synthetic interspecific hybrids obtained between two S. cerevisiae and S. uvarum strains. The isogenic nature of nuclear DNA of such hybrids allowed us to analyse the impact of mitochondrial DNA for fermentative and oxidative stress conditions. Under grape must conditions, the inheritance of mitochondrial DNA poorly impacted the fermentative performance of interspecific hybrids, unlike the hybrids with S. cerevisiae mitochondrial inheritance, which displayed increased tolerance to oxidative stress and dehydration, and showed an extended chronological longevity when cells were grown with aeration. In modern oenology, yeast starters are employed to inoculate grape juice, usually in the form of active dry yeast (ADY). The dehydration process implies stressful conditions that lead to oxidative damage. Other yeast species and interspecific hybrids other than Saccharomyces cerevisiae may be used to confer novel properties to the final product. However, these yeasts are usually more sensitive to drying. Understanding the causes of oxidative stress tolerance is therefore necessary for developing the use of these organisms in industry. This study indicates the impact of mitochondrial DNA inheritance for oxidative stress resistance in an interspecific context using
Mitochondrial transcription in mammalian cells
Shokolenko, Inna N.; Alexeyev, Mikhail F.
2017-01-01
As a consequence of recent discoveries of intimate involvement of mitochondria with key cellular processes, there has been a resurgence of interest in all aspects of mitochondrial biology, including the intricate mechanisms of mitochondrial DNA maintenance and expression. Despite four decades of research, there remains a lot to be learned about the processes that enable transcription of genetic information from mitochondrial DNA to RNA, as well as their regulation. These processes are vitally important, as evidenced by the lethality of inactivating the central components of mitochondrial transcription machinery. Here, we review the current understanding of mitochondrial transcription and its regulation in mammalian cells. We also discuss key theories in the field and highlight controversial subjects and future directions as we see them. PMID:27814650
Water treatment process and system for metals removal using Saccharomyces cerevisiae
Krauter, Paula A. W.; Krauter, Gordon W.
2002-01-01
A process and a system for removal of metals from ground water or from soil by bioreducing or bioaccumulating the metals using metal tolerant microorganisms Saccharomyces cerevisiae. Saccharomyces cerevisiae is tolerant to the metals, able to bioreduce the metals to the less toxic state and to accumulate them. The process and the system is useful for removal or substantial reduction of levels of chromium, molybdenum, cobalt, zinc, nickel, calcium, strontium, mercury and copper in water.
Dinwiddie, Darrell L.; Smith, Laurie D.; Miller, Neil A.; Atherton, Andrea M.; Farrow, Emily G.; Strenk, Meghan E.; Soden, Sarah E.; Saunders, Carol J.; Kingsmore, Stephen F.
2015-01-01
Mitochondrial diseases are notoriously difficult to diagnose due to extreme locus and allelic heterogeneity, with both nuclear and mitochondrial genomes potentially liable. Using exome sequencing we demonstrate the ability to rapidly and cost effectively evaluate both the nuclear and mitochondrial genomes to obtain a molecular diagnosis for four patients with three distinct mitochondrial disorders. One patient was found to have Leigh syndrome due to a mutation in MT-ATP6, two affected siblings were discovered to be compound heterozygous for mutations in the NDUFV1 gene, which causes mitochondrial complex I deficiency, and one patient was found to have coenzyme Q10 deficiency due to compound heterozygous mutations in COQ2. In all cases conventional diagnostic testing failed to identify a molecular diagnosis. We suggest that additional studies should be conducted to evaluate exome sequencing as a primary diagnostic test for mitochondrial diseases, including those due to mtDNA mutations. PMID:23631824
Mitochondrial Glutathione: Regulation and Functions.
Calabrese, Gaetano; Morgan, Bruce; Riemer, Jan
2017-11-20
Mitochondrial glutathione fulfills crucial roles in a number of processes, including iron-sulfur cluster biosynthesis and peroxide detoxification. Recent Advances: Genetically encoded fluorescent probes for the glutathione redox potential (E GSH ) have permitted extensive new insights into the regulation of mitochondrial glutathione redox homeostasis. These probes have revealed that the glutathione pools of the mitochondrial matrix and intermembrane space (IMS) are highly reduced, similar to the cytosolic glutathione pool. The glutathione pool of the IMS is in equilibrium with the cytosolic glutathione pool due to the presence of porins that allow free passage of reduced glutathione (GSH) and oxidized glutathione (GSSG) across the outer mitochondrial membrane. In contrast, limited transport of glutathione across the inner mitochondrial membrane ensures that the matrix glutathione pool is kinetically isolated from the cytosol and IMS. In contrast to the situation in the cytosol, there appears to be extensive crosstalk between the mitochondrial glutathione and thioredoxin systems. Further, both systems appear to be intimately involved in the removal of reactive oxygen species, particularly hydrogen peroxide (H 2 O 2 ), produced in mitochondria. However, a detailed understanding of these interactions remains elusive. We postulate that the application of genetically encoded sensors for glutathione in combination with novel H 2 O 2 probes and conventional biochemical redox state assays will lead to fundamental new insights into mitochondrial redox regulation and reinvigorate research into the physiological relevance of mitochondrial redox changes. Antioxid. Redox Signal. 27, 1162-1177.
Genetics of Mitochondrial Disease.
Saneto, Russell P
2017-01-01
Mitochondria are intracellular organelles responsible for adenosine triphosphate production. The strict control of intracellular energy needs require proper mitochondrial functioning. The mitochondria are under dual controls of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Mitochondrial dysfunction can arise from changes in either mtDNA or nDNA genes regulating function. There are an estimated ∼1500 proteins in the mitoproteome, whereas the mtDNA genome has 37 proteins. There are, to date, ∼275 genes shown to give rise to disease. The unique physiology of mitochondrial functioning contributes to diverse gene expression. The onset and range of phenotypic expression of disease is diverse, with onset from neonatal to seventh decade of life. The range of dysfunction is heterogeneous, ranging from single organ to multisystem involvement. The complexity of disease expression has severely limited gene discovery. Combining phenotypes with improvements in gene sequencing strategies are improving the diagnosis process. This chapter focuses on the interplay of the unique physiology and gene discovery in the current knowledge of genetically derived mitochondrial disease. Copyright © 2017 Elsevier Inc. All rights reserved.
Multifunctional Mitochondrial AAA Proteases
Glynn, Steven E.
2017-01-01
Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125
Multifunctional Mitochondrial AAA Proteases.
Glynn, Steven E
2017-01-01
Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.
Crouser, Elliott D; Julian, Mark W; Huff, Jennifer E; Joshi, Mandar S; Bauer, John A; Gadd, Martha E; Wewers, Mark D; Pfeiffer, Douglas R
2004-02-01
This study was designed to determine the role played by the mitochondrial permeability transition in the pathogenesis of mitochondrial damage and dysfunction in a representative systemic organ during the acute phase of endotoxemia. A well-established, normotensive feline model was employed to determine whether pretreatment with cyclosporine A, a potent inhibitor of the mitochondrial permeability transition, normalizes mitochondrial ultrastructural injury and dysfunction in the liver during acute endotoxemia. The Ohio State University Medical Center research laboratory. Random source, adult, male conditioned cats. Hemodynamic resuscitation and maintenance of acid-base balance and tissue oxygen availability were provided, as needed, to minimize the potentially confounding effects of tissue hypoxia and/or acidosis on the experimental results. Treatment groups received isotonic saline vehicle (control; n = 6), lipopolysaccharide (3.0 mg/kg, intravenously; n = 8), or cyclosporine A (6.0 mg/kg, intravenously; n = 6) or tacrolimus (FK506, 0.1 mg/kg, intravenously; n = 4) followed in 30 mins by lipopolysaccharide (3.0 mg/kg, intravenously). Liver samples were obtained 4 hrs posttreatment, and mitochondrial ultrastructure, function, and cytochrome c, Bax, and ceramide contents were assessed. As expected, significant mitochondrial injury was apparent in the liver 4 hrs after lipopolysaccharide treatment, despite maintenance of regional tissue oxygen availability. Namely, mitochondria demonstrated high-amplitude swelling and exhibited altered respiratory function. Cyclosporine A pretreatment attenuated lipopolysaccharide-induced mitochondrial ultrastructural abnormalities and normalized mitochondrial respiratory control, reflecting protection against inner mitochondrial membrane damage. However, an abnormal permeability of outer mitochondrial membranes to cytochrome c was observed in all lipopolysaccharide-treated groups and was associated with increased mitochondrial
Mechanisms of Ethanol Tolerance in Saccharomyces cerevisiae
USDA-ARS?s Scientific Manuscript database
Saccharomyces cerevisiae is a superb ethanol producer, yet is also sensitive to higher ethanol concentrations especially under high gravity or very high gravity fermentation conditions. Ethanol tolerance is associated with interplay of complex networks at the genome level. Although significant eff...
Genomics and Biochemistry of Saccharomyces cerevisiae Wine Yeast Strains.
Eldarov, M A; Kishkovskaia, S A; Tanaschuk, T N; Mardanov, A V
2016-12-01
Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term "unconscious" selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces. Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using "classical" and modern techniques for improving winemaking technology.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi, E-mail: harasima@pharm.hokudai.ac.jp
2010-06-25
Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we providemore » a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.« less
Dynamic tubulation of mitochondria drives mitochondrial network formation.
Wang, Chong; Du, Wanqing; Su, Qian Peter; Zhu, Mingli; Feng, Peiyuan; Li, Ying; Zhou, Yichen; Mi, Na; Zhu, Yueyao; Jiang, Dong; Zhang, Senyan; Zhang, Zerui; Sun, Yujie; Yu, Li
2015-10-01
Mitochondria form networks. Formation of mitochondrial networks is important for maintaining mitochondrial DNA integrity and interchanging mitochondrial material, whereas disruption of the mitochondrial network affects mitochondrial functions. According to the current view, mitochondrial networks are formed by fusion of individual mitochondria. Here, we report a new mechanism for formation of mitochondrial networks through KIF5B-mediated dynamic tubulation of mitochondria. We found that KIF5B pulls thin, highly dynamic tubules out of mitochondria. Fusion of these dynamic tubules, which is mediated by mitofusins, gives rise to the mitochondrial network. We further demonstrated that dynamic tubulation and fusion is sufficient for mitochondrial network formation, by reconstituting mitochondrial networks in vitro using purified fusion-competent mitochondria, recombinant KIF5B, and polymerized microtubules. Interestingly, KIF5B only controls network formation in the peripheral zone of the cell, indicating that the mitochondrial network is divided into subzones, which may be constructed by different mechanisms. Our data not only uncover an essential mechanism for mitochondrial network formation, but also reveal that different parts of the mitochondrial network are formed by different mechanisms.
Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast Saccharomyces cerevisiae.
Fišerová, Jindřiška; Richardson, Christine; Goldberg, Martin W
2016-01-01
Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events.
Song, Junna; Li, Yi; Song, Junmei; Hou, Fangjie; Liu, Baolin; Li, Aiying
2017-07-01
Hexokinase-II (HK-II) confers protection against cell death and this study was designed to investigate the effect of mangiferin on the regulation of mitochondrial HK-II. In vessel endothelial cells, saturated fatty acid palmitate (PA) stimulation induced HK-II detachment from mitochondria due to cellular acidification. Mangiferin reduced lactate accumulation by improving pyruvate dehydrogenase activity, promoted Akt translocation to HK-II and prevented HK-II detachment from mitochondria. Knockdown of Akt2 diminished the protective effect of mangiferin on mitochondrial HK-II, confirming the role of Akt in the regulation of HK-II. Mangiferin prevented mitochondrial permeability transition pore opening, restored mitochondrial membrane potential and thereby protected cell from apoptosis. In high-fat diet fed mice, oral administration of mangiferin induced Akt phosphorylation, increased HK-II binding to mitochondria and resultantly protected vessel endothelial function, demonstrating its protective effect on endothelial integrity in vivo. This finding provided a novel strategy for the protection of mitochondrial function in the endothelium. Copyright © 2017 Elsevier B.V. All rights reserved.
What Is Mitochondrial Disease?
... Review Mitochondrial Structure, Function and Diseases Review Cell Biology of Diagnosis and Treatment of Mitochondrial Diseases Review ... Factories and Much More The conventional teaching in biology and medicine is that mitochondria function only as “ ...
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.
Hyma, Katie E; Saerens, Sofie M; Verstrepen, Kevin J; Fay, Justin C
2011-01-01
The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea that wine making strains have been domesticated for wine production. In this study, we demonstrate that humans can distinguish between wines produced using wine strains and wild strains of S. cerevisiae as well as its sibling species, Saccharomyces paradoxus. Wine strains produced wine with fruity and floral characteristics, whereas wild strains produced wine with earthy and sulfurous characteristics. The differences that we observe between wine and wild strains provides further evidence that wine strains have evolved phenotypes that are distinct from their wild ancestors and relevant to their use in wine production. PMID:22093681
Analysis of non-Saccharomyces yeast populations isolated from grape musts from Sicily (Italy).
Romancino, D P; Di Maio, S; Muriella, R; Oliva, D
2008-12-01
The aim of this study was to identify the non-Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5.8S-internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. We isolated non-Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. This investigation is a first step in understanding the distribution of non-Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non-Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.
Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions
ERIC Educational Resources Information Center
Silva, Carlos M.; Neves, Patricia S.; Da Silva, Francisco A.; Xavier, Ana M. R. B.; Eusebio, M. F. J.
2008-01-01
Experimental work on ultrafiltration is presented to illustrate the practical and theoretical principles of this separation technique. The laboratory exercise comprises experiments with pure water and with aqueous "Saccharomyces cerevisiae" (from commercial Baker's yeast) suspensions. With this work students detect the characteristic phenomena…
Mitochondrial Energy and Redox Signaling in Plants
Schwarzländer, Markus
2013-01-01
Abstract Significance: For a plant to grow and develop, energy and appropriate building blocks are a fundamental requirement. Mitochondrial respiration is a vital source for both. The delicate redox processes that make up respiration are affected by the plant's changing environment. Therefore, mitochondrial regulation is critically important to maintain cellular homeostasis. This involves sensing signals from changes in mitochondrial physiology, transducing this information, and mounting tailored responses, by either adjusting mitochondrial and cellular functions directly or reprogramming gene expression. Recent Advances: Retrograde (RTG) signaling, by which mitochondrial signals control nuclear gene expression, has been a field of very active research in recent years. Nevertheless, no mitochondrial RTG-signaling pathway is yet understood in plants. This review summarizes recent advances toward elucidating redox processes and other bioenergetic factors as a part of RTG signaling of plant mitochondria. Critical Issues: Novel insights into mitochondrial physiology and redox-regulation provide a framework of upstream signaling. On the other end, downstream responses to modified mitochondrial function have become available, including transcriptomic data and mitochondrial phenotypes, revealing processes in the plant that are under mitochondrial control. Future Directions: Drawing parallels to chloroplast signaling and mitochondrial signaling in animal systems allows to bridge gaps in the current understanding and to deduce promising directions for future research. It is proposed that targeted usage of new technical approaches, such as quantitative in vivo imaging, will provide novel leverage to the dissection of plant mitochondrial signaling. Antioxid. Redox Signal. 18, 2122–2144. PMID:23234467
DOE Office of Scientific and Technical Information (OSTI.GOV)
Alán, Lukáš, E-mail: lukas.alan@fgu.cas.cz; Špaček
Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators.more » We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (> 48 h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity. - Highlights: • The mechanism for mitochondrial nucleoid clustering is proposed. • DNA intercalators (Doxorubicin or Ethidium Bromide) prevent
Industrial Relevance of Chromosomal Copy Number Variation in Saccharomyces Yeasts.
Gorter de Vries, Arthur R; Pronk, Jack T; Daran, Jean-Marc G
2017-06-01
Chromosomal copy number variation (CCNV) plays a key role in evolution and health of eukaryotes. The unicellular yeast Saccharomyces cerevisiae is an important model for studying the generation, physiological impact, and evolutionary significance of CCNV. Fundamental studies of this yeast have contributed to an extensive set of methods for analyzing and introducing CCNV. Moreover, these studies provided insight into the balance between negative and positive impacts of CCNV in evolutionary contexts. A growing body of evidence indicates that CCNV not only frequently occurs in industrial strains of Saccharomyces yeasts but also is a key contributor to the diversity of industrially relevant traits. This notion is further supported by the frequent involvement of CCNV in industrially relevant traits acquired during evolutionary engineering. This review describes recent developments in genome sequencing and genome editing techniques and discusses how these offer opportunities to unravel contributions of CCNV in industrial Saccharomyce s strains as well as to rationally engineer yeast chromosomal copy numbers and karyotypes. Copyright © 2017 Gorter de Vries et al.
Mitochondrial DNA transmission and confounding mitochondrial influences in cloned cattle and pigs.
Takeda, Kumiko
2013-04-01
Although somatic cell nuclear transfer (SCNT) is a powerful tool for production of cloned animals, SCNT embryos generally have low developmental competency and many abnormalities. The interaction between the donor nucleus and the enucleated ooplasm plays an important role in early embryonic development, but the underlying mechanisms that negatively impact developmental competency remain unclear. Mitochondria have a broad range of critical functions in cellular energy supply, cell signaling, and programmed cell death; thus, affect embryonic and fetal development. This review focuses on mitochondrial considerations influencing SCNT techniques in farm animals. Donor somatic cell mitochondrial DNA (mtDNA) can be transmitted through what has been considered a "bottleneck" in mitochondrial genetics via the SCNT maternal lineage. This indicates that donor somatic cell mitochondria have a role in the reconstructed cytoplasm. However, foreign somatic cell mitochondria may affect the early development of SCNT embryos. Nuclear-mitochondrial interactions in interspecies/intergeneric SCNT (iSCNT) result in severe problems. A major biological selective pressure exists against survival of exogenous mtDNA in iSCNT. Yet, mtDNA differences in SCNT animals did not reflect transfer of proteomic components following proteomic analysis. Further study of nuclear-cytoplasmic interactions is needed to illuminate key developmental characteristics of SCNT animals associated with mitochondrial biology.
Biocuration at the Saccharomyces Genome Database
Skrzypek, Marek S.; Nash, Robert S.
2015-01-01
Saccharomyces Genome Database is an online resource dedicated to managing information about the biology and genetics of the model organism, yeast (Saccharomyces cerevisiae). This information is derived primarily from scientific publications through a process of human curation that involves manual extraction of data and their organization into a comprehensive system of knowledge. This system provides a foundation for further analysis of experimental data coming from research on yeast as well as other organisms. In this review we will demonstrate how biocuration and biocurators add a key component, the biological context, to our understanding of how genes, proteins, genomes and cells function and interact. We will explain the role biocurators play in sifting through the wealth of biological data to incorporate and connect key information. We will also discuss the many ways we assist researchers with their various research needs. We hope to convince the reader that manual curation is vital in converting the flood of data into organized and interconnected knowledge, and that biocurators play an essential role in the integration of scientific information into a coherent model of the cell. PMID:25997651
Guo, Rui; Ren, Jun
2010-01-18
Binge drinking and alcohol toxicity are often associated with myocardial dysfunction possibly due to accumulation of the ethanol metabolite acetaldehyde although the underlying mechanism is unknown. This study was designed to examine the impact of accelerated ethanol metabolism on myocardial contractility, mitochondrial function and apoptosis using a murine model of cardiac-specific overexpression of alcohol dehydrogenase (ADH). ADH and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial contractility, mitochondrial damage and apoptosis (death receptor and mitochondrial pathways) were examined. Ethanol led to reduced cardiac contractility, enlarged cardiomyocyte, mitochondrial damage and apoptosis, the effects of which were exaggerated by ADH transgene. In particular, ADH exacerbated mitochondrial dysfunction manifested as decreased mitochondrial membrane potential and accumulation of mitochondrial O(2) (*-). Myocardium from ethanol-treated mice displayed enhanced Bax, Caspase-3 and decreased Bcl-2 expression, the effect of which with the exception of Caspase-3 was augmented by ADH. ADH accentuated ethanol-induced increase in the mitochondrial death domain components pro-caspase-9 and cytochrome C in the cytoplasm. Neither ethanol nor ADH affected the expression of ANP, total pro-caspase-9, cytosolic and total pro-caspase-8, TNF-alpha, Fas receptor, Fas L and cytosolic AIF. Taken together, these data suggest that enhanced acetaldehyde production through ADH overexpression following acute ethanol exposure exacerbated ethanol-induced myocardial contractile dysfunction, cardiomyocyte enlargement, mitochondrial damage and apoptosis, indicating a pivotal role of ADH in ethanol-induced cardiac dysfunction possibly through mitochondrial death pathway of apoptosis.
Saccharomyces cerevisiae Shuttle vectors.
Gnügge, Robert; Rudolf, Fabian
2017-05-01
Yeast shuttle vectors are indispensable tools in yeast research. They enable cloning of defined DNA sequences in Escherichia coli and their direct transfer into Saccharomyces cerevisiae cells. There are three types of commonly used yeast shuttle vectors: centromeric plasmids, episomal plasmids and integrating plasmids. In this review, we discuss the different plasmid systems and their characteristic features. We focus on their segregational stability and copy number and indicate how to modify these properties. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.
Mitochondrial Ion Channels in Cancer Transformation
Madamba, Stephen M.; Damri, Kevin N.; Dejean, Laurent M.; Peixoto, Pablo M.
2015-01-01
Cancer transformation involves reprograming of mitochondrial function to avert cell death mechanisms, monopolize energy metabolism, accelerate mitotic proliferation, and promote metastasis. Mitochondrial ion channels have emerged as promising therapeutic targets because of their connection to metabolic and apoptotic functions. This mini review discusses how mitochondrial channels may be associated with cancer transformation and expands on the possible involvement of mitochondrial protein import complexes in pathophysiological process. PMID:26090338
A test of the transcription model for biased inheritance of yeast mitochondrial DNA.
Lorimer, H E; Brewer, B J; Fangman, W L
1995-09-01
Two strand-specific origins of replication appear to be required for mammalian mitochondrial DNA (mtDNA) replication. Structural equivalents of these origins are found in the rep sequences of Saccharomyces cerevisiae mtDNA. These striking similarities have contributed to a universal model for the initiation of mtDNA replication in which a primer is created by cleavage of an origin region transcript. Consistent with this model are the properties of deletion mutants of yeast mtDNA ([rho-]) with a high density of reps (HS [rho-]). These mutant mtDNAs are preferentially inherited by the progeny resulting from the mating of HS [rho-] cells with cells containing wild-type mtDNA ([rho+]). This bias is presumed to result from a replication advantage conferred on HS [rho-] mtDNA by the high density of rep sequences acting as origins. To test whether transcription is indeed required for the preferential inheritance of HS [rho-] mtDNA, we deleted the nuclear gene (RPO41) for the mitochondrial RNA polymerase, reducing transcripts by at least 1000-fold. Since [rho-] genomes, but not [rho+] genomes, are stable when RPO41 is deleted, we examined matings between HS [rho-] and neutral [rho-] cells. Neutral [rho-] mtDNAs lack rep sequences and are not preferentially inherited in [rho-] x [rho+] crosses. In HS [rho-] x neutral [rho-] matings, the HS [rho-] mtDNA was preferentially inherited whether both parents were wild type or both were deleted for RPO41. Thus, transcription from the rep promoter does not appear to be necessary for biased inheritance. Our results, and analysis of the literature, suggest that priming by transcription is not a universal mechanism for mtDNA replication initiation.
Snf1-related kinase improves cardiac mitochondrial efficiency and decreases mitochondrial uncoupling
Rines, Amy K.; Chang, Hsiang-Chun; Wu, Rongxue; Sato, Tatsuya; Khechaduri, Arineh; Kouzu, Hidemichi; Shapiro, Jason; Shang, Meng; Burke, Michael A.; Abdelwahid, Eltyeb; Jiang, Xinghang; Chen, Chunlei; Rawlings, Tenley A.; Lopaschuk, Gary D.; Schumacker, Paul T.; Abel, E. Dale; Ardehali, Hossein
2017-01-01
Ischaemic heart disease limits oxygen and metabolic substrate availability to the heart, resulting in tissue death. Here, we demonstrate that the AMP-activated protein kinase (AMPK)-related protein Snf1-related kinase (SNRK) decreases cardiac metabolic substrate usage and mitochondrial uncoupling, and protects against ischaemia/reperfusion. Hearts from transgenic mice overexpressing SNRK have decreased glucose and palmitate metabolism and oxygen consumption, but maintained power and function. They also exhibit decreased uncoupling protein 3 (UCP3) and mitochondrial uncoupling. Conversely, Snrk knockout mouse hearts have increased glucose and palmitate oxidation and UCP3. SNRK knockdown in cardiac cells decreases mitochondrial efficiency, which is abolished with UCP3 knockdown. We show that Tribbles homologue 3 (Trib3) binds to SNRK, and downregulates UCP3 through PPARα. Finally, SNRK is increased in cardiomyopathy patients, and SNRK reduces infarct size after ischaemia/reperfusion. SNRK also decreases cardiac cell death in a UCP3-dependent manner. Our results suggest that SNRK improves cardiac mitochondrial efficiency and ischaemic protection. PMID:28117339
Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast.
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.
Gostimskaya, Irina; Grant, Chris M.
2016-01-01
Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron–sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron–sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own. PMID:26898146
Bioethanol production from sweet potato using Saccharomyces diastaticus
NASA Astrophysics Data System (ADS)
Abdullah, Suryani, Irma; Pradia Paundradewa, J.
2015-12-01
Sweet potato contains about 16 to 40% dry matter and about 70-90% of the dry matter is a carbohydrate made up of starch, sugar, cellulose, hemicellulose and pectin so suitable for used as raw material for bioethanol. In this study focused on the manufacture of bioethanol with changes in temperature and concentration variations of yeast with sweet potato raw materials used yeast Saccharomyces diastaticus. Operating variables used are at a temperature of 30°C; 31,475°C; 35°C; 38,525°C; and 40°C with a yeast concentration of 25.9%; 30%; 40%; 50% and 54.1%. The experimental results obtained, the optimum conditions of ethanol fermentation with yeast Saccharomyces diastaticus on 36,67 °C temperature and yeast concentration of 43,43 % v / v.
Duniere, L; Jin, L; Smiley, B; Qi, M; Rutherford, W; Wang, Y; McAllister, T
2015-05-01
Bacterial inoculants can improve the conservation and nutritional quality of silages. Inclusion of the yeast Saccharomyces in the diet of dairy cattle has also been reported to be beneficial. The present study assessed the ability of silage to be used as a means of delivering Saccharomyces strains to ruminants. Two strains of Saccharomyces cerevisiae (strain 1 and 3)and 1 strain of Saccharomyces paradoxus (strain 2) were inoculated (10(3) cfu/g) individually onto corn forage that was ensiled in mini silos for 90 d. Fermentation characteristics, aerobic stability, and nutritive value of silages were determined and real-time quantitative PCR (RT-qPCR) was used to quantify S. cerevisiae, S.paradoxus, total Saccharomyces, fungal, and bacterial populations. Fermentation characteristics of silage inoculated with S1 were similar to control silage. Although strain 3 inoculation increased ash and decreased OM contents of silage (P = 0.017), no differences were observed in nutrient composition or fermentation profiles after 90 d of ensiling. Inoculation with Saccharomyces had no detrimental effect on the aerobic stability of silage. In vitro DM disappearance, gas production, and microbial protein synthesis were not affected by yeast inoculation.Saccharomyces strain 1 was quantified throughout ensiling, whereas strain 2 was detected only immediately after inoculation. Saccharomyces cerevisiae strain 3 was quantified until d 7 and detectable 90 d after ensiling. All inoculants were detected and quantified during aerobic exposure. Inoculation with Saccharomyces did not alter lactobacilli populations. Saccharomycetales were detected by RT-qPCR throughout ensiling in all silages. Both S. cerevisiae and S. paradoxus populations increased during aerobic exposure, demonstrating that the density of these yeast strains would increase between the time that silage was removed from storage and the time it was fed.
Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii.
Datta, Suprama; Annapure, Uday S; Timson, David J
2017-04-30
Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met 177 , compared with the equivalent residue Leu 196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met 177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity. © 2017 The Author(s).
Melatonin and human mitochondrial diseases
Sharafati-Chaleshtori, Reza; Shirzad, Hedayatollah; Rafieian-Kopaei, Mahmoud; Soltani, Amin
2017-01-01
Mitochondrial dysfunction is one of the main causative factors in a wide variety of complications such as neurodegenerative disorders, ischemia/reperfusion, aging process, and septic shock. Decrease in respiratory complex activity, increase in free radical production, increase in mitochondrial synthase activity, increase in nitric oxide production, and impair in electron transport system and/or mitochondrial permeability are considered as the main factors responsible for mitochondrial dysfunction. Melatonin, the pineal gland hormone, is selectively taken up by mitochondria and acts as a powerful antioxidant, regulating the mitochondrial bioenergetic function. Melatonin increases the permeability of membranes and is the stimulator of antioxidant enzymes including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase. It also acts as an inhibitor of lipoxygenase. Melatonin can cause resistance to oxidation damage by fixing the microsomal membranes. Melatonin has been shown to retard aging and inhibit neurodegenerative disorders, ischemia/reperfusion, septic shock, diabetes, cancer, and other complications related to oxidative stress. The purpose of the current study, other than introducing melatonin, was to present the recent findings on clinical effects in diseases related to mitochondrial dysfunction including diabetes, cancer, gastrointestinal diseases, and diseases related to brain function. PMID:28400824
Mitochondrial Metabolism in Aging Heart
Lesnefsky, Edward J.; Chen, Qun; Hoppel, Charles L.
2016-01-01
Altered mitochondrial metabolism is the underlying basis for the increased sensitivity in the aged heart to stress. The aged heart exhibits impaired metabolic flexibility, with a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism. Aging impairs mitochondrial oxidative phosphorylation, with a greater role played by the mitochondria located between the myofibrils, the interfibrillar mitochondria. With aging, there is a decrease in activity of complexes III and IV, which account for the decrease in respiration. Furthermore, aging decreases mitochondrial content among the myofibrils. The end result is that in the interfibrillar area there is an approximate 50% decrease in mitochondrial function, affecting all substrates. The defective mitochondria persist in the aged heart, leading to enhanced oxidant production and oxidative injury and the activation of oxidant signaling for cell death. Aging defects in mitochondria represent new therapeutic targets, whether by manipulation of the mitochondrial proteome, modulation of electron transport, activation of biogenesis or mitophagy, or the regulation of mitochondrial fission and fusion. These mechanisms provide new ways to attenuate cardiac disease in elders by preemptive treatment of age-related defects, in contrast to the treatment of disease-induced dysfunction. PMID:27174952
Varela, Ana T; Gomes, Ana P; Simões, Anabela M; Teodoro, João S; Duarte, Filipe V; Rolo, Anabela P; Palmeira, Carlos M
2008-12-01
Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10microM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25microM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.
Mitochondrial medicine: to a new era of gene therapy for mitochondrial DNA mutations.
Cwerman-Thibault, Hélène; Sahel, José-Alain; Corral-Debrinski, Marisol
2011-04-01
Mitochondrial disorders can no longer be ignored in most medical disciplines. Such disorders include specific and widespread organ involvement, with tissue degeneration or tumor formation. Primary or secondary actors, mitochondrial dysfunctions also play a role in the aging process. Despite progresses made in identification of their molecular bases, nearly everything remains to be done as regards therapy. Research dealing with mitochondrial physiology and pathology has >20 years of history around the world. We are involved, as are many other laboratories, in the challenge of finding ways to fight these diseases. However, our main limitation is the scarcety of animal models required for both understanding the molecular mechanisms underlying the diseases and evaluating therapeutic strategies. This is especially true for diseases due to mutations in mitochondrial DNA (mtDNA), since an authentic genetic model of mtDNA mutations is technically a very difficult task due to both the inability of manipulating the mitochondrial genome of living mammalian cells and to its multicopy nature. This has led researchers in the field to consider the prospect of gene therapy approaches that can roughly be divided into three groups: (1) import of wild-type copies or relevant sections of DNA or RNA into mitochondria, (2) manipulation of mitochondrial genetic content, and (3) rescue of a defect by expression of an engineered gene product from the nucleus (allotopic or xenotropic expression). We briefly introduce these concepts and indicate where promising progress has been made in the last decade.
Chasseriaud, Laura; Coulon, Joana; Marullo, Philippe; Albertin, Warren; Bely, Marina
2018-04-01
Non-Saccharomyces yeast species, naturally found in grape must, may impact wine quality positively or negatively. In this study, a mixture of five non-Saccharomyces species (Torulaspora delbrueckii, Metschnikowia spp., Starmerella bacillaris (formerly called Candida zemplinina), Hanseniaspora uvarum, Pichia kluyveri), mimicking the composition of the natural non-Saccharomyces community found in grape must, was used for alcoholic fermentation. The impact of CO 2 saturation of the grape juice was studied first on this mixture alone, and then in the presence of Saccharomyces cerevisiae. Two isogenic strains of this species were used: the first with a short and the second a long fermentation lag phase. This study demonstrated that saturating grape juice with CO 2 had interesting potential as an oenological technique, inhibiting undesirable species (S. bacillaris and H. uvarum) and stimulating non-Saccharomyces of interest (T. delbrueckii and P. kluyveri). This stimulating effect was particularly marked when CO 2 saturation was associated with the presence of S. cerevisiae with long fermentation lag phase. The direct consequence of this association was an enhancement of 3-SH levels in the resulting wine.
Enhancing mitochondrial proteostasis reduces amyloid-β proteotoxicity.
Sorrentino, Vincenzo; Romani, Mario; Mouchiroud, Laurent; Beck, John S; Zhang, Hongbo; D'Amico, Davide; Moullan, Norman; Potenza, Francesca; Schmid, Adrien W; Rietsch, Solène; Counts, Scott E; Auwerx, Johan
2017-12-14
Alzheimer's disease is a common and devastating disease characterized by aggregation of the amyloid-β peptide. However, we know relatively little about the underlying molecular mechanisms or how to treat patients with Alzheimer's disease. Here we provide bioinformatic and experimental evidence of a conserved mitochondrial stress response signature present in diseases involving amyloid-β proteotoxicity in human, mouse and Caenorhabditis elegans that involves the mitochondrial unfolded protein response and mitophagy pathways. Using a worm model of amyloid-β proteotoxicity, GMC101, we recapitulated mitochondrial features and confirmed that the induction of this mitochondrial stress response was essential for the maintenance of mitochondrial proteostasis and health. Notably, increasing mitochondrial proteostasis by pharmacologically and genetically targeting mitochondrial translation and mitophagy increases the fitness and lifespan of GMC101 worms and reduces amyloid aggregation in cells, worms and in transgenic mouse models of Alzheimer's disease. Our data support the relevance of enhancing mitochondrial proteostasis to delay amyloid-β proteotoxic diseases, such as Alzheimer's disease.
Persistence of Two Non-Saccharomyces Yeasts (Hanseniaspora and Starmerella) in the Cellar
Grangeteau, Cédric; Gerhards, Daniel; von Wallbrunn, Christian; Alexandre, Hervé; Rousseaux, Sandrine; Guilloux-Benatier, Michèle
2016-01-01
Different genera and/or species of yeasts present on grape berries, in musts and wines are widely described. Nevertheless, the community of non-Saccharomyces yeasts present in the cellar is still given little attention. Thus it is not known if the cellar is a real ecological niche for these yeasts or if it is merely a transient habitat for populations brought in by grape berries during the winemaking period. This study focused on three species of non-Saccharomyces yeasts commonly encountered during vinification: Starmerella bacillaris (synonymy with Candida zemplinina), Hanseniaspora guilliermondii and Hanseniaspora uvarum. More than 1200 isolates were identified at the strain level by FT-IR spectroscopy (207 different FTIR strain pattern). Only a small proportion of non-Saccharomyces yeasts present in musts came directly from grape berries for the three species studied. Some strains were found in the must in two consecutive years and some of them were also found in the cellar environment before the arrival of the harvest of second vintage. This study demonstrates for the first time the persistence of non-Saccharomyces yeast strains from year to year in the cellar. Sulfur dioxide can affect yeast populations in the must and therefore their persistence in the cellar environment. PMID:27014199
Tsai, Han-Zen; Lin, Ren-Kuo; Hsieh, Tao-Shih
2016-04-12
Mitochondria play important roles in providing metabolic energy and key metabolites for synthesis of cellular building blocks. Mitochondria have additional functions in other cellular processes, including programmed cell death and aging. A previous study revealed Drosophila mitochondrial topoisomerase III alpha (Top3α) contributes to the maintenance of the mitochondrial genome and male germ-line stem cells. However, the involvement of mitochondrial Top3α in the mitochondrion-mediated aging process remains unclear. In this study, the M1L flies, in which Top3α protein lacks the mitochondrial import sequence and is thus present in cell nuclei but not in mitochondria, is used as a model system to examine the role of mitochondrial Top3α in the aging of fruit flies. Here, we reported that M1L flies exhibit mitochondrial defects which affect the aging process. First, we observed that M1L flies have a shorter life span, which was correlated with a significant reduction in the mitochondrial DNA copy number, the mitochondrial membrane potential, and ATP content compared with those of both wildtype and transgene-rescued flies of the same age. Second, we performed a mobility assay and electron microscopic analysis to demonstrate that the locomotion defect and mitophagy of M1L flies were enhanced with age, as compared with the controls. Finally, we showed that the correlation between the mtDNA deletion level and aging in M1L flies resembles what was reported in mammalian systems. The results reported here demonstrate that mitochondrial Top3α ablation results in mitochondrial genome instability and its dysfunction, thereby accelerating the aging process.
Sadoudi, Mohand; Tourdot-Maréchal, Raphaëlle; Rousseaux, Sandrine; Steyer, Damien; Gallardo-Chacón, Joan-Josep; Ballester, Jordi; Vichi, Stefania; Guérin-Schneider, Rémi; Caixach, Josep; Alexandre, Hervé
2012-12-01
There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the volatile profiles of several mono-culture and co-cultures. Candida zemplinina, Torulaspora delbrueckii and Metschnikowia pulcherrima were used to conduct fermentations either in mono-culture or in co-culture with S. cerevisiae. Up to 48 volatile compounds belonging to different chemical families were quantified. For the first time, we show that C. zemplinina is a strong producer of terpenes and lactones. We demonstrate by means of multivariate analysis that different interactions exist between the co-cultures studied. We observed a synergistic effect on aromatic compound production when M. pulcherrima was in co-culture with S. cerevisiae. However a negative interaction was observed between C. zemplinina and S. cerevisiae, which resulted in a decrease in terpene and lactone content. These interactions are independent of biomass production. The aromatic profiles of T. delbrueckii and S. cerevisiae in mono-culture and in co-culture are very close, and are biomass-dependent, reflecting a neutral interaction. This study reveals that a whole family of compounds could be altered by such interactions. These results suggest that the entire metabolic pathway is affected by these interactions. Copyright © 2012 Elsevier Ltd. All rights reserved.
Liou, Chia-Wei; Chen, Shang-Der; Wang, Pei-Wen; Chuang, Jiin-Haur; Tiao, Mao-Meng; Hsu, Te-Yao
2017-01-01
Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial disease affecting neuromuscular functions. Mt.8344A>G mutation in mitochondrial DNA (mtDNA) is the most common cause of MERRF syndrome and has been linked to an increase in reactive oxygen species (ROS) level and oxidative stress, as well as impaired mitochondrial bioenergetics. Here, we tested whether WJMSC has therapeutic potential for the treatment of MERRF syndrome through the transfer of mitochondria. The MERRF cybrid cells exhibited a high mt.8344A>G mutation ratio, enhanced ROS level and oxidative damage, impaired mitochondrial bioenergetics, defected mitochondria-dependent viability, exhibited an imbalance of mitochondrial dynamics, and are susceptible to apoptotic stress. Coculture experiments revealed that mitochondria were intercellularly conducted from the WJMSC to the MERRF cybrid. Furthermore, WJMSC transferred mitochondria exclusively to cells with defective mitochondria but not to cells with normal mitochondria. MERRF cybrid following WJMSC coculture (MF+WJ) demonstrated improvement of mt.8344A>G mutation ratio, ROS level, oxidative damage, mitochondrial bioenergetics, mitochondria-dependent viability, balance of mitochondrial dynamics, and resistance against apoptotic stress. WJMSC-derived mitochondrial transfer and its therapeutic effect were noted to be blocked by F-actin depolymerizing agent cytochalasin B. Collectively, the WJMSC ability to rescue cells with defective mitochondrial function through donating healthy mitochondria may lead to new insights into the development of more efficient strategies to treat diseases related to mitochondrial dysfunction. PMID:28607632
Li, Teng; Yang, Jie; Li, Yinwan; Cui, Ying; Xie, Qiang; Bu, Wenjun; Hillis, David M
2016-10-19
The Rhyparochromidae, the largest family of Lygaeoidea, encompasses more than 1,850 described species, but no mitochondrial genome has been sequenced to date. Here we describe the first mitochondrial genome for Rhyparochromidae: a complete mitochondrial genome of Panaorus albomaculatus (Scott, 1874). This mitochondrial genome is comprised of 16,345 bp, and contains the expected 37 genes and control region. The majority of the control region is made up of a large tandem-repeat region, which has a novel pattern not previously observed in other insects. The tandem-repeats region of P. albomaculatus consists of 53 tandem duplications (including one partial repeat), which is the largest number of tandem repeats among all the known insect mitochondrial genomes. Slipped-strand mispairing during replication is likely to have generated this novel pattern of tandem repeats. Comparative analysis of tRNA gene families in sequenced Pentatomomorpha and Lygaeoidea species shows that the pattern of nucleotide conservation is markedly higher on the J-strand. Phylogenetic reconstruction based on mitochondrial genomes suggests that Rhyparochromidae is not the sister group to all the remaining Lygaeoidea, and supports the monophyly of Lygaeoidea.
Escribano, R; González-Arenzana, L; Portu, J; Garijo, P; López-Alfaro, I; López, R; Santamaría, P; Gutiérrez, A R
2018-06-01
Twenty-five enological yeasts belonging to nine different species (Candida zeylanoides, Cryptococcus uzbekistanensis, Debaryomyces hansenii, Lachancea thermotolerans, Metschnikowia pulcherrima, Torulaspora delbrueckii, Williopsis pratensis, Zygosaccharomyces bailii and Saccharomyces cerevisiae) were screened for aroma formation and fermentative behaviour as part of a non-Saccharomyces yeast selection programme. Pure cultures were inoculated in pasteurized grape juice in order to perform alcoholic fermentations. Some non-Saccharomyces species did not ferment, others did not get established and none of them completed alcoholic fermentations. The physico-chemical parameters of the wines and the abundance of aromatic compounds at the end of alcoholic fermentation highlighted the notable differences in the aroma-forming ability and fermentative behaviour of the different non-Saccharomyces species, but not within clones. Lower diversity was detected within non-Saccharomyces species than that reported in S. cerevisiae with regard to enological behaviour and aromatic profiles. Metschnikowia pulcherrima and L. thermotolerans are the two species with higher possibilities to become an inoculum. Few significant differences were found within clones of the same species, but very important parameters in wine quality, such as volatile acidity, ethyl acetate and acetoin, which would justify selection programmes within those species. The results also demonstrated that T. delbrueckii and L. thermotolerans are two close species in their aromatic profiles. © 2018 The Society for Applied Microbiology.
Mitochondrial uncoupling, ROS generation and cardioprotection.
Cadenas, Susana
2018-05-31
Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase -a process known as proton leak- generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis. Copyright © 2018. Published by Elsevier B.V.
Aspirin increases mitochondrial fatty acid oxidation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Uppala, Radha; Dudiak, Brianne; Beck, Megan E.
The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse themore » mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 h incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders. - Highlights: • Aspirin increases mitochondrial—but inhibits peroxisomal—fatty acid oxidation. • Aspirin acetylates mitochondrial proteins including fatty acid oxidation enzymes. • SIRT3 does not influence the effect of aspirin on fatty acid oxidation. • Increased fatty acid oxidation is likely due to altered mitochondrial morphology and respiration.« less
Zabielski, Piotr; Lanza, Ian R.; Gopala, Srinivas; Holtz Heppelmann, Carrie J.; Bergen, H. Robert; Dasari, Surendra
2016-01-01
Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress. PMID:26718503
A Mitochondrial Mutator System in Maize1[w
Kuzmin, Evgeny V.; Duvick, Donald N.; Newton, Kathleen J.
2005-01-01
The P2 line of maize (Zea mays) is characterized by mitochondrial genome destabilization, initiated by recessive nuclear mutations. These alleles alter copy number control of mitochondrial subgenomes and disrupt normal transfer of mitochondrial genomic components to progeny, resulting in differences in mitochondrial DNA profiles among sibling plants and between parents and progeny. The mitochondrial DNA changes are often associated with variably defective phenotypes, reflecting depletion of essential mitochondrial genes. The P2 nuclear genotype can be considered a natural mutagenesis system for maize mitochondria. It dramatically accelerates mitochondrial genomic divergence by increasing low copy-number subgenomes, by rapidly amplifying aberrant recombination products, and by causing the random loss of normal components of the mitochondrial genomes. PMID:15681663
Bonnen, Penelope E.; Yarham, John W.; Besse, Arnaud; Wu, Ping; Faqeih, Eissa A.; Al-Asmari, Ali Mohammad; Saleh, Mohammad A.M.; Eyaid, Wafaa; Hadeel, Alrukban; He, Langping; Smith, Frances; Yau, Shu; Simcox, Eve M.; Miwa, Satomi; Donti, Taraka; Abu-Amero, Khaled K.; Wong, Lee-Jun; Craigen, William J.; Graham, Brett H.; Scott, Kenneth L.; McFarland, Robert; Taylor, Robert W.
2013-01-01
Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular etiology remains undiagnosed in the majority of cases. Through whole-exome sequencing, we identified recessive nonsense and splicing mutations in FBXL4 segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis, and severe mtDNA depletion in muscle. We show that FBXL4 is an F-box protein that colocalizes with mitochondria and that loss-of-function and splice mutations in this protein result in a severe respiratory chain deficiency, loss of mitochondrial membrane potential, and a disturbance of the dynamic mitochondrial network and nucleoid distribution in fibroblasts from affected individuals. Expression of the wild-type FBXL4 transcript in cell lines from two subjects fully rescued the levels of mtDNA copy number, leading to a correction of the mitochondrial biochemical deficit. Together our data demonstrate that mutations in FBXL4 are disease causing and establish FBXL4 as a mitochondrial protein with a possible role in maintaining mtDNA integrity and stability. PMID:23993193
Syndromes associated with mitochondrial DNA depletion
2014-01-01
Mitochondrial dysfunction accounts for a large group of inherited metabolic disorders most of which are due to a dysfunctional mitochondrial respiratory chain (MRC) and, consequently, deficient energy production. MRC function depends on the coordinated expression of both nuclear (nDNA) and mitochondrial (mtDNA) genomes. Thus, mitochondrial diseases can be caused by genetic defects in either the mitochondrial or the nuclear genome, or in the cross-talk between the two. This impaired cross-talk gives rise to so-called nuclear-mitochondrial intergenomic communication disorders, which result in loss or instability of the mitochondrial genome and, in turn, impaired maintenance of qualitative and quantitative mtDNA integrity. In children, most MRC disorders are associated with nuclear gene defects rather than alterations in the mtDNA itself. The mitochondrial DNA depletion syndromes (MDSs) are a clinically heterogeneous group of disorders with an autosomal recessive pattern of transmission that have onset in infancy or early childhood and are characterized by a reduced number of copies of mtDNA in affected tissues and organs. The MDSs can be divided into least four clinical presentations: hepatocerebral, myopathic, encephalomyopathic and neurogastrointestinal. The focus of this review is to offer an overview of these syndromes, listing the clinical phenotypes, together with their relative frequency, mutational spectrum, and possible insights for improving diagnostic strategies. PMID:24708634
Mitochondrial dysfunction and organophosphorus compounds
DOE Office of Scientific and Technical Information (OSTI.GOV)
Karami-Mohajeri, Somayyeh; Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Kerman University of Medical Sciences, Kerman; Abdollahi, Mohammad, E-mail: Mohammad.Abdollahi@UToronto.Ca
2013-07-01
Organophosphorous (OPs) pesticides are the most widely used pesticides in the agriculture and home. However, many acute or chronic poisoning reports about OPs have been published in the recent years. Mitochondria as a site of cellular oxygen consumption and energy production can be a target for OPs poisoning as a non-cholinergic mechanism of toxicity of OPs. In the present review, we have reviewed and criticized all the evidences about the mitochondrial dysfunctions as a mechanism of toxicity of OPs. For this purpose, all biochemical, molecular, and morphological data were retrieved from various studies. Some toxicities of OPs are arisen frommore » dysfunction of mitochondrial oxidative phosphorylation through alteration of complexes I, II, III, IV and V activities and disruption of mitochondrial membrane. Reductions of adenosine triphosphate (ATP) synthesis or induction of its hydrolysis can impair the cellular energy. The OPs disrupt cellular and mitochondrial antioxidant defense, reactive oxygen species generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome C released from mitochondria and consequent activation of caspases. The mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, electron donors, and through increasing the cytosolic ATP level. However, to elucidate many aspect of mitochondrial toxicity of Ops, further studies should be performed. - Highlights: • As a non-cholinergic mechanism of toxicity, mitochondria is a target for OPs. • OPs affect action of complexes I, II, III, IV and V in the mitochondria. • OPs reduce mitochondrial ATP. • OPs promote oxidative and genotoxic damage via release of cytochrome C from mitochondria. • OP-induced mitochondrial dysfunction can be restored by increasing the cytosolic ATP.« less
Mitochondrial Dysfunction in Cancer
Boland, Michelle L.; Chourasia, Aparajita H.; Macleod, Kay F.
2013-01-01
A mechanistic understanding of how mitochondrial dysfunction contributes to cell growth and tumorigenesis is emerging beyond Warburg as an area of research that is under-explored in terms of its significance for clinical management of cancer. Work discussed in this review focuses less on the Warburg effect and more on mitochondria and how dysfunctional mitochondria modulate cell cycle, gene expression, metabolism, cell viability, and other established aspects of cell growth and stress responses. There is increasing evidence that key oncogenes and tumor suppressors modulate mitochondrial dynamics through important signaling pathways and that mitochondrial mass and function vary between tumors and individuals but the significance of these events for cancer are not fully appreciated. We explore the interplay between key molecules involved in mitochondrial fission and fusion and in apoptosis, as well as in mitophagy, biogenesis, and spatial dynamics of mitochondria and consider how these distinct mechanisms are coordinated in response to physiological stresses such as hypoxia and nutrient deprivation. Importantly, we examine how deregulation of these processes in cancer has knock on effects for cell proliferation and growth. We define major forms of mitochondrial dysfunction and address the extent to which the functional consequences of such dysfunction can be determined and exploited for cancer diagnosis and treatment. PMID:24350057
Mitochondrial dysfunction in obesity.
de Mello, Aline Haas; Costa, Ana Beatriz; Engel, Jéssica Della Giustina; Rezin, Gislaine Tezza
2018-01-01
Obesity leads to various changes in the body. Among them, the existing inflammatory process may lead to an increase in the production of reactive oxygen species (ROS) and cause oxidative stress. Oxidative stress, in turn, can trigger mitochondrial changes, which is called mitochondrial dysfunction. Moreover, excess nutrients supply (as it commonly is the case with obesity) can overwhelm the Krebs cycle and the mitochondrial respiratory chain, causing a mitochondrial dysfunction, and lead to a higher ROS formation. This increase in ROS production by the respiratory chain may also cause oxidative stress, which may exacerbate the inflammatory process in obesity. All these intracellular changes can lead to cellular apoptosis. These processes have been described in obesity as occurring mainly in peripheral tissues. However, some studies have already shown that obesity is also associated with changes in the central nervous system (CNS), with alterations in the blood-brain barrier (BBB) and in cerebral structures such as hypothalamus and hippocampus. In this sense, this review presents a general view about mitochondrial dysfunction in obesity, including related alterations, such as inflammation, oxidative stress, and apoptosis, and focusing on the whole organism, covering alterations in peripheral tissues, BBB, and CNS. Copyright © 2017 Elsevier Inc. All rights reserved.
The expanding phenotype of mitochondrial myopathy.
DiMauro, Salvatore; Gurgel-Giannetti, Juliana
2005-10-01
Our understanding of mitochondrial diseases (defined restrictively as defects in the mitochondrial respiratory chain) continues to progress apace. In this review we provide an update of information regarding disorders that predominantly or exclusively affect skeletal muscle. Most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency, and mutations in genes that control mitochondrial DNA (mtDNA) abundance and structure such as POLG and TK2. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with altered lipid composition of the inner mitochondrial membrane, but a putative secondary impairment of the respiratory chain remains to be documented. Concerning the 'other genome', the role played by mutations in protein encoding genes of mtDNA in causing isolated myopathies has been confirmed. It has also been confirmed that mutations in tRNA genes of mtDNA can cause predominantly myopathic syndromes and - contrary to conventional wisdom - these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, myalgia, cramps, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.
Sato, Trey K.; Tremaine, Mary; Parreiras, Lucas S.; ...
2016-10-14
The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactionsmore » among genes encoding a xylose reductase ( GRE3), a component of MAP Kinase (MAPK) signaling ( HOG1), a regulator of Protein Kinase A (PKA) signaling ( IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis ( ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Lastly, our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.« less
Sato, Trey K; Tremaine, Mary; Parreiras, Lucas S; Hebert, Alexander S; Myers, Kevin S; Higbee, Alan J; Sardi, Maria; McIlwain, Sean J; Ong, Irene M; Breuer, Rebecca J; Avanasi Narasimhan, Ragothaman; McGee, Mick A; Dickinson, Quinn; La Reau, Alex; Xie, Dan; Tian, Mingyuan; Reed, Jennifer L; Zhang, Yaoping; Coon, Joshua J; Hittinger, Chris Todd; Gasch, Audrey P; Landick, Robert
2016-10-01
The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.
Tremaine, Mary; Hebert, Alexander S.; Myers, Kevin S.; Sardi, Maria; Dickinson, Quinn; Reed, Jennifer L.; Zhang, Yaoping; Coon, Joshua J.; Hittinger, Chris Todd; Gasch, Audrey P.; Landick, Robert
2016-01-01
The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism. PMID:27741250
Emerging Mitochondrial Therapeutic Targets in Optic Neuropathies.
Lopez Sanchez, M I G; Crowston, J G; Mackey, D A; Trounce, I A
2016-09-01
Optic neuropathies are an important cause of blindness worldwide. The study of the most common inherited mitochondrial optic neuropathies, Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA) has highlighted a fundamental role for mitochondrial function in the survival of the affected neuron-the retinal ganglion cell. A picture is now emerging that links mitochondrial dysfunction to optic nerve disease and other neurodegenerative processes. Insights gained from the peculiar susceptibility of retinal ganglion cells to mitochondrial dysfunction are likely to inform therapeutic development for glaucoma and other common neurodegenerative diseases of aging. Despite it being a fast-evolving field of research, a lack of access to human ocular tissues and limited animal models of mitochondrial disease have prevented direct retinal ganglion cell experimentation and delayed the development of efficient therapeutic strategies to prevent vision loss. Currently, there are no approved treatments for mitochondrial disease, including optic neuropathies caused by primary or secondary mitochondrial dysfunction. Recent advances in eye research have provided important insights into the molecular mechanisms that mediate pathogenesis, and new therapeutic strategies including gene correction approaches are currently being investigated. Here, we review the general principles of mitochondrial biology relevant to retinal ganglion cell function and provide an overview of the major optic neuropathies with mitochondrial involvement, LHON and ADOA, whilst highlighting the emerging link between mitochondrial dysfunction and glaucoma. The pharmacological strategies currently being trialed to improve mitochondrial dysfunction in these optic neuropathies are discussed in addition to emerging therapeutic approaches to preserve retinal ganglion cell function. Copyright © 2016 Elsevier Inc. All rights reserved.
Wang, Wang; Fernandez-Sanz, Celia; Sheu, Shey-Shing
2018-05-01
Recent advancement in mitochondrial research has significantly extended our knowledge on the role and regulation of mitochondria in health and disease. One important breakthrough is the delineation of how mitochondrial morphological changes, termed mitochondrial dynamics, are coupled to the bioenergetics and signaling functions of mitochondria. In general, it is believed that fusion leads to an increased mitochondrial respiration efficiency and resistance to stress-induced dysfunction while fission does the contrary. This concept seems not applicable to adult cardiomyocytes. The mitochondria in adult cardiomyocytes exhibit fragmented morphology (tilted towards fission) and show less networking and movement as compared to other cell types. However, being the most energy-demanding cells, cardiomyocytes in the adult heart possess vast number of mitochondria, high level of energy flow, and abundant mitochondrial dynamics proteins. This apparent discrepancy could be explained by recently identified new functions of the mitochondrial dynamics proteins. These "non-canonical" roles of mitochondrial dynamics proteins range from controlling inter-organelle communication to regulating cell viability and survival under metabolic stresses. Here, we summarize the newly identified non-canonical roles of mitochondrial dynamics proteins. We focus on how these fission and fusion independent roles of dynamics proteins regulate mitochondrial bioenergetics. We also discuss potential molecular mechanisms, unique intracellular location, and the cardiovascular disease relevance of these non-canonical roles of the dynamics proteins. We propose that future studies are warranted to differentiate the canonical and non-canonical roles of dynamics proteins and to identify new approaches for the treatment of heart diseases. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and
Liu, Jiankang
2008-01-01
We have identified a group of nutrients that can directly or indirectly protect mitochondria from oxidative damage and improve mitochondrial function and named them "mitochondrial nutrients". The direct protection includes preventing the generation of oxidants, scavenging free radicals or inhibiting oxidant reactivity, and elevating cofactors of defective mitochondrial enzymes with increased Michaelis-Menten constant to stimulate enzyme activity, and also protect enzymes from further oxidation, and the indirect protection includes repairing oxidative damage by enhancing antioxidant defense systems either through activation of phase 2 enzymes or through increase in mitochondrial biogenesis. In this review, we take alpha-lipoic acid (LA) as an example of mitochondrial nutrients by summarizing the protective effects and possible mechanisms of LA and its derivatives on age-associated cognitive and mitochondrial dysfunction of the brain. LA and its derivatives improve the age-associated decline of memory, improve mitochondrial structure and function, inhibit the age-associated increase of oxidative damage, elevate the levels of antioxidants, and restore the activity of key enzymes. In addition, co-administration of LA with other mitochondrial nutrients, such as acetyl-L: -carnitine and coenzyme Q10, appears more effective in improving cognitive dysfunction and reducing oxidative mitochondrial dysfunction. Therefore, administrating mitochondrial nutrients, such as LA and its derivatives in combination with other mitochondrial nutrients to aged people and patients suffering from neurodegenerative diseases, may be an effective strategy for improving mitochondrial and cognitive dysfunction.
Mitochondrial disorders: Challenges in diagnosis & treatment
Khan, Nahid Akhtar; Govindaraj, Periyasamy; Meena, Angamuthu Kannan; Thangaraj, Kumarasamy
2015-01-01
Mitochondrial dysfunctions are known to be responsible for a number of heterogenous clinical presentations with multi-systemic involvement. Impaired oxidative phosphorylation leading to a decrease in cellular energy (ATP) production is the most important cause underlying these disorders. Despite significant progress made in the field of mitochondrial medicine during the last two decades, the molecular mechanisms underlying these disorders are not fully understood. Since the identification of first mitochondrial DNA (mtDNA) mutation in 1988, there has been an exponential rise in the identification of mtDNA and nuclear DNA mutations that are responsible for mitochondrial dysfunction and disease. Genetic complexity together with ever widening clinical spectrum associated with mitochondrial dysfunction poses a major challenge in diagnosis and treatment. Effective therapy has remained elusive till date and is mostly efficient in relieving symptoms. In this review, we discuss the important clinical and genetic features of mitochondrials disorders with special emphasis on diagnosis and treatment. PMID:25857492
Fatal Saccharomyces Cerevisiae Aortic Graft Infection
NASA Technical Reports Server (NTRS)
Meyer, Michael (Technical Monitor); Smith, Davey; Metzgar, David; Wills, Christopher; Fierer, Joshua
2002-01-01
Saccharomyces cerevisiae is a yeast commonly used in baking and a frequent colonizer of human mucosal surfaces. It is considered relatively nonpathogenic in immunocompetent adults. We present a case of S. cerevisiae fungemia and aortic graft infection in an immunocompetent adult. This is the first reported case of S. cerevisiue fungemia where the identity of the pathogen was confirmed by rRNA sequencing.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Varela, Ana T.; Gomes, Ana P.; Simoes, Anabela M.
2008-12-01
Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10{mu}M, significantly depressesmore » the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25{mu}M stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.« less
Liu, Xuan; Trakooljul, Nares; Hadlich, Frieder; Murani, Eduard; Wimmers, Klaus; Ponsuksili, Siriluck
2017-10-25
Genes expressed in mitochondria work in concert with those expressed in the nucleus to mediate oxidative phosphorylation (OXPHOS), a process that is relevant for muscle metabolism and meat quality. Mitochondrial genome activity can be efficiently studied and compared in Duroc and Pietrain pigs, which harbor different mitochondrial haplotypes and distinct muscle fiber types, mitochondrial respiratory activities, and fat content. Pietrain pigs homozygous-positive for malignant hyperthermia susceptibility (PiPP) carried only haplotype 8 and showed the lowest absolute mtDNA copy number accompanied by a decrease transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6 and nuclear-encoded subunits NDUFA11 and NDUFB8. In contrast, we found that haplotype 4 of Duroc pigs had significantly higher mitochondrial DNA (mtDNA) copy numbers and an increase transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6. These results suggest that the variation in mitochondrial and nuclear genetic background among these animals has an effect on mitochondrial content and OXPHOS system subunit expression. We observed the co-expression pattern of mitochondrial and nuclear encoded OXPHOS subunits suggesting that the mitochondrial-nuclear crosstalk functionally involves in muscle metabolism. The findings provide valuable information for understanding muscle biology processes and energy metabolism, and may direct use for breeding strategies to improve meat quality and animal health.
Garcia Sanchez, Rosa; Solodovnikova, Natalia; Wendland, Jürgen
2012-08-01
Lager beer brewing relies on strains collectively known as Saccharomyces carlsbergensis, which are hybrids between S. cerevisiae and S. eubayanus-like strains. Lager yeasts are particularly adapted to low-temperature fermentations. Selection of new yeast strains for improved traits or fermentation performance is laborious, due to the allotetraploid nature of lager yeasts. Initially, we have generated new F1 hybrids by classical genetics, using spore clones of lager yeast and S. cerevisiae and complementation of auxotrophies of the single strains upon mating. These hybrids were improved on several parameters, including growth at elevated temperature and resistance against high osmolarity or high ethanol concentrations. Due to the uncertainty of chromosomal make-up of lager yeast spore clones, we introduced molecular markers to analyse mating-type composition by PCR. Based on these results, new hybrids between a lager and an ale yeast strain were isolated by micromanipulation. These hybrids were not subject to genetic modification. We generated and verified 13 hybrid strains. All of these hybrid strains showed improved stress resistance as seen in the ale parent, including improved survival at the end of fermentation. Importantly, some of the strains showed improved fermentation rates using 18° Plato at 18-25°C. Uniparental mitochondrial DNA inheritance was observed mostly from the S. cerevisiae parent. Copyright © 2012 John Wiley & Sons, Ltd.
Piló, Fernanda Barbosa; Carvajal-Barriga, Enrique Javier; Guamán-Burneo, Maria Cristina; Portero-Barahona, Patricia; Dias, Arthur Matoso Morato; Freitas, Larissa Falabella Daher de; Gomes, Fátima de Cássia Oliveira; Rosa, Carlos Augusto
2018-03-01
Chicha, a type of beer made mainly with maize or cassava, is a traditional fermented beverage of the Andean region. There have only been a few studies on yeasts associated with chicha fermentation, and the species diversity occurring during the production of this beverage is not known. The objective of this study was to determine the biodiversity of yeasts in chicha, and to characterize the Saccharomyces cerevisiae populations associated with the production of chicha de jora, seven-grain chicha, chicha de yuca, and chicha de morocho in Ecuador. The molecular diversity of S. cerevisiae populations was determined by restriction polymorphism mitochondrial profiles. The beverages were characterized based on their physicochemical parameters. Twenty-six species were identified, and the most prevalent species were S. cerevisiae and Torulaspora delbrueckii. Other yeast species were isolated at low frequencies. Among 121 isolates of S. cerevisiae, 68 different mtDNA molecular profiles were identified. These results showed that chichas are fermented by a high number of different strains of S. cerevisiae. Some other species provided a minor contribution to the fermentation process. The chicha presented generally similar physicochemical parameters to those observed for other traditional fermented beverages, and can be considered as an acid fermented beverage. Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.
Mei, Jie; Yang, Li-Yun; Lai, Lu; Xu, Zi-Qiang; Wang, Can; Zhao, Jie; Jin, Jian-Cheng; Jiang, Feng-Lei; Liu, Yi
2014-10-01
The interactions between quantum dots (QDs) and biological systems have attracted increasing attention due to concerns on possible toxicity of the nanoscale materials. The biological effects of CdSe QDs and CdSe/ZnS QDs with nearly identical hydrodynamic size on Saccharomyces cerevisiae were investigated via microcalorimetric, spectroscopic and microscopic methods, demonstrating a toxic order CdSe>CdSe/ZnS QDs. CdSe QDs damaged yeast cell wall and reduced the mitochondrial membrane potential. Noteworthy, adhesion of QDs to the yeast cell surface renders this work a good example of interaction site at cell surface, and the epitaxial coating of ZnS could greatly reduce the toxicity of Cd-containing QDs. These results will contribute to the safety evaluation of quantum dots, and provide valuable information for design of nanomaterials. Copyright © 2014 Elsevier Ltd. All rights reserved.
Jhun, Bong Sook; Xu, Shangcheng; Hurst, Stephen; Raffaello, Anna; Liu, Xiaoyun; Yi, Bing; Zhang, Huiliang; Gross, Polina; Mishra, Jyotsna; Ainbinder, Alina; Kettlewell, Sarah; Smith, Godfrey L.; Dirksen, Robert T.; Wang, Wang; Rizzuto, Rosario
2014-01-01
Abstract Aims: Mitochondrial Ca2+ homeostasis is crucial for balancing cell survival and death. The recent discovery of the molecular identity of the mitochondrial Ca2+ uniporter pore (MCU) opens new possibilities for applying genetic approaches to study mitochondrial Ca2+ regulation in various cell types, including cardiac myocytes. Basal tyrosine phosphorylation of MCU was reported from mass spectroscopy of human and mouse tissues, but the signaling pathways that regulate mitochondrial Ca2+ entry through posttranslational modifications of MCU are completely unknown. Therefore, we investigated α1-adrenergic-mediated signal transduction of MCU posttranslational modification and function in cardiac cells. Results: α1-adrenoceptor (α1-AR) signaling translocated activated proline-rich tyrosine kinase 2 (Pyk2) from the cytosol to mitochondrial matrix and accelerates mitochondrial Ca2+ uptake via Pyk2-dependent MCU phosphorylation and tetrametric MCU channel pore formation. Moreover, we found that α1-AR stimulation increases reactive oxygen species production at mitochondria, mitochondrial permeability transition pore activity, and initiates apoptotic signaling via Pyk2-dependent MCU activation and mitochondrial Ca2+ overload. Innovation: Our data indicate that inhibition of α1-AR-Pyk2-MCU signaling represents a potential novel therapeutic target to limit or prevent mitochondrial Ca2+ overload, oxidative stress, mitochondrial injury, and myocardial death during pathophysiological conditions, where chronic adrenergic stimulation is present. Conclusion: The α1-AR-Pyk2-dependent tyrosine phosphorylation of the MCU regulates mitochondrial Ca2+ entry and apoptosis in cardiac cells. Antioxid. Redox Signal. 21, 863–879. PMID:24800979
Mitochondrial oxidative stress in aging and healthspan
2014-01-01
The free radical theory of aging proposes that reactive oxygen species (ROS)-induced accumulation of damage to cellular macromolecules is a primary driving force of aging and a major determinant of lifespan. Although this theory is one of the most popular explanations for the cause of aging, several experimental rodent models of antioxidant manipulation have failed to affect lifespan. Moreover, antioxidant supplementation clinical trials have been largely disappointing. The mitochondrial theory of aging specifies more particularly that mitochondria are both the primary sources of ROS and the primary targets of ROS damage. In addition to effects on lifespan and aging, mitochondrial ROS have been shown to play a central role in healthspan of many vital organ systems. In this article we review the evidence supporting the role of mitochondrial oxidative stress, mitochondrial damage and dysfunction in aging and healthspan, including cardiac aging, age-dependent cardiovascular diseases, skeletal muscle aging, neurodegenerative diseases, insulin resistance and diabetes as well as age-related cancers. The crosstalk of mitochondrial ROS, redox, and other cellular signaling is briefly presented. Potential therapeutic strategies to improve mitochondrial function in aging and healthspan are reviewed, with a focus on mitochondrial protective drugs, such as the mitochondrial antioxidants MitoQ, SkQ1, and the mitochondrial protective peptide SS-31. PMID:24860647
Exercise training improves vascular mitochondrial function
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
Datta, Sandipan; Sahdeo, Sunil; Gray, Jennifer A.; Morriseau, Christophe; Hammock, Bruce D.; Cortopassi, Gino
2016-01-01
Mitochondrial toxicity is emerging as a major mechanism underlying serious human health consequences. This work performs a high-throughput screen (HTS) of 176 environmental chemicals for mitochondrial toxicity utilizing a previously reported biosensor platform. This established HTS confirmed known mitochondrial toxins and identified novel mitotochondrial uncouplers such as 2, 2′-Methylenebis(4-chlorophenol) and pentachlorophenol. It also identified a mitochondrial ‘structure activity relationship’ (SAR) in the sense that multiple environmental chlorophenols are mitochondrial inhibitors and uncouplers. This study demonstrates proof-of-concept that a mitochondrial HTS assay detects known and novel environmental mitotoxicants, and could be used to quickly evaluate human health risks from mitotoxicants in the environment. PMID:27717841
Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy (CIPN)
Canta, Annalisa; Pozzi, Eleonora; Carozzi, Valentina Alda
2015-01-01
The mitochondrial dysfunction has a critical role in several disorders including chemotherapy-induced peripheral neuropathies (CIPN). This is due to a related dysregulation of pathways involving calcium signalling, reactive oxygen species and apoptosis. Vincristine is able to affect calcium movement through the Dorsal Root Ganglia (DRG) neuronal mitochondrial membrane, altering its homeostasis and leading to abnormal neuronal excitability. Paclitaxel induces the opening of the mitochondrial permeability transition pore in axons followed by mitochondrial membrane potential loss, increased reactive oxygen species generation, ATP level reduction, calcium release and mitochondrial swelling. Cisplatin and oxaliplatin form adducts with mitochondrial DNA producing inhibition of replication, disruption of transcription and morphological abnormalities within mitochondria in DRG neurons, leading to a gradual energy failure. Bortezomib is able to modify mitochondrial calcium homeostasis and mitochondrial respiratory chain. Moreover, the expression of a certain number of genes, including those controlling mitochondrial functions, was altered in patients with bortezomib-induced peripheral neuropathy. PMID:29056658
Biocuration at the Saccharomyces genome database.
Skrzypek, Marek S; Nash, Robert S
2015-08-01
Saccharomyces Genome Database is an online resource dedicated to managing information about the biology and genetics of the model organism, yeast (Saccharomyces cerevisiae). This information is derived primarily from scientific publications through a process of human curation that involves manual extraction of data and their organization into a comprehensive system of knowledge. This system provides a foundation for further analysis of experimental data coming from research on yeast as well as other organisms. In this review we will demonstrate how biocuration and biocurators add a key component, the biological context, to our understanding of how genes, proteins, genomes and cells function and interact. We will explain the role biocurators play in sifting through the wealth of biological data to incorporate and connect key information. We will also discuss the many ways we assist researchers with their various research needs. We hope to convince the reader that manual curation is vital in converting the flood of data into organized and interconnected knowledge, and that biocurators play an essential role in the integration of scientific information into a coherent model of the cell. © 2015 Wiley Periodicals, Inc.
PCR on yeast colonies: an improved method for glyco-engineered Saccharomyces cerevisiae
2013-01-01
Background Saccharomyces cerevisiae is extensively used in bio-industries. However, its genetic engineering to introduce new metabolism pathways can cause unexpected phenotypic alterations. For example, humanisation of the glycosylation pathways is a high priority pharmaceutical industry goal for production of therapeutic glycoproteins in yeast. Genomic modifications can lead to several described physiological changes: biomass yields decrease, temperature sensitivity or cell wall structure modifications. We have observed that deletion of several N-mannosyltransferases in Saccharomyces cerevisiae, results in strains that can no longer be analyzed by classical PCR on yeast colonies. Findings In order to validate our glyco-engineered Saccharomyces cerevisiae strains, we developed a new protocol to carry out PCR directly on genetically modified yeast colonies. A liquid culture phase, combined with the use of a Hot Start DNA polymerase, allows a 3-fold improvement of PCR efficiency. The results obtained are repeatable and independent of the targeted sequence; as such the protocol is well adapted for intensive screening applications. Conclusions The developed protocol enables by-passing of many of the difficulties associated with PCR caused by phenotypic modifications brought about by humanisation of the glycosylation in yeast and allows rapid validation of glyco-engineered Saccharomyces cerevisiae cells. It has the potential to be extended to other yeast strains presenting cell wall structure modifications. PMID:23688076
Aspirin Increases Mitochondrial Fatty Acid Oxidation
Uppala, Radha; Dudiak, Brianne; Beck, Megan E.; Bharathi, Sivakama S.; Zhang, Yuxun; Stolz, Donna B.; Goetzman, Eric S.
2016-01-01
The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse the mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 hr incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders. PMID:27856258
Evolution of gastropod mitochondrial genome arrangements
2008-01-01
Background Gastropod mitochondrial genomes exhibit an unusually great variety of gene orders compared to other metazoan mitochondrial genome such as e.g those of vertebrates. Hence, gastropod mitochondrial genomes constitute a good model system to study patterns, rates, and mechanisms of mitochondrial genome rearrangement. However, this kind of evolutionary comparative analysis requires a robust phylogenetic framework of the group under study, which has been elusive so far for gastropods in spite of the efforts carried out during the last two decades. Here, we report the complete nucleotide sequence of five mitochondrial genomes of gastropods (Pyramidella dolabrata, Ascobulla fragilis, Siphonaria pectinata, Onchidella celtica, and Myosotella myosotis), and we analyze them together with another ten complete mitochondrial genomes of gastropods currently available in molecular databases in order to reconstruct the phylogenetic relationships among the main lineages of gastropods. Results Comparative analyses with other mollusk mitochondrial genomes allowed us to describe molecular features and general trends in the evolution of mitochondrial genome organization in gastropods. Phylogenetic reconstruction with commonly used methods of phylogenetic inference (ME, MP, ML, BI) arrived at a single topology, which was used to reconstruct the evolution of mitochondrial gene rearrangements in the group. Conclusion Four main lineages were identified within gastropods: Caenogastropoda, Vetigastropoda, Patellogastropoda, and Heterobranchia. Caenogastropoda and Vetigastropoda are sister taxa, as well as, Patellogastropoda and Heterobranchia. This result rejects the validity of the derived clade Apogastropoda (Caenogastropoda + Heterobranchia). The position of Patellogastropoda remains unclear likely due to long-branch attraction biases. Within Heterobranchia, the most heterogeneous group of gastropods, neither Euthyneura (because of the inclusion of P. dolabrata) nor Pulmonata
Kwolek-Mirek, Magdalena; Molon, Mateusz; Kaszycki, Pawel; Zadrag-Tecza, Renata
2016-08-01
Carnosine is an endogenous dipeptide composed of β-alanine and L-histidine, which occurs in vertebrates, including humans. It has a number of favorable properties including buffering, chelating, antioxidant, anti-glycation and anti-aging activities. In our study we used the Saccharomyces cerevisiae yeast as a model organism to examine the impact of L-carnosine on the cell lifespan. We demonstrated that L-carnosine slowed down the growth and decreased the metabolic activity of cells as well as prolonged their generation time. On the other hand, it allowed for enhancement of the yeast reproductive potential and extended its reproductive lifespan. These changes may be a result of the reduced mitochondrial membrane potential and decreased ATP content in the yeast cells. However, due to reduction of the post-reproductive lifespan, L-carnosine did not have an influence on the total lifespan of yeast. In conclusion, L-carnosine does not extend the total lifespan of S. cerevisiae but rather it increases the yeast's reproductive capacity by increasing the number of daughter cells produced.
Marmiroli, M; Pagano, L; Pasquali, F; Zappettini, A; Tosato, V; Bruschi, C V; Marmiroli, N
2016-01-01
The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics industry. Their size (1-10 nm in diameter) is, however, such that they can be taken up by living cells. Here, a bakers' yeast (Saccharomyces cerevisiae) deletion mutant collection has been exploited to provide a high-throughput means of revealing the genetic basis for tolerance/susceptibility to CdS QD exposure. The deletion of 112 genes, some associated with the abiotic stress response, some with various metabolic processes, some with mitochondrial organization, some with transport and some with DNA repair, reduced the level of tolerance to CdS QDs. A gene ontology analysis highlighted the role of oxidative stress in determining the cellular response. The transformation of sensitive mutants with centromeric plasmids harbouring DNA from a wild type strain restored the wild type growth phenotype when the complemented genes encoded either HSC82, DSK2 or ALD3. The use of these simple eukaryote knock-out mutants for functional toxicogenomic analysis will inform studies focusing on higher organisms.
Loss of Mitochondrial Function Impairs Lysosomes.
Demers-Lamarche, Julie; Guillebaud, Gérald; Tlili, Mouna; Todkar, Kiran; Bélanger, Noémie; Grondin, Martine; Nguyen, Angela P; Michel, Jennifer; Germain, Marc
2016-05-06
Alterations in mitochondrial function, as observed in neurodegenerative diseases, lead to disrupted energy metabolism and production of damaging reactive oxygen species. Here, we demonstrate that mitochondrial dysfunction also disrupts the structure and function of lysosomes, the main degradation and recycling organelle. Specifically, inhibition of mitochondrial function, following deletion of the mitochondrial protein AIF, OPA1, or PINK1, as well as chemical inhibition of the electron transport chain, impaired lysosomal activity and caused the appearance of large lysosomal vacuoles. Importantly, our results show that lysosomal impairment is dependent on reactive oxygen species. Given that alterations in both mitochondrial function and lysosomal activity are key features of neurodegenerative diseases, this work provides important insights into the etiology of neurodegenerative diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Mitochondrial dynamics in mammalian health and disease.
Liesa, Marc; Palacín, Manuel; Zorzano, Antonio
2009-07-01
The meaning of the word mitochondrion (from the Greek mitos, meaning thread, and chondros, grain) illustrates that the heterogeneity of mitochondrial morphology has been known since the first descriptions of this organelle. Such a heterogeneous morphology is explained by the dynamic nature of mitochondria. Mitochondrial dynamics is a concept that includes the movement of mitochondria along the cytoskeleton, the regulation of mitochondrial architecture (morphology and distribution), and connectivity mediated by tethering and fusion/fission events. The relevance of these events in mitochondrial and cell physiology has been partially unraveled after the identification of the genes responsible for mitochondrial fusion and fission. Furthermore, during the last decade, it has been identified that mutations in two mitochondrial fusion genes (MFN2 and OPA1) cause prevalent neurodegenerative diseases (Charcot-Marie Tooth type 2A and Kjer disease/autosomal dominant optic atrophy). In addition, other diseases such as type 2 diabetes or vascular proliferative disorders show impaired MFN2 expression. Altogether, these findings have established mitochondrial dynamics as a consolidated area in cellular physiology. Here we review the most significant findings in the field of mitochondrial dynamics in mammalian cells and their implication in human pathologies.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Serasinghe, Madhavika N.; Mitochondrial Research and Innovation Group, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; Yoon, Yisang
2008-11-15
Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six {alpha}-helices ({alpha}1-{alpha}6) out of which {alpha}2-{alpha}5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found thatmore » hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the {alpha}1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that {alpha}1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the {alpha}5 helix and the linker between {alpha}3 and {alpha}4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by {alpha}1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.« less
DeBalsi, Karen L.; Hoff, Kirsten E.; Copeland, William C.
2016-01-01
As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined. PMID:27143693
Development of pharmacological strategies for mitochondrial disorders
Kanabus, M; Heales, S J; Rahman, S
2014-01-01
Mitochondrial diseases are an unusually genetically and phenotypically heterogeneous group of disorders, which are extremely challenging to treat. Currently, apart from supportive therapy, there are no effective treatments for the vast majority of mitochondrial diseases. Huge scientific effort, however, is being put into understanding the mechanisms underlying mitochondrial disease pathology and developing potential treatments. To date, a variety of treatments have been evaluated by randomized clinical trials, but unfortunately, none of these has delivered breakthrough results. Increased understanding of mitochondrial pathways and the development of many animal models, some of which are accurate phenocopies of human diseases, are facilitating the discovery and evaluation of novel prospective treatments. Targeting reactive oxygen species has been a treatment of interest for many years; however, only in recent years has it been possible to direct antioxidant delivery specifically into the mitochondria. Increasing mitochondrial biogenesis, whether by pharmacological approaches, dietary manipulation or exercise therapy, is also currently an active area of research. Modulating mitochondrial dynamics and mitophagy and the mitochondrial membrane lipid milieu have also emerged as possible treatment strategies. Recent technological advances in gene therapy, including allotopic and transkingdom gene expression and mitochondrially targeted transcription activator-like nucleases, have led to promising results in cell and animal models of mitochondrial diseases, but most of these techniques are still far from clinical application. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8 PMID:24116962
Mitochondrial dysfunction in autism.
Giulivi, Cecilia; Zhang, Yi-Fan; Omanska-Klusek, Alicja; Ross-Inta, Catherine; Wong, Sarah; Hertz-Picciotto, Irva; Tassone, Flora; Pessah, Isaac N
2010-12-01
Impaired mitochondrial function may influence processes highly dependent on energy, such as neurodevelopment, and contribute to autism. No studies have evaluated mitochondrial dysfunction and mitochondrial DNA (mtDNA) abnormalities in a well-defined population of children with autism. To evaluate mitochondrial defects in children with autism. Observational study using data collected from patients aged 2 to 5 years who were a subset of children participating in the Childhood Autism Risk From Genes and Environment study in California, which is a population-based, case-control investigation with confirmed autism cases and age-matched, genetically unrelated, typically developing controls, that was launched in 2003 and is still ongoing. Mitochondrial dysfunction and mtDNA abnormalities were evaluated in lymphocytes from 10 children with autism and 10 controls. Oxidative phosphorylation capacity, mtDNA copy number and deletions, mitochondrial rate of hydrogen peroxide production, and plasma lactate and pyruvate. The reduced nicotinamide adenine dinucleotide (NADH) oxidase activity (normalized to citrate synthase activity) in lymphocytic mitochondria from children with autism was significantly lower compared with controls (mean, 4.4 [95% confidence interval {CI}, 2.8-6.0] vs 12 [95% CI, 8-16], respectively; P = .001). The majority of children with autism (6 of 10) had complex I activity below control range values. Higher plasma pyruvate levels were found in children with autism compared with controls (0.23 mM [95% CI, 0.15-0.31 mM] vs 0.08 mM [95% CI, 0.04-0.12 mM], respectively; P = .02). Eight of 10 cases had higher pyruvate levels but only 2 cases had higher lactate levels compared with controls. These results were consistent with the lower pyruvate dehydrogenase activity observed in children with autism compared with controls (1.0 [95% CI, 0.6-1.4] nmol × [min × mg protein](-1) vs 2.3 [95% CI, 1.7-2.9] nmol × [min × mg protein](-1), respectively; P = .01
Pharmacological modulation of mitochondrial calcium homeostasis.
Arduino, Daniela M; Perocchi, Fabiana
2018-01-10
Mitochondria are pivotal organelles in calcium (Ca 2+ ) handling and signalling, constituting intracellular checkpoints for numerous processes that are vital for cell life. Alterations in mitochondrial Ca 2+ homeostasis have been linked to a variety of pathological conditions and are critical in the aetiology of several human diseases. Efforts have been taken to harness mitochondrial Ca 2+ transport mechanisms for therapeutic intervention, but pharmacological compounds that direct and selectively modulate mitochondrial Ca 2+ homeostasis are currently lacking. New avenues have, however, emerged with the breakthrough discoveries on the genetic identification of the main players involved in mitochondrial Ca 2+ influx and efflux pathways and with recent hints towards a deep understanding of the function of these molecular systems. Here, we review the current advances in the understanding of the mechanisms and regulation of mitochondrial Ca 2+ homeostasis and its contribution to physiology and human disease. We also introduce and comment on the recent progress towards a systems-level pharmacological targeting of mitochondrial Ca 2+ homeostasis. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.
Atypical mitochondrial fission upon bacterial infection
Stavru, Fabrizia; Palmer, Amy E.; Wang, Chunxin; Youle, Richard J.; Cossart, Pascale
2013-01-01
We recently showed that infection by Listeria monocytogenes causes mitochondrial network fragmentation through the secreted pore-forming toxin listeriolysin O (LLO). Here, we examine factors involved in canonical fusion and fission. Strikingly, LLO-induced mitochondrial fragmentation does not require the traditional fission machinery, as Drp1 oligomers are absent from fragmented mitochondria following Listeria infection or LLO treatment, as the dynamin-like protein 1 (Drp1) receptor Mff is rapidly degraded, and as fragmentation proceeds efficiently in cells with impaired Drp1 function. LLO does not cause processing of the fusion protein optic atrophy protein 1 (Opa1), despite inducing a decrease in the mitochondrial membrane potential, suggesting a unique Drp1- and Opa1-independent fission mechanism distinct from that triggered by uncouplers or the apoptosis inducer staurosporine. We show that the ER marks LLO-induced mitochondrial fragmentation sites even in the absence of functional Drp1, demonstrating that the ER activity in regulating mitochondrial fission can be induced by exogenous agents and that the ER appears to regulate fission by a mechanism independent of the canonical mitochondrial fission machinery. PMID:24043775
DOE Office of Scientific and Technical Information (OSTI.GOV)
Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester
Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessedmore » by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kobashigawa, Shinko, E-mail: kobashin@nagasaki-u.ac.jp; Suzuki, Keiji; Yamashita, Shunichi
2011-11-04
Highlights: Black-Right-Pointing-Pointer We report first time that ionizing radiation induces mitochondrial dynamic changes. Black-Right-Pointing-Pointer Radiation-induced mitochondrial fission was caused by Drp1 localization. Black-Right-Pointing-Pointer We found that radiation causes delayed ROS from mitochondria. Black-Right-Pointing-Pointer Down regulation of Drp1 rescued mitochondrial dysfunction after radiation exposure. -- Abstract: Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial O{submore » 2}{sup {center_dot}-} production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial O{sub 2}{sup {center_dot}-}. Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.« less
Molecular mechanisms of ethanol tolerance in Saccharomyces cerevisiae
USDA-ARS?s Scientific Manuscript database
The yeast Saccharomyces cerevisiae is a superb ethanol producer, yet sensitive to ethanol at higher concentrations especially under high gravity or very high gravity fermentation conditions. Although significant efforts have been made to study ethanol-stress response in past decades, molecular mecha...
High quality de novo sequencing and assembly of the Saccharomyces arboricolus genome
2013-01-01
Background Comparative genomics is a formidable tool to identify functional elements throughout a genome. In the past ten years, studies in the budding yeast Saccharomyces cerevisiae and a set of closely related species have been instrumental in showing the benefit of analyzing patterns of sequence conservation. Increasing the number of closely related genome sequences makes the comparative genomics approach more powerful and accurate. Results Here, we report the genome sequence and analysis of Saccharomyces arboricolus, a yeast species recently isolated in China, that is closely related to S. cerevisiae. We obtained high quality de novo sequence and assemblies using a combination of next generation sequencing technologies, established the phylogenetic position of this species and considered its phenotypic profile under multiple environmental conditions in the light of its gene content and phylogeny. Conclusions We suggest that the genome of S. arboricolus will be useful in future comparative genomics analysis of the Saccharomyces sensu stricto yeasts. PMID:23368932
Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi
2017-08-01
Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study. Copyright © 2017 Elsevier Ltd. All rights reserved.
Mitochondrial recombination increases with age in Podospora anserina.
van Diepeningen, Anne D; Goedbloed, Daniël J; Slakhorst, S Marijke; Koopmanschap, A Bertha; Maas, Marc F P M; Hoekstra, Rolf F; Debets, Alfons J M
2010-05-01
With uniparental inheritance of mitochondria, there seems little reason for homologous recombination in mitochondria, but the machinery for mitochondrial recombination is quite well-conserved in many eukaryote species. In fungi and yeasts heteroplasmons may be formed when strains fuse and transfer of organelles takes place, making it possible to study mitochondrial recombination when introduced mitochondria contain different markers. A survey of wild-type isolates from a local population of the filamentous fungus Podospora anserina for the presence of seven optional mitochondrial introns indicated that mitochondrial recombination does take place in nature. Moreover the recombination frequency appeared to be correlated with age: the more rapidly ageing fraction of the population had a significantly lower linkage disequilibrium indicating more recombination. Direct confrontation experiments with heterokaryon incompatible strains with different mitochondrial markers at different (relative) age confirmed that mitochondrial recombination increases with age. We propose that with increasing mitochondrial damage over time, mitochondrial recombination - even within a homoplasmic population of mitochondria - is a mechanism that may restore mitochondrial function. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.
Lin, Hung-Yu; Liou, Chia-Wei; Chen, Shang-Der; Hsu, Te-Yao; Chuang, Jiin-Haur; Wang, Pei-Wen; Huang, Sheng-Teng; Tiao, Mao-Meng; Chen, Jin-Bor; Lin, Tsu-Kung; Chuang, Yao-Chung
2015-05-01
Adult mesenchymal stem cell (MSC)-conducted mitochondrial transfer has been recently shown to rescue cellular bioenergetics and prevent cell death caused by mitochondrial dysfunction. Wharton's jelly-derived MSCs (WJMSCs) harvested from postpartum umbilical cords are an accessible and abundant source of stem cells. This study aimed to determine the capability of WJMSCs to transfer their own mitochondria and rescue impaired oxidative phosphorylation (OXPHOS) and bioenergetics caused by mitochondrial DNA defects. To do this, WJMSCs were co-cultured with mitochondrial DNA (mtDNA)-depleted ρ(0) cells and the recapture of mitochondrial function was evaluated. WJMSCs were shown to be capable of transferring their own mitochondria into ρ(0) cells and underwent interorganellar mixture within these cells. Permissive culture media (BrdU-containing and pyruvate- and uridine-free) sieved out a survival cell population from the co-cultured WJMSCs (BrdU-sensitive) and ρ(0) cells (pyruvate/uridine-free). The survival cells had mtDNA identical to that of WJMSCs, whereas they expressed cellular markers identical to that of ρ(0) cells. Importantly, these ρ(0)-plus -WJMSC-mtDNA (ρ(+W)) cells recovered the expression of mtDNA-encoded proteins and exhibited functional oxygen consumption and respiratory control, as well as the activity of electron transport chain (ETC) complexes I, II, III and IV. In addition, ETC complex V-inhibitor-sensitive ATP production and metabolic shifting were also recovered. Furthermore, cellular behaviors including attachment-free proliferation, aerobic viability and OXPHOS-reliant cellular motility were also regained after mitochondrial transfer by WJMSCs. The therapeutic effect of WJMSCs-derived mitochondrial transfer was able to stably sustain for at least 45 passages. In conclusion, this study suggests that WJMSCs may serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction through the donation of healthy
Boczonadi, Veronika; King, Martin S; Smith, Anthony C; Olahova, Monika; Bansagi, Boglarka; Roos, Andreas; Eyassu, Filmon; Borchers, Christoph; Ramesh, Venkateswaran; Lochmüller, Hanns; Polvikoski, Tuomo; Whittaker, Roger G; Pyle, Angela; Griffin, Helen; Taylor, Robert W; Chinnery, Patrick F; Robinson, Alan J; Kunji, Edmund R S; Horvath, Rita
2018-03-08
PurposeTo understand the role of the mitochondrial oxodicarboxylate carrier (SLC25A21) in the development of spinal muscular atrophy-like disease.MethodsWe identified a novel pathogenic variant in a patient by whole-exome sequencing. The pathogenicity of the mutation was studied by transport assays, computer modeling, followed by targeted metabolic testing and in vitro studies in human fibroblasts and neurons.ResultsThe patient carries a homozygous pathogenic variant c.695A>G; p.(Lys232Arg) in the SLC25A21 gene, encoding the mitochondrial oxodicarboxylate carrier, and developed spinal muscular atrophy and mitochondrial myopathy. Transport assays show that the mutation renders SLC25A21 dysfunctional and 2-oxoadipate cannot be imported into the mitochondrial matrix. Computer models of central metabolism predicted that impaired transport of oxodicarboxylate disrupts the pathways of lysine and tryptophan degradation, and causes accumulation of 2-oxoadipate, pipecolic acid, and quinolinic acid, which was confirmed in the patient's urine by targeted metabolomics. Exposure to 2-oxoadipate and quinolinic acid decreased the level of mitochondrial complexes in neuronal cells (SH-SY5Y) and induced apoptosis.ConclusionMitochondrial oxodicarboxylate carrier deficiency leads to mitochondrial dysfunction and the accumulation of oxoadipate and quinolinic acid, which in turn cause toxicity in spinal motor neurons leading to spinal muscular atrophy-like disease.GENETICS in MEDICINE advance online publication, 8 March 2018; doi:10.1038/gim.2017.251.
Charron, Guillaume; Leducq, Jean-Baptiste; Bertin, Chloé; Dubé, Alexandre K; Landry, Christian R
2014-03-01
We examined the northern limit of Saccharomyces cerevisiae and Saccharomyces paradoxus in northeast America. We collected 876 natural samples at 29 sites and applied enrichment methods for the isolation of mesophilic yeasts. We uncovered a large diversity of yeasts, in some cases, associated with specific substrates. Sequencing of the ITS1, 5.8S and ITS2 loci allowed to assign 226 yeast strains at the species level, including 41 S. paradoxus strains. Our intensive sampling suggests that if present, S. cerevisiae is rare at these northern latitudes. Our sampling efforts spread across several months of the year revealed that successful sampling increases throughout the summer and diminishes significantly at the beginning of the fall. The data obtained on the ecological context of yeasts corroborate what was previously reported on Pichiaceae, Saccharomycodaceae, Debaryomycetaceae and Phaffomycetaceae yeast families. We identified 24 yeast isolates that could not be assigned to any known species and that may be of taxonomic, medical, or biotechnological importance. Our study reports new data on the taxonomic diversity of yeasts and new resources for studying the evolution and ecology of S. paradoxus. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
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.