Gabriel, Frédéric; Accoceberry, Isabelle; Bessoule, Jean-Jacques; Salin, Bénédicte; Lucas-Guérin, Marine; Manon, Stephen; Dementhon, Karine; Noël, Thierry
2014-01-01
It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying 14Cα-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.
Bessoule, Jean-Jacques; Salin, Bénédicte; Lucas-Guérin, Marine; Manon, Stephen; Dementhon, Karine; Noël, Thierry
2014-01-01
It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying 14Cα-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner. PMID:25486052
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.
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
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
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.
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.
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.
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.
Nickel Inhibits Mitochondrial Fatty Acid Oxidation
Uppala, Radha; McKinney, Richard W.; Brant, Kelly A.; Fabisiak, James P.; Goetzman, Eric S.
2015-01-01
Nickel exposure is associated with changes in cellular energy metabolism which may contribute to its carcinogenic properties. Here, we demonstrate that nickel strongly represses mitochondrial fatty acid oxidation—the pathway by which fatty acids are catabolized for energy—in both primary human lung fibroblasts and mouse embryonic fibroblasts. At the concentrations used, nickel suppresses fatty acid oxidation without globally suppressing mitochondrial function as evidenced by increased glucose oxidation to CO2. Pre-treatment with L-carnitine, previously shown to prevent nickel-induced mitochondrial dysfunction in neuroblastoma cells, did not prevent the inhibition of fatty acid oxidation. The effect of nickel on fatty acid oxidation occurred only with prolonged exposure (>5 hr), suggesting that direct inhibition of the active sites of metabolic enzymes is not the mechanism of action. Nickel is a known hypoxia-mimetic that activates hypoxia inducible factor-1α (HIF1α). Nickel-induced inhibition of fatty acid oxidation was blunted in HIF1α knockout fibroblasts, implicating HIF1α as one contributor to the mechanism. Additionally, nickel down-regulated the protein levels of the key fatty acid oxidation enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) in a dose-dependent fashion. In conclusion, inhibition of fatty acid oxidation by nickel, concurrent with increased glucose metabolism, represents a form of metabolic reprogramming that may contribute to nickel-induced carcinogenesis. PMID:26051273
Nickel inhibits mitochondrial fatty acid oxidation.
Uppala, Radha; McKinney, Richard W; Brant, Kelly A; Fabisiak, James P; Goetzman, Eric S
2015-08-07
Nickel exposure is associated with changes in cellular energy metabolism which may contribute to its carcinogenic properties. Here, we demonstrate that nickel strongly represses mitochondrial fatty acid oxidation-the pathway by which fatty acids are catabolized for energy-in both primary human lung fibroblasts and mouse embryonic fibroblasts. At the concentrations used, nickel suppresses fatty acid oxidation without globally suppressing mitochondrial function as evidenced by increased glucose oxidation to CO2. Pre-treatment with l-carnitine, previously shown to prevent nickel-induced mitochondrial dysfunction in neuroblastoma cells, did not prevent the inhibition of fatty acid oxidation. The effect of nickel on fatty acid oxidation occurred only with prolonged exposure (>5 h), suggesting that direct inhibition of the active sites of metabolic enzymes is not the mechanism of action. Nickel is a known hypoxia-mimetic that activates hypoxia inducible factor-1α (HIF1α). Nickel-induced inhibition of fatty acid oxidation was blunted in HIF1α knockout fibroblasts, implicating HIF1α as one contributor to the mechanism. Additionally, nickel down-regulated the protein levels of the key fatty acid oxidation enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) in a dose-dependent fashion. In conclusion, inhibition of fatty acid oxidation by nickel, concurrent with increased glucose metabolism, represents a form of metabolic reprogramming that may contribute to nickel-induced carcinogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.
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
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
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.
Mitochondrial metabolism and stress response of yeast: Applications in fermentation technologies.
Kitagaki, Hiroshi; Takagi, Hiroshi
2014-04-01
Mitochondria are sites of oxidative respiration. During sake brewing, sake yeasts are exposed to long periods of hypoxia; the structure, role, and metabolism of mitochondria of sake yeasts have not been studied in detail. It was first elucidated that the mitochondrial structure of sake yeast transforms from filamentous to dotted structure during sake brewing, which affects malate metabolism. Based on the information of yeast mitochondria during sake brewing, practical technologies have been developed; (i) breeding pyruvate-underproducing sake yeast by the isolation of a mutant resistant to an inhibitor of mitochondrial pyruvate transport; and (ii) modifying malate and succinate production by manipulating mitochondrial activity. During the bread-making process, baker's yeast cells are exposed to a variety of baking-associated stresses, such as freeze-thaw, air-drying, and high sucrose concentrations. These treatments induce oxidative stress generating reactive oxygen species due to mitochondrial damage. A novel metabolism of proline and arginine catalyzed by N-acetyltransferase Mpr1 in the mitochondria eventually leads to synthesis of nitric oxide, which confers oxidative stress tolerance on yeast cells. The enhancement of proline and arginine metabolism could be promising for breeding novel baker's yeast strains that are tolerant to multiple baking-associated stresses. These new and practical methods provide approaches to improve the processes in the field of industrial fermentation technologies. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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
[Genetic system for maintaining the mitochondrial human genome in yeast Yarrowia lipolytica].
Isakova, E P; Deryabina, Yu I; Velyakova, A V; Biryukova, J K; Teplova, V V; Shevelev, A B
2016-01-01
For the first time, the possibility of maintaining an intact human mitochondrial genome in a heterologous system in the mitochondria of yeast Yarrowia lipolytica is shown. A method for introducing directional changes into the structure of the mitochondrial human genome replicating in Y. lipolytica by an artificially induced ability of yeast mitochondria for homologous recombination is proposed. A method of introducing and using phenotypic selection markers for the presence or absence of defects in genes tRNA-Lys and tRNA-Leu of the mitochondrial genome is developed. The proposed system can be used to correct harmful mutations of the human mitochondrial genome associated with mitochondrial diseases and for preparative amplification of intact mitochondrial DNA with an adjusted sequence in yeast cells. The applicability of the new system for the correction of mutations in the genes of Lys- and Leu-specific tRNAs of the human mitochondrial genome associated with serious and widespread human mitochondrial diseases such as myoclonic epilepsy with lactic acidosis (MELAS) and myoclonic epilepsy with ragged-red fibers (MERRF) is shown.
Homocysteine regulates fatty acid and lipid metabolism in yeast.
Visram, Myriam; Radulovic, Maja; Steiner, Sabine; Malanovic, Nermina; Eichmann, Thomas O; Wolinski, Heimo; Rechberger, Gerald N; Tehlivets, Oksana
2018-04-13
S -Adenosyl-l-homocysteine hydrolase (AdoHcy hydrolase; Sah1 in yeast/AHCY in mammals) degrades AdoHcy, a by-product and strong product inhibitor of S -adenosyl-l-methionine (AdoMet)-dependent methylation reactions, to adenosine and homocysteine (Hcy). This reaction is reversible, so any elevation of Hcy levels, such as in hyperhomocysteinemia (HHcy), drives the formation of AdoHcy, with detrimental consequences for cellular methylation reactions. HHcy, a pathological condition linked to cardiovascular and neurological disorders, as well as fatty liver among others, is associated with a deregulation of lipid metabolism. Here, we developed a yeast model of HHcy to identify mechanisms that dysregulate lipid metabolism. Hcy supplementation to wildtype cells up-regulated cellular fatty acid and triacylglycerol content and induced a shift in fatty acid composition, similar to changes observed in mutants lacking Sah1. Expression of the irreversible bacterial pathway for AdoHcy degradation in yeast allowed us to dissect the impact of AdoHcy accumulation on lipid metabolism from the impact of elevated Hcy. Expression of this pathway fully suppressed the growth deficit of sah1 mutants as well as the deregulation of lipid metabolism in both the sah1 mutant and Hcy-exposed wildtype, showing that AdoHcy accumulation mediates the deregulation of lipid metabolism in response to elevated Hcy in yeast. Furthermore, Hcy supplementation in yeast led to increased resistance to cerulenin, an inhibitor of fatty acid synthase, as well as to a concomitant decline of condensing enzymes involved in very long-chain fatty acid synthesis, in line with the observed shift in fatty acid content and composition. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Homocysteine regulates fatty acid and lipid metabolism in yeast
Visram, Myriam; Radulovic, Maja; Steiner, Sabine; Malanovic, Nermina; Eichmann, Thomas O.; Wolinski, Heimo; Rechberger, Gerald N.; Tehlivets, Oksana
2018-01-01
S-Adenosyl-l-homocysteine hydrolase (AdoHcy hydrolase; Sah1 in yeast/AHCY in mammals) degrades AdoHcy, a by-product and strong product inhibitor of S-adenosyl-l-methionine (AdoMet)-dependent methylation reactions, to adenosine and homocysteine (Hcy). This reaction is reversible, so any elevation of Hcy levels, such as in hyperhomocysteinemia (HHcy), drives the formation of AdoHcy, with detrimental consequences for cellular methylation reactions. HHcy, a pathological condition linked to cardiovascular and neurological disorders, as well as fatty liver among others, is associated with a deregulation of lipid metabolism. Here, we developed a yeast model of HHcy to identify mechanisms that dysregulate lipid metabolism. Hcy supplementation to wildtype cells up-regulated cellular fatty acid and triacylglycerol content and induced a shift in fatty acid composition, similar to changes observed in mutants lacking Sah1. Expression of the irreversible bacterial pathway for AdoHcy degradation in yeast allowed us to dissect the impact of AdoHcy accumulation on lipid metabolism from the impact of elevated Hcy. Expression of this pathway fully suppressed the growth deficit of sah1 mutants as well as the deregulation of lipid metabolism in both the sah1 mutant and Hcy-exposed wildtype, showing that AdoHcy accumulation mediates the deregulation of lipid metabolism in response to elevated Hcy in yeast. Furthermore, Hcy supplementation in yeast led to increased resistance to cerulenin, an inhibitor of fatty acid synthase, as well as to a concomitant decline of condensing enzymes involved in very long-chain fatty acid synthesis, in line with the observed shift in fatty acid content and composition. PMID:29414770
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.
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.
Douglas, M G; Butow, R A
1976-04-01
Products of mitochondrial protein synthesis in yeast have been labeled in vivo with 35SO42-. More than 20 polypeptide species fulfilling the criteria of mitochondrial translation products have been detected by analysis on sodium dodecyl sulfate-exponential polyacrylamide slab gels. A comparison of mitochondrial translation products in two wild-type strains has revealed variant forms of some polypeptide species which show genetic behavior consistent with the location of their structural genes on mtDNA. Our results demonstrate the feasibility of performing genetic analysis on putative gene products of mtDNA in wild-type yeast by direct examination of the segregation and recombination behavior of specific polypeptide species.
Wu, Baojun; Buljic, Adnan; Hao, Weilong
2015-10-01
The frequency of horizontal gene transfer (HGT) in mitochondrial DNA varies substantially. In plants, HGT is relatively common, whereas in animals it appears to be quite rare. It is of considerable importance to understand mitochondrial HGT across the major groups of eukaryotes at a genome-wide level, but so far this has been well studied only in plants. In this study, we generated ten new mitochondrial genome sequences and analyzed 40 mitochondrial genomes from the Saccharomycetaceae to assess the magnitude and nature of mitochondrial HGT in yeasts. We provide evidence for extensive, homologous-recombination-mediated, mitochondrial-to-mitochondrial HGT occurring throughout yeast mitochondrial genomes, leading to genomes that are highly chimeric evolutionarily. This HGT has led to substantial intraspecific polymorphism in both sequence content and sequence divergence, which to our knowledge has not been previously documented in any mitochondrial genome. The unexpectedly high frequency of mitochondrial HGT in yeast may be driven by frequent mitochondrial fusion, relatively low mitochondrial substitution rates and pseudohyphal fusion to produce heterokaryons. These findings suggest that mitochondrial HGT may play an important role in genome evolution of a much broader spectrum of eukaryotes than previously appreciated and that there is a critical need to systematically study the frequency, extent, and importance of mitochondrial HGT across eukaryotes. © The Author 2015. 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.
Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul
2010-01-01
Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the
Metabolic Engineering of Oleaginous Yeasts for Fatty Alcohol Production
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wang, Wei; Wei, Hui; Knoshaug, Eric
To develop pathways for advanced biological upgrading of sugars to hydrocarbons, we are seeking biological approaches to produce high carbon efficiency intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels. In this study, we successfully demonstrated fatty alcohol production by oleaginous yeasts Yarrowia lipolytica and Lipomyces starkeyi by expressing a bacteria-derived fatty acyl-CoA reductase (FAR). Moreover, we find higher extracellular distribution of fatty alcohols produced by FAR-expressing L. starkeyi strain as compared to Y. lipolytica strain, which would benefit the downstream product recovery process. In both oleaginous yeasts, long chain length saturated fatty alcohols were predominant, accounting for moremore » than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Taken together, our work demonstrates that in addition to Y. lipolytica, L. starkeyi can also serve as a platform organism for production of fatty acid-derived biofuels and bioproducts via metabolic engineering. We believe strain and process development both will significantly contribute to our goal of producing scalable and cost-effective fatty alcohols from renewable biomass.« less
Song, Byoung-Joon; Moon, Kwan-Hoon; Olsson, Nils U.; Salem, Norman
2008-01-01
Background/Aims We reported that reduced dietary intake of polyunsaturated fatty acids (PUFA) such as arachidonic (AA,20:4n6, omega-6) and docosahexaenoic (DHA,22:6n3, omega-3) acids led to alcohol-induced fatty liver and fibrosis. This study was aimed at studying the mechanisms by which a DHA/AA-supplemented diet prevents alcohol-induced fatty liver. Methods Male Long-Evans rats were fed an ethanol or control liquid-diet with or without DHA/AA for 9 weeks. Plasma transaminase levels, liver histology, oxidative/nitrosative stress markers, and activities of oxidatively-modified mitochondrial proteins were evaluated. Results Chronic alcohol administration increased the degree of fatty liver but fatty liver decreased significantly in rats fed the alcohol-DHA/AA-supplemented diet. Alcohol exposure increased oxidative/nitrosative stress with elevated levels of ethanol-inducible CYP2E1, nitric oxide synthase, nitrite and mitochondrial hydrogen peroxide. However, these increments were normalized in rats fed the alcohol-DHA/AA-supplemented diet. The number of oxidatively-modified mitochondrial proteins was markedly increased following alcohol exposure but significantly reduced in rats fed the alcohol-DHA/AA-supplemented diet. The suppressed activities of mitochondrial aldehyde dehydrogenase, ATP synthase, and 3-ketoacyl-CoA thiolase in ethanol-exposed rats were also recovered in animals fed the ethanol-DHA/AA-supplemented diet. Conclusions Addition of DHA/AA prevents alcohol-induced fatty liver and mitochondrial dysfunction in an animal model by protecting various mitochondrial enzymes most likely through reducing oxidative/nitrosative stress. PMID:18571270
Lipotoxicity, fatty acid uncoupling and mitochondrial carrier function.
Rial, Eduardo; Rodríguez-Sánchez, Leonor; Gallardo-Vara, Eunate; Zaragoza, Pilar; Moyano, Eva; González-Barroso, M Mar
2010-01-01
Diseases like obesity, diabetes or generalized lipodystrophy cause a chronic elevation of circulating fatty acids that can become cytotoxic, a condition known as lipotoxicity. Fatty acids cause oxidative stress and alterations in mitochondrial structure and function. The uncoupling of the oxidative phosphorylation is one of the most recognized deleterious fatty acid effects and several metabolite transporters are known to mediate in their action. The fatty acid interaction with the carriers leads to membrane depolarization and/or the conversion of the carrier into a pore. The result is the opening of the permeability transition pore and the initiation of apoptosis. Unlike the other members of the mitochondrial carrier superfamily, the eutherian uncoupling protein UCP1 has evolved to achieve its heat-generating capacity in the physiological context provided by the brown adipocyte and therefore it is activated by the low fatty acid concentrations generated by the noradrenaline-stimulated lipolysis. Copyright © 2010 Elsevier B.V. All rights reserved.
A Mitochondrial Pyruvate Carrier Required for Pyruvate Uptake in Yeast, Drosophila, and Humans
Bricker, Daniel K.; Taylor, Eric B.; Schell, John C.; Orsak, Thomas; Boutron, Audrey; Chen, Yu-Chan; Cox, James E.; Cardon, Caleb M.; Van Vranken, Jonathan G.; Dephoure, Noah; Redin, Claire; Boudina, Sihem; Gygi, Steven P.; Brivet, Michèle; Thummel, Carl S.; Rutter, Jared
2013-01-01
Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast, Drosophila, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the inner mitochondrial membrane. Yeast and Drosophila mutants lacking MPC1 display impaired pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial pyruvate uptake, and silencing of MPC1 or MPC2 in mammalian cells impairs pyruvate oxidation. A point mutation in MPC1 provides resistance to a known inhibitor of the mitochondrial pyruvate carrier. Human genetic studies of three families with children suffering from lactic acidosis and hyperpyruvatemia revealed a causal locus that mapped to MPC1, changing single amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1 and Mpc2 form an essential part of the mitochondrial pyruvate carrier. PMID:22628558
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
Nosek, Jozef; Tomáška, L'ubomír; Ryčovská, Adriana; Fukuhara, Hiroshi
2002-01-01
Recent studies have demonstrated that a large number of organisms carry linear mitochondrial DNA molecules possessing specialized telomeric structures at their ends. Based on this specific structural feature of linear mitochondrial genomes, we have developed an approach for identification of the opportunistic yeast pathogen Candida parapsilosis. The strategy for identification of C. parapsilosis strains is based on PCR amplification of specific DNA sequences derived from the mitochondrial telomere region. This assay is complemented by immunodetection of a protein component of mitochondrial telomeres. The results demonstrate that mitochondrial telomeres represent specific molecular markers with potential applications in yeast diagnostics and taxonomy. PMID:11923346
Tambellini, Nicolas P; Zaremberg, Vanina; Krishnaiah, Saikumari; Turner, Raymond J; Weljie, Aalim M
2017-10-06
The nonmetabolizable lysophosphatidylcholine (LysoPC) analogue edelfosine is the prototype of a class of compounds being investigated for their potential as selective chemotherapeutic agents. Edelfosine targets membranes, disturbing cellular homeostasis. Is not clear at this point how membrane alterations are communicated between intracellular compartments leading to growth inhibition and eventual cell death. In the present study, a combined metabolomics/lipidomics approach for the unbiased identification of metabolic pathways altered in yeast treated with sublethal concentrations of the LysoPC analogue was employed. Mass spectrometry of polar metabolites, fatty acids, and lipidomic profiling was used to study the effects of edelfosine on yeast metabolism. Amino acid and sugar metabolism, the Krebs cycle, and fatty acid profiles were most disrupted, with polar metabolites and short-medium chain fatty acid changes preceding long and very long-chain fatty acid variations. Initial increases in metabolites such as trehalose, proline, and γ-amino butyric acid with a concomitant decrease in metabolites of the Krebs cycle, citrate and fumarate, are interpreted as a cellular attempt to offset oxidative stress in response to mitochondrial dysfunction induced by the treatment. Notably, alanine, inositol, and myristoleic acid showed a steady increase during the period analyzed (2, 4, and 6 h after treatment). Of importance was the finding that edelfosine induced significant alterations in neutral glycerolipid metabolism resulting in a significant increase in the signaling lipid diacylglycerol.
Phadnis, Naina; Mehta, Reema; Meednu, Nida; Sia, Elaine A
2006-07-13
Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.
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
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
Triptolide-induced mitochondrial damage dysregulates fatty acid metabolism in mouse sertoli cells.
Cheng, Yisen; Chen, Gaojian; Wang, Li; Kong, Jiamin; Pan, Ji; Xi, Yue; Shen, Feihai; Huang, Zhiying
2018-08-01
Triptolide is a major active ingredient of tripterygium glycosides, used for the therapy of immune and inflammatory diseases. However, its clinical applications are limited by severe male fertility toxicity associated with decreased sperm count, mobility and testicular injures. In this study, we determined that triptoide-induced mitochondrial dysfunction triggered reduction of lactate and dysregulation of fatty acid metabolism in mouse Sertoli cells. First, triptolide induced mitochondrial damage through the suppressing of proliferator-activated receptor coactivator-1 alpha (PGC-1α) activity and protein. Second, mitochondrial damage decreased lactate production and dysregulated fatty acid metabolism. Finally, mitochondrial dysfunction was initiated by the inhibition of sirtuin 1 (SIRT1) with the regulation of AMP-activated protein kinase (AMPK) in Sertoli cells after triptolide treatment. Meanwhile, triptolide induced mitochondrial fatty acid oxidation dysregulation by increasing AMPK phosphorylation. Taken together, we provide evidence that the mechanism of triptolide-induced testicular toxicity under mitochondrial injury may involve a metabolic change. Copyright © 2018 Elsevier B.V. All rights reserved.
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.
Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories
Zhou, Yongjin J.; Buijs, Nicolaas A.; Zhu, Zhiwei; Qin, Jiufu; Siewers, Verena; Nielsen, Jens
2016-01-01
Sustainable production of oleochemicals requires establishment of cell factory platform strains. The yeast Saccharomyces cerevisiae is an attractive cell factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast cell factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l−1 of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l−1) and fatty alcohols (1.5 g l−1), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value. PMID:27222209
Fillmore, N; Mori, J; Lopaschuk, G D
2014-01-01
Heart disease is a leading cause of death worldwide. In many forms of heart disease, including heart failure, ischaemic heart disease and diabetic cardiomyopathies, changes in cardiac mitochondrial energy metabolism contribute to contractile dysfunction and to a decrease in cardiac efficiency. Specific metabolic changes include a relative increase in cardiac fatty acid oxidation rates and an uncoupling of glycolysis from glucose oxidation. In heart failure, overall mitochondrial oxidative metabolism can be impaired while, in ischaemic heart disease, energy production is impaired due to a limitation of oxygen supply. In both of these conditions, residual mitochondrial fatty acid oxidation dominates over mitochondrial glucose oxidation. In diabetes, the ratio of cardiac fatty acid oxidation to glucose oxidation also increases, although primarily due to an increase in fatty acid oxidation and an inhibition of glucose oxidation. Recent evidence suggests that therapeutically regulating cardiac energy metabolism by reducing fatty acid oxidation and/or increasing glucose oxidation can improve cardiac function of the ischaemic heart, the failing heart and in diabetic cardiomyopathies. In this article, we review the cardiac mitochondrial energy metabolic changes that occur in these forms of heart disease, what role alterations in mitochondrial fatty acid oxidation have in contributing to cardiac dysfunction and the potential for targeting fatty acid oxidation to treat these forms of heart disease. 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:24147975
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.
Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast *
Clauser, Karl R.; Shen, Hongying; Kamer, Kimberli J.; Wells, James A.
2017-01-01
The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of ∼20–60 amino acids, a net charge between +3 to +6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, ∼80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage. PMID:28122942
Chen, Cong; Han, Xiao; Zou, Xuan; Li, Yuan; Yang, Liang; Cao, Ke; Xu, Jie; Long, Jiangang; Liu, Jiankang; Feng, Zhihui
2014-01-01
4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) is a synthetic fatty-acid synthase (FASN) inhibitor with potential therapeutic effects in several cancer models. Human mitochondrial β-ketoacyl-acyl carrier protein synthase (HsmtKAS) is a key enzyme in the newly discovered mitochondrial fatty acid synthesis pathway that can produce the substrate for lipoic acid (LA) synthesis. HsmtKAS shares conserved catalytic domains with FASN, which are responsible for binding to C75. In our study, we explored the possible effect of C75 on HsmtKAS and mitochondrial function. C75 treatment decreased LA content, impaired mitochondrial function, increased reactive oxygen species content, and reduced cell viability. HsmtKAS but not FASN knockdown had an effect that was similar to C75 treatment. In addition, an LA supplement efficiently inhibited C75-induced mitochondrial dysfunction and oxidative stress. Overexpression of HsmtKAS showed cellular protection against low dose C75 addition, whereas there was no protective effect upon high dose C75 addition. In summary, the mitochondrial fatty acid synthesis pathway has a vital role in mitochondrial function. Besides FASN, C75 might also inhibit HsmtKAS, thereby reducing LA production, impairing mitochondrial function, and potentially having toxic effects. LA supplements sufficiently ameliorated the toxicity of C75, showing that a combination of C75 and LA may be a reliable cancer treatment. PMID:24784139
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.
Silibinin triggers yeast apoptosis related to mitochondrial Ca2+ influx in Candida albicans.
Yun, Dae Gyu; Lee, Dong Gun
2016-11-01
Candida albicans is a common yeast that resides in the human body, but can occasionally cause systemic fungal infection, namely candidiasis. As this infection rate is gradually increasing, it is becoming a major problem to public health. Accordingly, we for the first time investigated the antifungal activity and mode of action of silibinin, a natural product extracted from Silybum marianum (milk thistle), against C. albicans. On treatment with 100μM silibinin, generation of reactive oxygen species (ROS) from mitochondria, which can cause yeast apoptosis via oxidative stress, was increased by 24.17% compared to that in untreated cells. Subsequently, we found disturbances in ion homeostasis such as release of intracellular K + and accumulation of cytoplasmic and mitochondrial Ca 2+ . Among these phenomena, mitochondrial Ca 2+ overload particularly plays a crucial role in the process of apoptosis, promoting the activation of pro-apoptotic factors. Therefore, we investigated the significance of mitochondrial Ca 2+ in apoptosis by employing 20mM ruthenium red (RR). Additional apoptosis hallmarks such as mitochondrial membrane depolarization, cytochrome c release, caspase activation, phosphatidylserine (PS) exposure, and DNA damage were observed in response to silibinin treatment, whereas RR pre-treatment seemed to block these responses. In summary, our results suggest that silibinin induces yeast apoptosis mediated by mitochondrial Ca 2+ signaling in C. albicans. Copyright © 2016 Elsevier Ltd. All rights reserved.
Mitochondrial origin of extracelullar transferred electrons in yeast-based biofuel cells.
Hubenova, Yolina; Mitov, Mario
2015-12-01
The influence of mitochondrial electron transport chain inhibitors on the electricity outputs of Candida melibiosica yeast-based biofuel cell was investigated. The addition of 30 μM rotenone or antimycin A to the yeast suspension results in a decrease in the current generation, corresponding to 25.7±1.3%, respectively 38.8±1.9% reduction in the electric charge passed through the bioelectrochemical system. The latter percentage coincides with the share of aerobic respiration in the yeast catabolic processes, determined by the decrease of the ethanol production during cultivation in the presence of oxygen compared with that obtained under strict anaerobic conditions. It was established that the presence of both inhibitors leads to almost complete mitochondrial dysfunction, expressed by inactivation of cytochrome c oxidase and NADH:ubiquinone oxidoreductase as well as reduced electrochemical activity of isolated yeast mitochondria. It was also found that methylene blue partially neutralized the rotenone poisoning, probably serving as alternative intracellular electron shuttle for by-passing the complex I blockage. Based on the obtained results, we suppose that electrons generated through the aerobic respiration processes in the mitochondria participate in the extracellular electron transfer from the yeast cells to the biofuel cell anode, which contributes to higher current outputs at aerobic conditions. Copyright © 2014 Elsevier B.V. All rights reserved.
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.
FABP4 reversed the regulation of leptin on mitochondrial fatty acid oxidation in mice adipocytes
Gan, Lu; Liu, Zhenjiang; Cao, Weina; Zhang, Zhenzhen; Sun, Chao
2015-01-01
Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation, and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation, and the inhibition of Akt/mTOR signal pathway played a key role in this process. PMID:26310911
FABP4 reversed the regulation of leptin on mitochondrial fatty acid oxidation in mice adipocytes.
Gan, Lu; Liu, Zhenjiang; Cao, Weina; Zhang, Zhenzhen; Sun, Chao
2015-08-27
Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation, and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation, and the inhibition of Akt/mTOR signal pathway played a key role in this process.
MOXI Is a Mitochondrial Micropeptide That Enhances Fatty Acid β-Oxidation.
Makarewich, Catherine A; Baskin, Kedryn K; Munir, Amir Z; Bezprozvannaya, Svetlana; Sharma, Gaurav; Khemtong, Chalermchai; Shah, Akansha M; McAnally, John R; Malloy, Craig R; Szweda, Luke I; Bassel-Duby, Rhonda; Olson, Eric N
2018-06-26
Micropeptide regulator of β-oxidation (MOXI) is a conserved muscle-enriched protein encoded by an RNA transcript misannotated as non-coding. MOXI localizes to the inner mitochondrial membrane where it associates with the mitochondrial trifunctional protein, an enzyme complex that plays a critical role in fatty acid β-oxidation. Isolated heart and skeletal muscle mitochondria from MOXI knockout mice exhibit a diminished ability to metabolize fatty acids, while transgenic MOXI overexpression leads to enhanced β-oxidation. Additionally, hearts from MOXI knockout mice preferentially oxidize carbohydrates over fatty acids in an isolated perfused heart system compared to wild-type (WT) animals. MOXI knockout mice also exhibit a profound reduction in exercise capacity, highlighting the role of MOXI in metabolic control. The functional characterization of MOXI provides insight into the regulation of mitochondrial metabolism and energy homeostasis and underscores the regulatory potential of additional micropeptides that have yet to be identified. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Selection of oleaginous yeasts for fatty acid production.
Lamers, Dennis; van Biezen, Nick; Martens, Dirk; Peters, Linda; van de Zilver, Eric; Jacobs-van Dreumel, Nicole; Wijffels, René H; Lokman, Christien
2016-05-27
Oleaginous yeast species are an alternative for the production of lipids or triacylglycerides (TAGs). These yeasts are usually non-pathogenic and able to store TAGs ranging from 20 % to 70 % of their cell mass depending on culture conditions. TAGs originating from oleaginous yeasts can be used as the so-called second generation biofuels, which are based on non-food competing "waste carbon sources". In this study the selection of potentially new interesting oleaginous yeast strains is described. Important selection criteria were: a broad maximum temperature and pH range for growth (robustness of the strain), a broad spectrum of carbon sources that can be metabolized (preferably including C-5 sugars), a high total fatty acid content in combination with a low glycogen content and genetic accessibility. Based on these selection criteria, among 24 screened species, Schwanniomyces occidentalis (Debaromyces occidentalis) CBS2864 was selected as a promising strain for the production of high amounts of lipids.
Live cell imaging of mitochondrial movement along actin cables in budding yeast.
Fehrenbacher, Kammy L; Yang, Hyeong-Cheol; Gay, Anna Card; Huckaba, Thomas M; Pon, Liza A
2004-11-23
Mitochondrial inheritance is essential for cell division. In budding yeast, mitochondrial movement from mother to daughter requires (1) actin cables, F-actin bundles that undergo retrograde movement during elongation from buds into mother cells; (2) the mitochore, a mitochondrial protein complex implicated in linking mitochondria to actin cables; and (3) Arp2/3 complex-mediated force generation on mitochondria. We observed three new classes of mitochondrial motility: anterograde movement at velocities of 0.2-0.33 microm/s, retrograde movement at velocities of 0.26-0.51 microm/s, and no net anterograde or retrograde movement. In all cases, motile mitochondria were associated with actin cables undergoing retrograde flow at velocities of 0.18-0.62 microm/s. Destabilization of actin cables or mutations of the mitochore blocked all mitochondrial movements. In contrast, mutations in the Arp2/3 complex affected anterograde but not retrograde mitochondrial movements. Actin cables are required for movement of mitochondria, secretory vesicles, mRNA, and spindle alignment elements in yeast. We provide the first direct evidence that one of the proposed cargos use actin cables as tracks. In the case of mitochondrial inheritance, anterograde movement drives transfer of the organelle from mothers to buds, while retrograde movement contributes to retention of the organelle in mother cells. Interaction of mitochondria with actin cables is required for anterograde and retrograde movement. In contrast, force generation on mitochondria is required only for anterograde movement. Finally, we propose a novel mechanism in which actin cables serve as "conveyor belts" that drive retrograde organelle movement.
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
Qian, Yufeng; Kachroo, Aashiq H.; Yellman, Christopher M.; Marcotte, Edward M.; Johnson, Kenneth A.
2014-01-01
Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans. PMID:24398692
High-oleate yeast oil without polyunsaturated fatty acids.
Tsakraklides, Vasiliki; Kamineni, Annapurna; Consiglio, Andrew L; MacEwen, Kyle; Friedlander, Jonathan; Blitzblau, Hannah G; Hamilton, Maureen A; Crabtree, Donald V; Su, Austin; Afshar, Jonathan; Sullivan, John E; LaTouf, W Greg; South, Colin R; Greenhagen, Emily H; Shaw, A Joe; Brevnova, Elena E
2018-01-01
Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements. We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content. High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.
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
Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart.
Thapa, Dharendra; Zhang, Manling; Manning, Janet R; Guimarães, Danielle A; Stoner, Michael W; O'Doherty, Robert M; Shiva, Sruti; Scott, Iain
2017-08-01
Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1. NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice. Copyright © 2017 the American Physiological Society.
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.
Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.
Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J
2003-10-01
Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.
Sullivan, E Madison; Pennington, Edward Ross; Green, William D; Beck, Melinda A; Brown, David A; Shaikh, Saame Raza
2018-05-01
Mitochondria are the energy-producing organelles within a cell. Furthermore, mitochondria have a role in maintaining cellular homeostasis and proper calcium concentrations, building critical components of hormones and other signaling molecules, and controlling apoptosis. Structurally, mitochondria are unique because they have 2 membranes that allow for compartmentalization. The composition and molecular organization of these membranes are crucial to the maintenance and function of mitochondria. In this review, we first present a general overview of mitochondrial membrane biochemistry and biophysics followed by the role of different dietary saturated and unsaturated fatty acids in modulating mitochondrial membrane structure-function. We focus extensively on long-chain n-3 (ω-3) polyunsaturated fatty acids and their underlying mechanisms of action. Finally, we discuss implications of understanding molecular mechanisms by which dietary n-3 fatty acids target mitochondrial structure-function in metabolic diseases such as obesity, cardiac-ischemia reperfusion injury, obesity, type 2 diabetes, nonalcoholic fatty liver disease, and select cancers.
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
Mühlenhoff, Ulrich; Stadler, Jochen A; Richhardt, Nadine; Seubert, Andreas; Eickhorst, Thomas; Schweyen, Rudolf J; Lill, Roland; Wiesenberger, Gerlinde
2003-10-17
The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.
Bjørndal, Bodil; Alterås, Eva Katrine; Lindquist, Carine; Svardal, Asbjørn; Skorve, Jon; Berge, Rolf K
2018-01-01
The 4-thia fatty acid tetradecylthiopropionic acid (TTP) is known to inhibit mitochondrial β-oxidation, and can be used as chemically induced hepatic steatosis-model in rodents, while 3-thia fatty acid tetradecylthioacetic acid (TTA) stimulates fatty acid oxidation through activation of peroxisome proliferator activated receptor alpha (PPARα). We wished to determine how these two compounds affected in vivo respiration and mitochondrial efficiency, with an additional goal to elucidate whether mitochondrial function is reflected in plasma acylcarnitine levels. C57BL/6 mice were divided in 4 groups of 10 mice and fed a control low-fat diet, low-fat diets with 0.4% ( w /w) TTP, 0.4% TTA or a combination of these two fatty acids for three weeks ( n = 10). At sacrifice, β-oxidation and oxidative phosphorylation (OXPHOS) capacity was analysed in fresh liver samples. Hepatic mitochondria were studied using transmission electron microscopy. Lipid classes were measured in plasma, heart and liver, acylcarnitines were measured in plasma, and gene expression was measured in liver. The TTP diet resulted in hepatic lipid accumulation, plasma L-carnitine and acetylcarnitine depletion and elevated palmitoylcarnitine and non-esterified fatty acid levels. No significant lipid accumulation was observed in heart. The TTA supplement resulted in enhanced hepatic β-oxidation, accompanied by an increased level of acetylcarnitine and palmitoylcarnitine in plasma. Analysis of mitochondrial respiration showed that TTP reduced oxidative phosphorylation, while TTA increased the maximum respiratory capacity of the electron transport system. Combined treatment with TTP and TTA resulted in a profound stimulation of genes involved in the PPAR-response and L-carnitine metabolism, and partly prevented triacylglycerol accumulation in the liver concomitant with increased peroxisomal β-oxidation and depletion of plasma acetylcarnitines. Despite an increased number of mitochondria in the liver
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.
Dong, Tao; Yu, Liang; Gao, Difeng; Yu, Xiaochen; Miao, Chao; Zheng, Yubin; Lian, Jieni; Li, Tingting; Chen, Shulin
2015-12-01
Accurate determination of fatty acid contents is routinely required in microalgal and yeast biofuel studies. A method of rapid in situ fatty acid methyl ester (FAME) derivatization directly from wet fresh microalgal and yeast biomass was developed in this study. This method does not require prior solvent extraction or dehydration. FAMEs were prepared with a sequential alkaline hydrolysis (15 min at 85 °C) and acidic esterification (15 min at 85 °C) process. The resulting FAMEs were extracted into n-hexane and analyzed using gas chromatography. The effects of each processing parameter (temperature, reaction time, and water content) upon the lipids quantification in the alkaline hydrolysis step were evaluated with a full factorial design. This method could tolerate water content up to 20% (v/v) in total reaction volume, which equaled up to 1.2 mL of water in biomass slurry (with 0.05-25 mg of fatty acid). There were no significant differences in FAME quantification (p>0.05) between the standard AOAC 991.39 method and the proposed wet in situ FAME preparation method. This fatty acid quantification method is applicable to fresh wet biomass of a wide range of microalgae and yeast species.
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.
Regulation of mitochondrial trifunctional protein modulates nonalcoholic fatty liver disease in mice
Nassir, Fatiha; Arndt, Justin J.; Johnson, Sarah A.
2018-01-01
Mitochondrial trifunctional protein (MTP) plays a critical role in the oxidation of long-chain fatty acids. We previously reported that aging mice (>9 months old) heterozygous for an MTP defect (MTP+/−) develop nonalcoholic fatty liver disease (NAFLD). We tested whether a high-fat diet (HFD) accelerates NAFLD in young MTP+/−mice, and whether overexpression of the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 3 (SIRT3) deacetylates MTP and improves mitochondrial function and NAFLD. Three-month-old WT and MTP+/− mice were fed HFD (60% cal fat) for 16 weeks and livers were assessed for fatty acid oxidation (FAO) and NAFLD. Compared with WT, MTP+/− mice displayed reduced hepatic SIRT3 levels and reduced FAO, with increased hepatic steatosis and the inflammatory marker CD68. Hepatic overexpression of SIRT3 in HFD-fed MTP+/− mice increased hepatic MTP protein levels at the posttranscriptional level. Immunoprecipitation of MTP from liver mitochondria followed by Western blot with acetyl-lysine antibody showed higher acetylation of MTP in MTP+/− compared with WT mice. Overexpression of SIRT3 in MTP+/− mice significantly reduced the acetylation of MTP compared with β-galactosidase controls, increased mitochondrial FAO, and reduced hepatic steatosis, CD68, and serum ALT levels. Taken together, our data indicate that deacetylation of MTP by SIRT3 improves mitochondrial function and rescues NAFLD in MTP+/− mice. PMID:29581157
Sitepu, Irnayuli R; Sestric, Ryan; Ignatia, Laura; Levin, David; German, J Bruce; Gillies, Laura A; Almada, Luis A G; Boundy-Mills, Kyria L
2013-09-01
Oleaginous yeasts have been studied for oleochemical production for over 80 years. Only a few species have been studied intensely. To expand the diversity of oleaginous yeasts available for lipid research, we surveyed a broad diversity of yeasts with indicators of oleaginicity including known oleaginous clades, and buoyancy. Sixty-nine strains representing 17 genera and 50 species were screened for lipid production. Yeasts belonged to Ascomycota families, Basidiomycota orders, and the yeast-like algal genus Prototheca. Total intracellular lipids and fatty acid composition were determined under different incubation times and nitrogen availability. Thirteen new oleaginous yeast species were discovered, representing multiple ascomycete and basidiomycete clades. Nitrogen starvation generally increased intracellular lipid content. The fatty acid profiles varied with the growth conditions regardless of taxonomic affiliation. The dominant fatty acids were oleic acid, palmitic acid, linoleic acid, and stearic acid. Yeasts and culture conditions that produced fatty acids appropriate for biodiesel were identified. Copyright © 2013 Elsevier Ltd. All rights reserved.
Sitepu, Irnayuli R.; Sestric, Ryan; Ignatia, Laura; Levin, David; German, J. Bruce; Gillies, Laura A.; Almada, Luis A.G.; Boundy-Mills, Kyria L.
2013-01-01
Oleaginous yeasts have been studied for oleochemical production for over 80 years. Only a few species have been studied intensely. To expand the diversity of oleaginous yeasts available for lipid research, we surveyed a broad diversity of yeasts with indicators of oleaginicity including known oleaginous clades, and buoyancy. Sixty-nine strains representing 17 genera and 50 species were screened for lipid production. Yeasts belonged to Ascomycota families, Basidiomycota orders, and the yeast-like algal genus Prototheca. Total intracellular lipids and fatty acid composition were determined under different incubation times and nitrogen availability. Thirteen new oleaginous yeast species were discovered, representing multiple ascomycete and basidiomycete clades. Nitrogen starvation generally increased intracellular lipid content. The fatty acid profiles varied with the growth conditions regardless of taxonomic affiliation. The dominant fatty acids were oleic acid, palmitic acid, linoleic acid, and stearic acid. Yeasts and culture conditions that produced fatty acids appropriate for biodiesel were identified. PMID:23891835
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.
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.
Pulmonary fatty acid synthesis. I. Mitochondrial acetyl transfer by rat lung in vitro.
Evans, R M; Scholz, R W
1977-04-01
Incorporation of tritiated water into fatty acids by rat adipose tissue and lung tissue slices incubated with 5 mM glucose indicated a level of fatty acid synthesis in rat lung approximately 15% that observed in adipose tissue in vitro. (-)-Hydroxycitrate, and inhibitor of ATP citrate lyase, markedly reduced tritiated water incorporation into fatty acids by lung tissue slices. The effects of (-)-hydroxycitrate and n-butymalonate on the incorporation of 14C-labeled glucose, pyruvate, acetate, and citrate suggested that citrate is a major acetyl carrier for de novo fatty acid synthesis in lung tissue. Alternative mechanisms to citrate as an acetyl carrier were also considered. Lung mitochondrial preparations formed significant levels of acetylcarnitine in the presence of pyruvate and carnitine. However, the effect of carnitine on the incorporation of 14C-labeled glucose, pyruvate, acetate, and citrate into fatty acids by lung tissue slices indicated that acetylcarnitine may not be a significant acetyl carrier for fatty acid synthesis but may serve as an acetyl "buffer" in the control of mitochondrial acetyl-CoA levels. Additionally, it appears unlikely that either acetylaspartate or acetoacetate are of major importance in acetyl transfer in lung tissue.
Baleva, M V; Meyer, M; Entelis, N; Tarassov, I; Kamenski, P; Masquida, B
2017-11-01
In yeast, the import of tRNA Lys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.
Li, Hui; Xiao, Yang; Tang, Lin; Zhong, Feng; Huang, Gan; Xu, Jun-Mei; Xu, Ai-Min; Dai, Ru-Ping; Zhou, Zhi-Guang
2018-01-01
A high level of circulating free fatty acids (FFAs) is known to be an important trigger for macrophage apoptosis during the development of atherosclerosis. However, the underlying mechanism by which FFAs result in macrophage apoptosis is not well understood. In cultured human macrophage Thp-1 cells, we showed that palmitate (PA), the most abundant FFA in circulation, induced excessive reactive oxidative substance production, increased malondialdehyde concentration, and decreased adenosine triphosphate levels. Furthermore, PA treatment also led to mitochondrial dysfunction, including the decrease of mitochondrial number, the impairment of respiratory complex IV and succinate dehydrogenase activity, and the reduction of mitochondrial membrane potential. Mitochondrial apoptosis was also detected after PA treatment, indicated by a decrease in cytochrome c release, downregulation of Bcl-2, upregulation of Bax, and increased caspase-3 activity. PA treatment upregulated the expression of adipocyte fatty acid-binding protein (A-FABP), a critical regulator of fatty acid trafficking and lipid metabolism. Inhibition of A-FABP with BMS309403, a small-molecule A-FABP inhibitor, almost reversed all of these indexes. Thus, this study suggested that PA-mediated macrophage apoptosis through A-FABP upregulation, which subsequently resulted in mitochondrial dysfunction and reactive oxidative stress. Inhibition of A-FABP may be a potential therapeutic target for macrophage apoptosis and to delay the progress of atherosclerosis. PMID:29441065
Galloway, Chad A; Lee, Hakjoo; Brookes, Paul S; Yoon, Yisang
2014-09-15
Mitochondria produce the majority of cellular ATP through oxidative phosphorylation, and their capacity to do so is influenced by many factors. Mitochondrial morphology is recently suggested as an important contributor in controlling mitochondrial bioenergetics. Mitochondria divide and fuse continuously, which is affected by environmental factors, including metabolic alterations. Underscoring its bioenergetic influence, altered mitochondrial morphology is reported in tissues of patients and in animal models of metabolic dysfunction. In this study, we found that mitochondrial fission plays a vital role in the progression of nonalcoholic fatty liver disease (NAFLD). The development of hepatic steatosis, oxidative/nitrative stress, and hepatic tissue damage, induced by a high-fat diet, were alleviated in genetically manipulated mice suppressing mitochondrial fission. The alleviation of steatosis was recapitulated in primary hepatocytes with the inhibition of mitochondrial fission. Mechanistically, our study indicates that fission inhibition enhances proton leak under conditions of free fatty acid incubation, implicating bioenergetic change through manipulating mitochondrial fission. Taken together, our results suggest a mechanistic role for mitochondrial fission in the etiology of NAFLD. The efficacy of decreasing mitochondrial fission in the suppression of NAFLD suggests that mitochondrial fission represents a novel target for therapeutic treatment of NAFLD. Copyright © 2014 the American Physiological Society.
Sriprakash, K S; Batum, C
1981-09-01
Using a protoplast fusion technique we have been able to locate to the mitochondrial genome of the asporogenous yeast Torulopsis glabrata mutations conferring resistance to oligomycin, antimycin and diuron. When two strains differing in the size of their mtDNAs were fused the mitochondrial markers from the parent with the larger mtDNA (71-91) were transmitted predominantly among the fusion products. Both genetical and physical evidence support the occurrence of recombination in T. glabrata mitochondrial genome. Segregation of the mitochondrial genome appears to take place before the separation of the first bud from the fusion product.
Isakova, E P; Epova, E Yu; Sekova, V Yu; Trubnikova, E V; Kudykina, Yu K; Zylkova, M V; Guseva, M A; Deryabina, Yu I
2015-01-01
None of the studied eukaryotic species has a natural system for homologous recombination of the mitochondrial genome. We propose an integrated genetic construct pQ-SRUS, which allows introduction of the recA gene from Bacillus subtilis into the nuclear genome of an extremophilic yeast, Yarrowia lipolytica. The targeting of recombinant RecA to the yeast mitochondria is provided by leader sequences (5'-UTR and 3'-UTR) derived from the SOD2 gene mRNA, which exhibits affinity to the outer mitochondrial membrane and thus provides cotranslational transport of RecA to the inner space of the mitochondria. The Y. lipolytica strain bearing the pQ-SRUS construct has the unique ability to integrate DNA constructs into the mitochondrial genome. This fact was confirmed using a tester construct, pQ-NIHN, intended for the introduction of the EYFP gene into the translation initiation region of the Y. lipolytica ND1 mitochondrial gene. The Y. lipolytica strain bearing pQ-SRUS makes it possible to engineer recombinant producers based on Y. lipolytica bearing transgenes in the mitochondrial genome. They are promising for the construction of a genetic system for in vivo replication and modification of the human mitochondrial genome. These strains may be used as a tool for the treatment of human mitochondrial diseases (including genetically inherited ones).
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
Seifert, Erin L; Bézaire, Véronic; Estey, Carmen; Harper, Mary-Ellen
2008-09-12
Uncoupling protein-3 (UCP3) is a mitochondrial inner membrane protein expressed most abundantly in skeletal muscle and to a lesser extent in heart and brown adipose tissue. Evidence supports a role for UCP3 in fatty acid oxidation (FAO); however, the underlying mechanism has not been explored. In 2001 we proposed a role for UCP3 in fatty acid export, leading to higher FAO rates (Himms-Hagen, J., and Harper, M. E. (2001) Exp. Biol. Med. (Maywood) 226, 78-84). Specifically, this widely held hypothesis states that during elevated FAO rates, UCP3 exports fatty acid anions, thereby maintaining mitochondrial co-enzyme A availability; reactivation of exported fatty acid anions would ultimately enable increased FAO. Here we tested mechanistic aspects of this hypothesis as well as its functional implications, namely increased FAO rates. Using complementary mechanistic approaches in mitochondria from wild-type and Ucp3(-/-) mice, we find that UCP3 is not required for FAO regardless of substrate type or supply rate covering a 20-fold range. Fatty acid anion export and reoxidation during elevated FAO, although present in skeletal muscle mitochondria, are independent of UCP3 abundance. Interestingly, UCP3 was found to be necessary for the fasting-induced enhancement of FAO rate and capacity, possibly via mitigated mitochondrial oxidative stress. Thus, although our observations indicate that UCP3 can impact FAO rates, the mechanistic basis is not via an integral function for UCP3 in the FAO machinery. Overall our data indicate a function for UCP3 in mitochondrial adaptation to perturbed cellular energy balance and integrate previous observations that have linked UCP3 to reduced oxidative stress and FAO.
Feng, Yuan Z; Nikolić, Nataša; Bakke, Siril S; Boekschoten, Mark V; Kersten, Sander; Kase, Eili T; Rustan, Arild C; Thoresen, G Hege
2014-02-01
The role of peroxisome proliferator-activated receptor δ (PPARδ) activation on global gene expression and mitochondrial fuel utilization were investigated in human myotubes. Only 21 genes were up-regulated and 3 genes were down-regulated after activation by the PPARδ agonist GW501516. Pathway analysis showed up-regulated mitochondrial fatty acid oxidation, TCA cycle and cholesterol biosynthesis. GW501516 increased oleic acid oxidation and mitochondrial oxidative capacity by 2-fold. Glucose uptake and oxidation were reduced, but total substrate oxidation was not affected, indicating a fuel switch from glucose to fatty acid. Cholesterol biosynthesis was increased, but lipid biosynthesis and mitochondrial content were not affected. This study confirmed that the principal effect of PPARδ activation was to increase mitochondrial fatty acid oxidative capacity. Our results further suggest that PPARδ activation reduced glucose utilization through a switch in mitochondrial substrate preference by up-regulating pyruvate dehydrogenase kinase isozyme 4 and genes involved in lipid metabolism and fatty acid oxidation.
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
Panozzo, C; Laleve, A; Tribouillard-Tanvier, D; Ostojić, J; Sellem, C H; Friocourt, G; Bourand-Plantefol, A; Burg, A; Delahodde, A; Blondel, M; Dujardin, G
2017-12-01
Bcs1p is a chaperone that is required for the incorporation of the Rieske subunit within complex III of the mitochondrial respiratory chain. Mutations in the human gene BCS1L (BCS1-like) are the most frequent nuclear mutations resulting in complex III-related pathologies. In yeast, the mimicking of some pathogenic mutations causes a respiratory deficiency. We have screened chemical libraries and found that two antibiotics, pentamidine and clarithromycin, can compensate two bcs1 point mutations in yeast, one of which is the equivalent of a mutation found in a human patient. As both antibiotics target the large mtrRNA of the mitoribosome, we focused our analysis on mitochondrial translation. We found that the absence of non-essential translation factors Rrf1 or Mif3, which act at the recycling/initiation steps, also compensates for the respiratory deficiency of yeast bcs1 mutations. At compensating concentrations, both antibiotics, as well as the absence of Rrf1, cause an imbalanced synthesis of respiratory subunits which impairs the assembly of the respiratory complexes and especially that of complex IV. Finally, we show that pentamidine also decreases the assembly of complex I in nematode mitochondria. It is well known that complexes III and IV exist within the mitochondrial inner membrane as supramolecular complexes III 2 /IV in yeast or I/III 2 /IV in higher eukaryotes. Therefore, we propose that the changes in mitochondrial translation caused by the drugs or by the absence of translation factors, can compensate for bcs1 mutations by modifying the equilibrium between illegitimate, and thus inactive, and active supercomplexes. Copyright © 2017. Published by Elsevier B.V.
Lunetti, Paola; Damiano, Fabrizio; De Benedetto, Giuseppe; Siculella, Luisa; Pennetta, Antonio; Muto, Luigina; Paradies, Eleonora; Marobbio, Carlo Marya Thomas; Dolce, Vincenza
2016-01-01
Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25Δ mutant manifests a defect in the biosynthesis of δ-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia. PMID:27476175
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
Yeast exonuclease 5 is essential for mitochondrial genome maintenance.
Burgers, Peter M; Stith, Carrie M; Yoder, Bonita L; Sparks, Justin L
2010-03-01
Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Delta strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5' exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5' double-stranded DNA or 5' RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.
Characterization of the respiration-induced yeast mitochondrial permeability transition pore.
Bradshaw, Patrick C; Pfeiffer, Douglas R
2013-12-01
When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.
Xu, Jianping; Yan, Zhun; Guo, Hong
2009-06-01
The inheritance of mitochondrial genes and genomes are uniparental in most sexual eukaryotes. This pattern of inheritance makes mitochondrial genomes in natural populations effectively clonal. Here, we examined the mitochondrial population genetics of the emerging human pathogenic fungus Cryptococcus gattii. The DNA sequences for five mitochondrial DNA fragments were obtained from each of 50 isolates belonging to two evolutionary divergent lineages, VGI and VGII. Our analyses revealed a greater sequence diversity within VGI than that within VGII, consistent with observations of the nuclear genes. The combined analyses of all five gene fragments indicated significant divergence between VGI and VGII. However, the five individual genealogies showed different relationships among the isolates, consistent with recent hybridization and mitochondrial gene transfer between the two lineages. Population genetic analyses of the multilocus data identified evidence for predominantly clonal mitochondrial population structures within both lineages. Interestingly, there were clear signatures of recombination among mitochondrial genes within the VGII lineage. Our analyses suggest historical mitochondrial genome divergence within C. gattii, but there is evidence for recent hybridization and recombination in the mitochondrial genome of this important human yeast pathogen.
Schrauwen, Patrick; Hinderling, Vera; Hesselink, Matthijs K C; Schaart, Gert; Kornips, Esther; Saris, Wim H M; Westerterp-Plantenga, Margriet; Langhans, Wolfgang
2002-10-01
The physiological function of human uncoupling protein-3 is still unknown. Uncoupling protein-3 is increased during fasting and high-fat feeding. In these situations the availability of fatty acids to the mitochondria exceeds the capacity to metabolize fatty acids, suggesting a role for uncoupling protein-3 in handling of non-metabolizable fatty acids. To test the hypothesis that uncoupling protein-3 acts as a mitochondrial exporter of non-metabolizable fatty acids from the mitochondrial matrix, we gave human subjects Etomoxir (which blocks mitochondrial entry of fatty acids) or placebo in a cross-over design during a 36-h stay in a respiration chamber. Etomoxir inhibited 24-h fat oxidation and fat oxidation during exercise by approximately 14-19%. Surprisingly, uncoupling protein-3 content in human vastus lateralis muscle was markedly up-regulated within 36 h of Etomoxir administration. Up-regulation of uncoupling protein-3 was accompanied by lowered fasting blood glucose and increased translocation of glucose transporter-4. These data support the hypothesis that the physiological function of uncoupling protein-3 is to facilitate the outward transport of non-metabolizable fatty acids from the mitochondrial matrix and thus prevents mitochondria from the potential deleterious effects of high fatty acid levels. In addition our data show that up-regulation of uncoupling protein-3 can be beneficial in the treatment of type 2 diabetes.
Heimer, Gali; Kerätär, Juha M; Riley, Lisa G; Balasubramaniam, Shanti; Eyal, Eran; Pietikäinen, Laura P; Hiltunen, J Kalervo; Marek-Yagel, Dina; Hamada, Jeffrey; Gregory, Allison; Rogers, Caleb; Hogarth, Penelope; Nance, Martha A; Shalva, Nechama; Veber, Alvit; Tzadok, Michal; Nissenkorn, Andreea; Tonduti, Davide; Renaldo, Florence; Kraoua, Ichraf; Panteghini, Celeste; Valletta, Lorella; Garavaglia, Barbara; Cowley, Mark J; Gayevskiy, Velimir; Roscioli, Tony; Silberstein, Jonathon M; Hoffmann, Chen; Raas-Rothschild, Annick; Tiranti, Valeria; Anikster, Yair; Christodoulou, John; Kastaniotis, Alexander J; Ben-Zeev, Bruria; Hayflick, Susan J
2016-12-01
Mitochondrial fatty acid synthesis (mtFAS) is an evolutionarily conserved pathway essential for the function of the respiratory chain and several mitochondrial enzyme complexes. We report here a unique neurometabolic human disorder caused by defective mtFAS. Seven individuals from five unrelated families presented with childhood-onset dystonia, optic atrophy, and basal ganglia signal abnormalities on MRI. All affected individuals were found to harbor recessive mutations in MECR encoding the mitochondrial trans-2-enoyl-coenzyme A-reductase involved in human mtFAS. All six mutations are extremely rare in the general population, segregate with the disease in the families, and are predicted to be deleterious. The nonsense c.855T>G (p.Tyr285 ∗ ), c.247_250del (p.Asn83Hisfs ∗ 4), and splice site c.830+2_830+3insT mutations lead to C-terminal truncation variants of MECR. The missense c.695G>A (p.Gly232Glu), c.854A>G (p.Tyr285Cys), and c.772C>T (p.Arg258Trp) mutations involve conserved amino acid residues, are located within the cofactor binding domain, and are predicted by structural analysis to have a destabilizing effect. Yeast modeling and complementation studies validated the pathogenicity of the MECR mutations. Fibroblast cell lines from affected individuals displayed reduced levels of both MECR and lipoylated proteins as well as defective respiration. These results suggest that mutations in MECR cause a distinct human disorder of the mtFAS pathway. The observation of decreased lipoylation raises the possibility of a potential therapeutic strategy. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Uprety, Bijaya K; Rakshit, Sudip K
2017-12-01
Tailoring lipids from oleaginous yeasts to contain specific types of fatty acid is of considerable interest to food, fuel, and pharmaceutical industries. In this study, the essential oil obtained from Citrus sinesus L. has been used to alter the fatty acid composition of two common oleaginous yeasts, Rhodosporidium toruloides and Cryptococcus curvatus. With increasing levels of essential oil in the medium, the metabolic flux of the fatty acid biosynthesis pathway shifted towards saturated fatty acid production. Essential oil reduced the activities of elongase and ∆9 desaturase. This made the lipid obtained from both these yeasts rich in saturated fatty acids. At certain specific concentrations of the essential oil in the medium, the lipid obtained from R. toruloides and C. curvatus cultures was similar to mahuwa butter and palm oil, respectively. Limonene is the major constituents of orange essential oil. Its effect on one of the oleaginous yeasts, R. toruloides, was also studied separately. Effects similar to orange essential oil were obtained with limonene. Thus, we can conclude that limonene in orange essential oil brings about compositional change of microbial lipid produced in this organism.
Clay, Hayley B.; Parl, Angelika K.; Mitchell, Sabrina L.; Singh, Larry; Bell, Lauren N.; Murdock, Deborah G.
2016-01-01
Despite the presence of a cytosolic fatty acid synthesis pathway, mitochondria have retained their own means of creating fatty acids via the mitochondrial fatty acid synthesis (mtFASII) pathway. The reason for its conservation has not yet been elucidated. Therefore, to better understand the role of mtFASII in the cell, we used thin layer chromatography to characterize the contribution of the mtFASII pathway to the fatty acid composition of selected mitochondrial lipids. Next, we performed metabolomic analysis on HeLa cells in which the mtFASII pathway was either hypofunctional (through knockdown of mitochondrial acyl carrier protein, ACP) or hyperfunctional (through overexpression of mitochondrial enoyl-CoA reductase, MECR). Our results indicate that the mtFASII pathway contributes little to the fatty acid composition of mitochondrial lipid species examined. Additionally, loss of mtFASII function results in changes in biochemical pathways suggesting alterations in glucose utilization and redox state. Interestingly, levels of bioactive lipids, including lysophospholipids and sphingolipids, directly correlate with mtFASII function, indicating that mtFASII may be involved in the regulation of bioactive lipid levels. Regulation of bioactive lipid levels by mtFASII implicates the pathway as a mediator of intracellular signaling. PMID:26963735
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.
Koltovaya, N A; Guerasimova, A S; Tchekhouta, I A; Devin, A B
2003-08-01
An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast. Copyright 2003 John Wiley & Sons, Ltd.
Frayn, K N; Langin, D; Karpe, F
2008-03-01
The release of fatty acids from white adipose tissue is regulated at several levels. We have examined the suggestion that fatty acid release might be diminished by upregulation of mitochondrial fatty acid oxidation in the adipocyte, through increasing mitochondrial uncoupling. The intrinsic oxidative capacity of white adipose tissue is low, and older studies suggest that there is little fatty acid oxidation in white adipocytes, human or rodent. We have examined data on fatty acid metabolism and O(2) consumption in human white adipose tissue in vivo, and conclude that increasing fatty acid oxidation within the oxidative capacity of the tissue would produce only small changes (a few percent) in fatty acid release. The major locus of control of fatty acid release beyond the stimulation of lipolysis is the pathway of fatty acid esterification, already probably targeted by the thiazolidinedione insulin-sensitising agents. An alternative approach would be to upregulate the mitochondrial capacity of the adipocyte. We review proof-of-concept studies in which the phenotype of the white adipocyte has been changed to resemble that of the brown adipocyte by expression of peroxisome proliferator-activated receptor coactivator-1alpha. This increases oxidative capacity and also leads to fatty acid retention through upregulation of glycerol-3-phosphate production, and hence increased fatty acid re-esterification. We conclude that prevention or treatment of insulin resistance through alteration of adipocyte fatty acid handling will require more than a simple alteration of the activity of mitochondrial beta-oxidation within normal limits.
Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly
Cook, Jeremy D.; Kondapalli, Kalyan C.; Rawat, Swati; Childs, William C.; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L.
2010-01-01
Frataxin, a conserved nuclear encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich’s ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two: Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone n the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to better understand the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry (ITC). Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into a Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly. PMID:20815377
Molecular Details of the Yeast Frataxin-Isu1 Interaction during Mitochondrial Fe-S Cluster Assembly
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cook, J.; Kondapalli, K; Rawat, S
2010-01-01
Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural modulemore » to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.« less
Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.
Cook, Jeremy D; Kondapalli, Kalyan C; Rawat, Swati; Childs, William C; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L
2010-10-12
Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.
Salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA.
Gao, Qiuqiang; Liou, Liang-Chun; Ren, Qun; Bao, Xiaoming; Zhang, Zhaojie
2014-03-03
The yeast cell wall plays an important role in maintaining cell morphology, cell integrity and response to environmental stresses. Here, we report that salt stress causes cell wall damage in yeast cells lacking mitochondrial DNA (ρ 0 ). Upon salt treatment, the cell wall is thickened, broken and becomes more sensitive to the cell wall-perturbing agent sodium dodecyl sulfate (SDS). Also, SCW11 mRNA levels are elevated in ρ 0 cells. Deletion of SCW11 significantly decreases the sensitivity of ρ 0 cells to SDS after salt treatment, while overexpression of SCW11 results in higher sensitivity. In addition, salt stress in ρ 0 cells induces high levels of reactive oxygen species (ROS), which further damages the cell wall, causing cells to become more sensitive towards the cell wall-perturbing agent.
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
Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger
2017-01-01
Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570
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
2016-03-01
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).
Bellanti, Francesco; Villani, Rosanna; Tamborra, Rosanna; Blonda, Maria; Iannelli, Giuseppina; di Bello, Giorgia; Facciorusso, Antonio; Poli, Giuseppe; Iuliano, Luigi; Avolio, Carlo; Vendemiale, Gianluigi; Serviddio, Gaetano
2018-05-01
The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD) has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids (FFAs) and cholesterol accumulation. Excess cholesterol autoxidizes to oxysterols during oxidative stress conditions. We hypothesize that interaction of FAs and cholesterol derivatives may primarily impair mitochondrial function and affect biogenesis adaptation during NAFLD progression. We demonstrated that the accumulation of specific non-enzymatic oxysterols in the liver of animals fed high-fat+high-cholesterol diet induces mitochondrial damage and depletion of proteins of the respiratory chain complexes. When tested in vitro, 5α-cholestane-3β,5,6β-triol (triol) combined to FFAs was able to reduce respiration in isolated liver mitochondria, induced apoptosis in primary hepatocytes, and down-regulated transcription factors involved in mitochondrial biogenesis. Finally, a lower protein content in the mitochondrial respiratory chain complexes was observed in human non-alcoholic steatohepatitis. In conclusion, hepatic accumulation of FFAs and non-enzymatic oxysterols synergistically facilitates development and progression of NAFLD by impairing mitochondrial function, energy balance and biogenesis adaptation to chronic injury. Copyright © 2017. Published by Elsevier B.V.
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.
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
Lohr, Kerstin; Pachl, Fiona; Moghaddas Gholami, Amin; Geillinger, Kerstin E; Daniel, Hannelore; Kuster, Bernhard; Klingenspor, Martin
2016-10-01
Nonalcoholic fatty liver disease (NAFLD) is a major health burden in the aging society with an urging medical need for a better understanding of the underlying mechanisms. Mitochondrial fatty acid oxidation and mitochondrial-derived reactive oxygen species (ROS) are considered critical in the development of hepatic steatosis, the hallmark of NAFLD. Our study addressed in C57BL/6J mice the effect of high fat diet feeding and age on liver mitochondria at an early stage of NAFLD development. We therefore analyzed functional characteristics of hepatic mitochondria and associated alterations in the mitochondrial proteome in response to high fat feeding in adolescent, young adult, and middle-aged mice. Susceptibility to diet-induced obesity increased with age. Young adult and middle-aged mice developed fatty liver, but not adolescent mice. Fat accumulation was negatively correlated with an age-related reduction in mitochondrial mass and aggravated by a reduced capacity of fatty acid oxidation in high fat-fed mice. Irrespective of age, high fat diet increased ROS production in hepatic mitochondria associated with a balanced nuclear factor erythroid-derived 2 like 2 (NFE2L2) dependent antioxidative response, most likely triggered by reduced tethering of NFE2L2 to mitochondrial phosphoglycerate mutase 5. Age indirectly influenced mitochondrial function by reducing mitochondrial mass, thus exacerbating diet-induced fat accumulation. Therefore, consideration of age in metabolic studies must be emphasized. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.
Mitochondrial-nuclear genome interactions in nonalcoholic fatty liver disease in mice
Betancourt, Angela M.; King, Adrienne L.; Fetterman, Jessica L.; Millender-Swain, Telisha; Finley, Rachel D.; Oliva, Claudia R.; Crowe, David Ralph; Ballinger, Scott W.; Bailey, Shannon M.
2014-01-01
Nonalcoholic fatty liver disease (NAFLD) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation, and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. Herein, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, Mitochondrial-Nuclear eXchange (MNX) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared to wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation, and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD. PMID:24758559
Lanaspa, Miguel A; Sanchez-Lozada, Laura G; Choi, Yea-Jin; Cicerchi, Christina; Kanbay, Mehmet; Roncal-Jimenez, Carlos A; Ishimoto, Takuji; Li, Nanxing; Marek, George; Duranay, Murat; Schreiner, George; Rodriguez-Iturbe, Bernardo; Nakagawa, Takahiko; Kang, Duk-Hee; Sautin, Yuri Y; Johnson, Richard J
2012-11-23
Uric acid is an independent risk factor in fructose-induced fatty liver, but whether it is a marker or a cause remains unknown. Hepatocytes exposed to uric acid developed mitochondrial dysfunction and increased de novo lipogenesis, and its blockade prevented fructose-induced lipogenesis. Rather than a consequence, uric acid induces fatty liver Hyperuricemic people are more prone to develop fructose-induced fatty liver. Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states.
Vergeade, Aurelia; Bertram, Clinton C.; Bikineyeva, Alfiya T.; Zackert, William E.; Zinkel, Sandra S.; May, James M.; Dikalov, Sergey I.; Roberts, L. Jackson; Boutaud, Olivier
2016-01-01
Modifications of cardiolipin (CL) levels or compositions are associated with changes in mitochondrial function in a wide range of pathologies. We have made the discovery that acetaminophen remodels CL fatty acids composition from tetralinoleoyl to linoleoyltrioleoyl-CL, a remodeling that is associated with decreased mitochondrial respiration. Our data show that CL remodeling causes a shift in electron entry from complex II to the β-oxidation electron transfer flavoprotein quinone oxidoreductase (ETF/QOR) pathway. These data demonstrate that electron entry in the respiratory chain is regulated by CL fatty acid composition and provide proof-of-concept that pharmacological intervention can be used to modify CL composition. PMID:27085476
Skorin, C; Necochea, C; Johow, V; Soto, U; Grau, A M; Bremer, J; Leighton, F
1992-01-01
Fatty acid oxidation was studied in the presence of inhibitors of carnitine palmitoyltransferase I (CPT I), in normal and in peroxisome-proliferated rat hepatocytes. The oxidation decreased in mitochondria, as expected, but in peroxisomes it increased. These two effects were seen, in variable proportions, with (+)-decanoylcarnitine, 2-tetradecylglycidic acid (TDGA) and etomoxir. The decrease in mitochondrial oxidation (ketogenesis) affected saturated fatty acids with 12 or more carbon atoms, whereas the increase in peroxisomal oxidation (H2O2 production) affected saturated fatty acids with 8 or more carbon atoms. The peroxisomal increase was sensitive to chlorpromazine, a peroxisomal inhibitor. To study possible mechanisms, palmitoyl-, octanoyl- and acetyl-carnitine acyltransferase activities were measured, in homogenates and in subcellular fractions from control and TDGA-treated cells. The palmitoylcarnitine acyltransferase was inhibited, as expected, but the octanoyltransferase activity also decreased. The CoA derivative of TDGA was synthesized and tentatively identified as being responsible for inhibition of the octanoylcarnitine acyltransferase. These results show that inhibitors of the mitochondrial CPT I may also inhibit the peroxisomal octanoyl transferase; they also support the hypothesis that the octanoyltransferase has the capacity to control or regulate peroxisomal fatty acid oxidation. PMID:1736904
Sunny, Nishanth E.; Parks, Elizabeth J.; Browning, Jeffrey D.; Burgess, Shawn C.
2013-01-01
Summary Approximately one-third of the U.S. population has nonalcoholic fatty liver disease (NAFLD), a condition closely associated with insulin resistance and increased risk of liver injury. Dysregulated mitochondrial metabolism is central in these disorders, but the manner and degree of dysregulation are disputed. This study tested whether humans with NAFLD have abnormal in vivo hepatic mitochondrial metabolism. Subjects with low (3.0%) and high (17%) intrahepatic triglyceride (IHTG) were studied using 2H and 13C tracers to evaluate systemic lipolysis, hepatic glucose production, and mitochondrial pathways (TCA cycle, anaplerosis, and ketogenesis). Individuals with NAFLD had 50% higher rates of lipolysis and 30% higher rates of gluconeogenesis. There was a positive correlation between IHTG content and both mitochondrial oxidative and anaplerotic fluxes. These data indicate that mitochondrial oxidative metabolism is ∼2-fold greater in those with NAFLD, providing a potential link between IHTG content, oxidative stress, and liver damage. PMID:22152305
Tonin, Anelise M; Amaral, Alexandre U; Busanello, Estela N B; Grings, Mateus; Castilho, Roger F; Wajner, Moacir
2013-02-01
Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid β-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.
Bui, Huyen T.; Karren, Mary A.; Bhar, Debjani
2012-01-01
To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms. PMID:23148233
Khoomrung, Sakda; Chumnanpuen, Pramote; Jansa-ard, Suwanee; Nookaew, Intawat; Nielsen, Jens
2012-06-01
We present a fast and accurate method for preparation of fatty acid methyl esters (FAMEs) using microwave-assisted derivatization of fatty acids present in yeast samples. The esterification of free/bound fatty acids to FAMEs was completed within 5 min, which is 24 times faster than with conventional heating methods. The developed method was validated in two ways: (1) through comparison with a conventional method (hot plate) and (2) through validation with the standard reference material (SRM) 3275-2 omega-3 and omega-6 fatty acids in fish oil (from the Nation Institute of Standards and Technology, USA). There were no significant differences (P>0.05) in yields of FAMEs with both validations. By performing a simple modification of closed-vessel microwave heating, it was possible to carry out the esterification in Pyrex glass tubes kept inside the closed vessel. Hereby, we are able to increase the number of sample preparations to several hundred samples per day as the time for preparation of reused vessels was eliminated. Pretreated cell disruption steps are not required, since the direct FAME preparation provides equally quantitative results. The new microwave-assisted derivatization method facilitates the preparation of FAMEs directly from yeast cells, but the method is likely to also be applicable for other biological samples.
Yazawa, Hisashi; Iwahashi, Hitoshi; Kamisaka, Yasushi; Kimura, Kazuyoshi; Uemura, Hiroshi
2009-03-01
Saccharomyces cerevisiae produces saturated and monounsaturated fatty acids of 16- and 18-carbon atoms and no polyunsaturated fatty acids (PUFAs) with more than two double bonds. To study the biological significance of PUFAs in yeast, we introduced Kluyveromyces lactis Delta12 fatty acid desaturase (KlFAD2) and omega3 fatty acid desaturase (KlFAD3) genes into S. cerevisiae to produce linoleic and alpha-linolenic acids in S. cerevisiae. The strain producing linoleic and alpha-linolenic acids showed an alkaline pH-tolerant phenotype. DNA microarray analyses showed that the transcription of a set of genes whose expressions are under the repression of Rim101p were downregulated in this strain, suggesting that Rim101p, a transcriptional repressor which governs the ion tolerance, was activated. In line with this activation, the strain also showed elevated resistance to Li(+) and Na(+) ions and to zymolyase, a yeast lytic enzyme preparation containing mainly beta-1,3-glucanase, indicating that the cell wall integrity was also strengthened in this strain. Our findings demonstrate a novel influence of PUFA production on transcriptional control that is likely to play an important role in the early stage of alkaline stress response. The Accession No. for microarray data in the Center for Information Biology Gene Expression database is CBX68.
Timón-Gómez, Alba; Proft, Markus; Pascual-Ahuir, Amparo
2013-01-01
Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.
Actin dynamics affect mitochondrial quality control and aging in budding yeast.
Higuchi, Ryo; Vevea, Jason D; Swayne, Theresa C; Chojnowski, Robert; Hill, Vanessa; Boldogh, Istvan R; Pon, Liza A
2013-12-02
Actin cables of budding yeast are bundles of F-actin that extend from the bud tip or neck to the mother cell tip, serve as tracks for bidirectional cargo transport, and undergo continuous movement from buds toward mother cells [1]. This movement, retrograde actin cable flow (RACF), is similar to retrograde actin flow in lamellipodia, growth cones, immunological synapses, dendritic spines, and filopodia [2-5]. In all cases, actin flow is driven by the push of actin polymerization and assembly at the cell cortex, and myosin-driven pulling forces deeper within the cell [6-10]. Therefore, for movement and inheritance from mothers to buds, mitochondria must "swim upstream" against the opposing force of RACF [11]. We find that increasing RACF rates results in increased fitness of mitochondria inherited by buds and that the increase in mitochondrial fitness leads to extended replicative lifespan and increased cellular healthspan. The sirtuin SIR2 is required for normal RACF and mitochondrial fitness, and increasing RACF rates in sir2Δ cells increases mitochondrial fitness and cellular healthspan but does not affect replicative lifespan. These studies support the model that RACF serves as a filter for segregation of fit from less-fit mitochondria during inheritance, which controls cellular lifespan and healthspan. They also support a role for Sir2p in these processes. Copyright © 2013 Elsevier Ltd. All rights reserved.
Gharwalova, Lucia; Sigler, Karel; Dolezalova, Jana; Masak, Jan; Rezanka, Tomas; Kolouchova, Irena
2017-11-03
Mid-exponential cultures of two traditional biotechnological yeast species, winery Saccharomyces cerevisiae and the less ethanol tolerant bottom-fermenting brewery Saccharomyces pastorianus, were exposed to different concentrations of added ethanol (3, 5 and 8%) The degree of ethanol-induced cell stress was assessed by measuring the cellular activity of superoxide dismutase (SOD), level of lipid peroxidation products, changes in cell lipid content and fatty acid profile. The resveratrol as an antioxidant was found to decrease the ethanol-induced rise of SOD activity and suppress the ethanol-induced decrease in cell lipids. A lower resveratrol concentration (0.5 mg/l) even reduced the extent of lipid peroxidation in cells. Resveratrol also alleviated ethanol-induced changes in cell lipid composition in both species by strongly enhancing the proportion of saturated fatty acids and contributing thereby to membrane stabilization. Lower resveratrol concentrations could thus diminish the negative effects of ethanol stress on yeast cells and improve their physiological state. These effects may be utilized to enhance yeast vitality in high-ethanol-producing fermentations or to increase the number of yeast generations in brewery.
Mitochondrial-nuclear genome interactions in non-alcoholic fatty liver disease in mice.
Betancourt, Angela M; King, Adrienne L; Fetterman, Jessica L; Millender-Swain, Telisha; Finley, Rachel D; Oliva, Claudia R; Crowe, David R; Ballinger, Scott W; Bailey, Shannon M
2014-07-15
NAFLD (non-alcoholic fatty liver disease) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. In the present study, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, MNX (mitochondrial-nuclear exchange) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared with wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR (respiratory control ratio) when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD.
Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Fiehn, Oliver; Cathcart, Erin; Fry, Russell W; Kanti, Atit; Joko Nugroho, Agustinus; Faulina, Sarah Asih; Stephanandra, Sira; German, J Bruce; Boundy-Mills, Kyria L
2017-06-01
Polyol esters of fatty acids (PEFA) are amphiphilic glycolipids produced by yeast that could play a role as natural, environmentally friendly biosurfactants. We recently reported discovery of a new PEFA-secreting yeast species, Rhodotorula babjevae, a basidiomycetous yeast to display this behavior, in addition to a few other Rhodotorula yeasts reported on the 1960s. Additional yeast species within the taxonomic order Sporidiobolales were screened for secreted glycolipid production. PEFA production equal or above 1 g L -1 were detected in 19 out of 65 strains of yeast screened, belonging to 6 out of 30 yeast species tested. Four of these species were not previously known to secrete glycolipids. These results significantly increase the number of yeast species known to secrete PEFA, holding promise for expanding knowledge of PEFA synthesis and secretion mechanisms, as well as setting the groundwork towards commercialization.
Electron transport chain in a thermotolerant yeast.
Mejía-Barajas, Jorge A; Martínez-Mora, José A; Salgado-Garciglia, Rafael; Noriega-Cisneros, Ruth; Ortiz-Avila, Omar; Cortés-Rojo, Christian; Saavedra-Molina, Alfredo
2017-04-01
Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨ m ), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca 2+ ions (Ca 2+ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30 o , while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.
Yalçin, Sakine; Yalçin, Suzan; Cakin, Kemal; Eltan, Onder; Dağaşan, Levent
2010-08-15
The objective of this study was to determine the effects of dietary yeast autolysate on performance, egg traits, egg cholesterol content, egg yolk fatty acid composition, lipid oxidation of egg yolk, some blood parameters and humoral immune response of laying hens during a 16 week period. A total of 225 Hyline Brown laying hens, 22 weeks of age, were allocated equally to one control group and four treatment groups. Yeast autolysate (Saccharomyces cerevisiae, InteWall) was used at levels of 1, 2, 3 and 4 g kg(-1) in the diets of the first, second, third and fourth treatment groups respectively. Dietary treatments did not significantly affect body weight, feed intake and egg traits. Yeast autolysate supplementation increased egg production (P < 0.001) and egg weight (P < 0.001) and improved feed efficiency (P < 0.05). Yeast autolysate at levels of 2, 3 and 4 g kg(-1) decreased egg yolk cholesterol level as mg g(-1) yolk (P < 0.01) and blood serum levels of cholesterol and triglyceride (P < 0.05) and increased antibody titres to sheep red blood cells (P < 0.01). Total saturated fatty acids and the ratio of saturated/unsaturated fatty acids increased (P < 0.01) and total monounsaturated fatty acids (P < 0.001) decreased with yeast autolysate supplementation. Dietary yeast autolysate at levels of 2, 3 and 4 g kg(-1) had beneficial effects on performance, egg cholesterol content and humoral immune response. It is concluded that 2 g kg(-1) yeast autolysate will be enough to have beneficial effects in laying hens. Copyright (c) 2010 Society of Chemical Industry.
Bhattacharya, Arunabh; Lustgarten, Michael; Shi, Yun; Liu, Yuhong; Jang, Youngmok C; Pulliam, Daniel; Jernigan, Amanda L; Van Remmen, Holly
2013-01-01
Previous studies have shown that muscle atrophy is associated with mitochondrial dysfunction and an increased rate of mitochondrial reactive oxygen species production. We recently demonstrated that fatty acid hydroperoxides (FA-OOH) are significantly elevated in mitochondria isolated from atrophied muscles. The purpose of the current study is to determine whether FA-OOH can alter skeletal muscle mitochondrial function. We found that FA-OOH (at low micromolar concentrations) induces mitochondrial dysfunction assessed by decrease in the rate of ATP production, oxygen consumption and activity of respiratory chain complexes I and III. Using methods to distinguish superoxide release towards the matrix and inter-membrane space, we demonstrate that FA-OOH significantly elevates oxidative stress in the mitochondrial matrix (and not the inter-membrane space) with complex I as the major site of superoxide production (most likely from a site upstream of the ubiquinone binding site but downstream from the flavin binding site-the iron sulfur clusters). Our results are the first to indicate that FA-OOH’s are important modulators of mitochondrial function and oxidative stress in skeletal muscle mitochondria and may play an important role in muscle atrophies that are associated with increased generation of FA-OOH’s, e.g., denervation-induced muscle atrophy. PMID:21172427
Borengasser, Sarah J.; Lau, Franchesca; Kang, Ping; Blackburn, Michael L.; Ronis, Martin J. J.; Badger, Thomas M.; Shankar, Kartik
2011-01-01
In utero exposure to maternal obesity increases the offspring's risk of obesity in later life. We have also previously reported that offspring of obese rat dams develop hepatic steatosis, mild hyperinsulinemia, and a lipogenic gene signature in the liver at postnatal day (PND)21. In the current study, we examined systemic and hepatic adaptations in male Sprague-Dawley offspring from lean and obese dams at PND21. Indirect calorimetry revealed decreases in energy expenditure (p<0.001) and increases in RER values (p<0.001), which were further exacerbated by high fat diet (45% kcals from fat) consumption indicating an impaired ability to utilize fatty acids in offspring of obese dams as analyzed by PRCF. Mitochondrial function is known to be associated with fatty acid oxidation (FAO) in the liver. Several markers of hepatic mitochondrial function were reduced in offspring of obese dams. These included SIRT3 mRNA (p = 0.012) and mitochondrial protein content (p = 0.002), electron transport chain complexes (II, III, and ATPase), and fasting PGC-1α mRNA expression (p<0.001). Moreover, hepatic LCAD, a SIRT3 target, was not only reduced 2-fold (p<0.001) but was also hyperacetylated in offspring of obese dams (p<0.005) suggesting decreased hepatic FAO. In conclusion, exposure to maternal obesity contributes to early perturbations in whole body and liver energy metabolism. Mitochondrial dysfunction may be an underlying event that reduces hepatic fatty acid oxidation and precedes the development of detrimental obesity associated co-morbidities such as insulin resistance and NAFLD. PMID:21901160
Sanjuán Szklarz, Luiza K; Kozjak-Pavlovic, Vera; Vögtle, F-Nora; Chacinska, Agnieszka; Milenkovic, Dusanka; Vogel, Sandra; Dürr, Mark; Westermann, Benedikt; Guiard, Bernard; Martinou, Jean-Claude; Borner, Christoph; Pfanner, Nikolaus; Meisinger, Chris
2007-04-20
The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.
Unveiling the mystery of mitochondrial DNA replication in yeasts.
Chen, Xin Jie; Clark-Walker, George Desmond
2018-01-01
Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Marinho, Polyana C; Vieira, Aline B; Pereira, Priscila G; Rabelo, Kíssila; Ciambarella, Bianca T; Nascimento, Ana L R; Cortez, Erika; Moura, Aníbal S; Guimarães, Fernanda V; Martins, Marco A; Barquero, Gonzalo; Ferreira, Rodrigo N; de Carvalho, Jorge J
2018-01-01
Nonalcoholic fatty liver disease (NAFLD) is recognized as the most common cause of liver dysfunction worldwide and is commonly associated with obesity. Evidences suggest that NAFLD might be a mitochondrial disease, which contributes to the hepatic steatosis, oxidative stress, cytokine release, and cell death. Capybara oil (CO) is a rich source of polyunsaturated fatty acids (PUFA), which is known to improve inflammation and oxidative stress. In order to determine the effects of CO on NAFLD, C57Bl/6 mice were divided into 3 groups and fed a high-fat diet (HFD) (NAFLD group and NAFLD + CO group) or a control diet (CG group) during 16 weeks. The CO (1.5 g/kg/daily) was administered by gavage during the last 4 weeks of the diet protocol. We evaluated plasma liver enzymes, hepatic steatosis, and cytokine expression in liver as well as hepatocyte ultrastructural morphology and mitochondrial function. CO treatment suppressed hepatic steatosis, attenuated inflammatory response, and decreased plasma alanine aminotransferase (ALT) in mice with NAFLD. CO was also capable of restoring mitochondrial ultrastructure and function as well as balance superoxide dismutase and catalase levels. Our findings indicate that CO treatment has positive effects on NAFLD improving mitochondrial dysfunction, steatosis, acute inflammation, and oxidative stress.
Sultana, Shemaila; Solotchi, Mihai; Ramachandran, Aparna; Patel, Smita S
2017-11-03
Single-subunit RNA polymerases (RNAPs) are present in phage T7 and in mitochondria of all eukaryotes. This RNAP class plays important roles in biotechnology and cellular energy production, but we know little about its fidelity and error rates. Herein, we report the error rates of three single-subunit RNAPs measured from the catalytic efficiencies of correct and all possible incorrect nucleotides. The average error rates of T7 RNAP (2 × 10 -6 ), yeast mitochondrial Rpo41 (6 × 10 -6 ), and human mitochondrial POLRMT (RNA polymerase mitochondrial) (2 × 10 -5 ) indicate high accuracy/fidelity of RNA synthesis resembling those of replicative DNA polymerases. All three RNAPs exhibit a distinctly high propensity for GTP misincorporation opposite dT, predicting frequent A→G errors in RNA with rates of ∼10 -4 The A→C, G→A, A→U, C→U, G→U, U→C, and U→G errors mostly due to pyrimidine-purine mismatches were relatively frequent (10 -5 -10 -6 ), whereas C→G, U→A, G→C, and C→A errors from purine-purine and pyrimidine-pyrimidine mismatches were rare (10 -7 -10 -10 ). POLRMT also shows a high C→A error rate on 8-oxo-dG templates (∼10 -4 ). Strikingly, POLRMT shows a high mutagenic bypass rate, which is exacerbated by TEFM (transcription elongation factor mitochondrial). The lifetime of POLRMT on terminally mismatched elongation substrate is increased in the presence of TEFM, which allows POLRMT to efficiently bypass the error and continue with transcription. This investigation of nucleotide selectivity on normal and oxidatively damaged DNA by three single-subunit RNAPs provides the basic information to understand the error rates in mitochondria and, in the case of T7 RNAP, to assess the quality of in vitro transcribed RNAs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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.
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.
Huang, Tai-Yu; Zheng, Donghai; Houmard, Joseph A; Brault, Jeffrey J; Hickner, Robert C; Cortright, Ronald N
2017-04-01
Peroxisomes are indispensable organelles for lipid metabolism in humans, and their biogenesis has been assumed to be under regulation by peroxisome proliferator-activated receptors (PPARs). However, recent studies in hepatocytes suggest that the mitochondrial proliferator PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1α) also acts as an upstream transcriptional regulator for enhancing peroxisomal abundance and associated activity. It is unknown whether the regulatory mechanism(s) for enhancing peroxisomal function is through the same node as mitochondrial biogenesis in human skeletal muscle (HSkM) and whether fatty acid oxidation (FAO) is affected. Primary myotubes from vastus lateralis biopsies from lean donors (BMI = 24.0 ± 0.6 kg/m 2 ; n = 6) were exposed to adenovirus encoding human PGC-1α or GFP control. Peroxisomal biogenesis proteins (peroxins) and genes ( PEXs ) responsible for proliferation and functions were assessed by Western blotting and real-time qRT-PCR, respectively. [1- 14 C]palmitic acid and [1- 14 C]lignoceric acid (exclusive peroxisomal-specific substrate) were used to assess mitochondrial oxidation of peroxisomal-derived metabolites. After overexpression of PGC-1α, 1 ) peroxisomal membrane protein 70 kDa (PMP70), PEX19, and mitochondrial citrate synthetase protein content were significantly elevated ( P < 0.05), 2 ) PGC-1α , PMP70 , key PEXs , and peroxisomal β-oxidation mRNA expression levels were significantly upregulated ( P < 0.05), and 3 ) a concomitant increase in lignoceric acid oxidation by both peroxisomal and mitochondrial activity was observed ( P < 0.05). These novel findings demonstrate that, in addition to the proliferative effect on mitochondria, PGC-1α can induce peroxisomal activity and accompanying elevations in long-chain and very-long-chain fatty acid oxidation by a peroxisomal-mitochondrial functional cooperation, as observed in HSkM cells. Copyright © 2017 the American Physiological Society.
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.
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.
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.
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
Aulds, Jason; Wierzbicki, Sara; McNairn, Adrian; Schmitt, Mark E.
2012-01-01
RNase mitochondrial RNA processing (MRP) is an essential, evolutionarily conserved endoribonuclease composed of 10 different protein subunits and a single RNA. RNase MRP has established roles in multiple pathways including ribosome biogenesis, cell cycle regulation, and mitochondrial DNA replication. Although each of these functions is important to cell growth, additional functions may exist given the essential nature of the complex. To identify novel RNase MRP substrates, we utilized RNA immunoprecipitation and microarray chip analysis to identify RNA that physically associates with RNase MRP. We identified several new potential substrates for RNase MRP including a cell cycle-regulated transcript, CTS1; the yeast homolog of the mammalian p27Kip1, SIC1; and the U2 RNA component of the spliceosome. In addition, we found RNase MRP to be involved in the regulation of the Ty1 transposon RNA. These results reinforce and broaden the role of RNase MRP in cell cycle regulation and help to identify new roles of this endoribonuclease. PMID:22977255
Aulds, Jason; Wierzbicki, Sara; McNairn, Adrian; Schmitt, Mark E
2012-10-26
RNase mitochondrial RNA processing (MRP) is an essential, evolutionarily conserved endoribonuclease composed of 10 different protein subunits and a single RNA. RNase MRP has established roles in multiple pathways including ribosome biogenesis, cell cycle regulation, and mitochondrial DNA replication. Although each of these functions is important to cell growth, additional functions may exist given the essential nature of the complex. To identify novel RNase MRP substrates, we utilized RNA immunoprecipitation and microarray chip analysis to identify RNA that physically associates with RNase MRP. We identified several new potential substrates for RNase MRP including a cell cycle-regulated transcript, CTS1; the yeast homolog of the mammalian p27(Kip1), SIC1; and the U2 RNA component of the spliceosome. In addition, we found RNase MRP to be involved in the regulation of the Ty1 transposon RNA. These results reinforce and broaden the role of RNase MRP in cell cycle regulation and help to identify new roles of this endoribonuclease.
Transcription profile of brewery yeast under fermentation conditions.
James, T C; Campbell, S; Donnelly, D; Bond, U
2003-01-01
Yeast strains, used in the brewing industry, experience distinctive physiological conditions. During a brewing fermentation, yeast are exposed to anaerobic conditions, high pressure, high specific gravity and low temperatures. The purpose of this study was to examine the global gene expression profile of yeast subjected to brewing stress. We have carried out a microarray analysis of a typical brewer's yeast during the course of an 8-day fermentation in 15 degrees P wort. We used the probes derived from Saccharomyces cerevisiae genomic DNA on the chip and RNA isolated from three stages of brewing. This analysis shows a high level of expression of genes involved in fatty acid and ergosterol biosynthesis early in fermentation. Furthermore, genes involved in respiration and mitochondrial protein synthesis also show higher levels of expression. Surprisingly, we observed a complete repression of many stress response genes and genes involved in protein synthesis throughout the 8-day period compared with that at the start of fermentation. This microarray data set provides an analysis of gene expression under brewing fermentation conditions. The data provide an insight into the various metabolic processes altered or activated by brewing conditions of growth. This study leads to future experiments whereby selective alterations in brewing conditions could be introduced to take advantage of the changing transcript profile to improve the quality of the brew.
Bradshaw, Elliot; Yoshida, Minoru; Ling, Feng
2012-04-24
In budding yeast, the mitochondrial DNA (mtDNA) replication pathway involving the homologous DNA pairing protein Mhr1 promotes mitochondrial allele segregation. Mitochondrial fusion facilitates the recombination-mediated replication pathway; however, the role of fission remains largely unknown. By monitoring mitochondrial allele segregation during zygotic division, we found that the absence of fission proteins Fis1 or Mdv1, but not Dnm1, resulted in increased initial homoplasmy levels and decreased mtDNA copy number. However, decreases in mtDNA copy number alone were not sufficient for rapid establishment of homoplasmy, suggesting that inhibiting the activities of certain fission proteins promotes homoplasmy by reducing the number of mtDNA segregation units. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Heude, M; Chanet, R
1975-04-01
The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) anc chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the (see article) genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) process for the (see article) induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressed or derepressed state of the mitochondria.
Penetrating cation/fatty acid anion pair as a mitochondria-targeted protonophore
Severin, Fedor F.; Severina, Inna I.; Antonenko, Yury N.; Rokitskaya, Tatiana I.; Cherepanov, Dmitry A.; Mokhova, Elena N.; Vyssokikh, Mikhail Yu.; Pustovidko, Antonina V.; Markova, Olga V.; Yaguzhinsky, Lev S.; Korshunova, Galina A.; Sumbatyan, Nataliya V.; Skulachev, Maxim V.; Skulachev, Vladimir P.
2010-01-01
A unique phenomenon of mitochondria-targeted protonophores is described. It consists in a transmembrane H+-conducting fatty acid cycling mediated by penetrating cations such as 10-(6’-plastoquinonyl)decyltriphenylphosphonium (SkQ1) or dodecyltriphenylphosphonium (C12TPP). The phenomenon has been modeled by molecular dynamics and directly proved by experiments on bilayer planar phospholipid membrane, liposomes, isolated mitochondria, and yeast cells. In bilayer planar phospholipid membrane, the concerted action of penetrating cations and fatty acids is found to result in conversion of a pH gradient (ΔpH) to a membrane potential (Δψ) of the Nernstian value (about 60 mV Δψ at ΔpH = 1). A hydrophobic cation with localized charge (cetyltrimethylammonium) failed to substitute for hydrophobic cations with delocalized charge. In isolated mitochondria, SkQ1 and C12TPP, but not cetyltrimethylammonium, potentiated fatty acid-induced (i) uncoupling of respiration and phosphorylation, and (ii) inhibition of H2O2 formation. In intact yeast cells, C12TPP stimulated respiration regardless of the extracellular pH value, whereas a nontargeted protonophorous uncoupler (trifluoromethoxycarbonylcyanide phenylhydrazone) stimulated respiration at pH 5 but not at pH 3. Hydrophobic penetrating cations might be promising to treat obesity, senescence, and some kinds of cancer that require mitochondrial hyperpolarization. PMID:20080732
Luz, Anthony L; Lagido, Cristina; Hirschey, Matthew D; Meyer, Joel N
2016-08-01
Mitochondria are a target of many drugs and environmental toxicants; however, how toxicant-induced mitochondrial dysfunction contributes to the progression of human disease remains poorly understood. To address this issue, in vivo assays capable of rapidly assessing mitochondrial function need to be developed. Here, using the model organism Caenorhabditis elegans, we describe how to rapidly assess the in vivo role of the electron transport chain, glycolysis, or fatty acid oxidation in energy metabolism following toxicant exposure, using a luciferase-expressing ATP reporter strain. Alterations in mitochondrial function subsequent to toxicant exposure are detected by depleting steady-state ATP levels with inhibitors of the mitochondrial electron transport chain, glycolysis, or fatty acid oxidation. Differential changes in ATP following short-term inhibitor exposure indicate toxicant-induced alterations at the site of inhibition. Because a microplate reader is the only major piece of equipment required, this is a highly accessible method for studying toxicant-induced mitochondrial dysfunction in vivo. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.
Thevenet, Jonathan; De Marchi, Umberto; Domingo, Jaime Santo; Christinat, Nicolas; Bultot, Laurent; Lefebvre, Gregory; Sakamoto, Kei; Descombes, Patrick; Masoodi, Mojgan; Wiederkehr, Andreas
2016-05-01
Medium-chain triglycerides have been used as part of a ketogenic diet effective in reducing epileptic episodes. The health benefits of the derived medium-chain fatty acids (MCFAs) are thought to result from the stimulation of liver ketogenesis providing fuel for the brain. We tested whether MCFAs have direct effects on energy metabolism in induced pluripotent stem cell-derived human astrocytes and neurons. Using single-cell imaging, we observed an acute pronounced reduction of the mitochondrial electrical potential and a concomitant drop of the NAD(P)H signal in astrocytes, but not in neurons. Despite the observed effects on mitochondrial function, MCFAs did not lower intracellular ATP levels or activate the energy sensor AMP-activated protein kinase. ATP concentrations in astrocytes were unaltered, even when blocking the respiratory chain, suggesting compensation through accelerated glycolysis. The MCFA decanoic acid (300 μM) promoted glycolysis and augmented lactate formation by 49.6%. The shorter fatty acid octanoic acid (300 μM) did not affect glycolysis but increased the rates of astrocyte ketogenesis 2.17-fold compared with that of control cells. MCFAs may have brain health benefits through the modulation of astrocyte metabolism leading to activation of shuttle systems that provide fuel to neighboring neurons in the form of lactate and ketone bodies.-Thevenet, J., De Marchi, U., Santo Domingo, J., Christinat, N., Bultot, L., Lefebvre, G., Sakamoto, K., Descombes, P., Masoodi, M., Wiederkehr, A. Medium-chain fatty acids inhibit mitochondrial metabolism in astrocytes promoting astrocyte-neuron lactate and ketone body shuttle systems. © FASEB.
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.
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.
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
Nguyen, Dan; Samson, Susan L; Reddy, Vasumathi T; Gonzalez, Erica V; Sekhar, Rajagopal V
2013-06-01
Aging is associated with impaired fasted oxidation of nonesterified fatty acids (NEFA) suggesting a mitochondrial defect. Aging is also associated with deficiency of glutathione (GSH), an important mitochondrial antioxidant, and with insulin resistance. This study tested whether GSH deficiency in aging contributes to impaired mitochondrial NEFA oxidation and insulin resistance, and whether GSH restoration reverses these defects. Three studies were conducted: (i) in 82-week-old C57BL/6 mice, the effect of naturally occurring GSH deficiency and its restoration on mitochondrial (13) C1 -palmitate oxidation and glucose metabolism was compared with 22-week-old C57BL/6 mice; (ii) in 20-week C57BL/6 mice, the effect of GSH depletion on mitochondrial oxidation of (13) C1 -palmitate and glucose metabolism was studied; (iii) the effect of GSH deficiency and its restoration on fasted NEFA oxidation and insulin resistance was studied in GSH-deficient elderly humans, and compared with GSH-replete young humans. Chronic GSH deficiency in old mice and elderly humans was associated with decreased fasted mitochondrial NEFA oxidation and insulin resistance, and these defects were reversed with GSH restoration. Acute depletion of GSH in young mice resulted in lower mitochondrial NEFA oxidation, but did not alter glucose metabolism. These data suggest that GSH is a novel regulator of mitochondrial NEFA oxidation and insulin resistance in aging. Chronic GSH deficiency promotes impaired NEFA oxidation and insulin resistance, and GSH restoration reverses these defects. Supplementing diets of elderly humans with cysteine and glycine to correct GSH deficiency could provide significant metabolic benefits. © 2013 John Wiley & Sons Ltd and the Anatomical Society.
Podholová, Kristýna; Plocek, Vítězslav; Rešetárová, Stanislava; Kučerová, Helena; Hlaváček, Otakar; Váchová, Libuše; Palková, Zdena
2016-03-29
Mitochondrial retrograde signaling mediates communication from altered mitochondria to the nucleus and is involved in many normal and pathophysiological changes, including cell metabolic reprogramming linked to cancer development and progression in mammals. The major mitochondrial retrograde pathway described in yeast includes three activators, Rtg1p, Rtg2p and Rtg3p, and repressors, Mks1p and Bmh1p/Bmh2p. Using differentiated yeast colonies, we show that Mks1p-Rtg pathway regulation is complex and includes three branches that divergently regulate the properties and fate of three specifically localized cell subpopulations via signals from differently altered mitochondria. The newly identified RTG pathway-regulated genes ATO1/ATO2 are expressed in colonial upper (U) cells, the cells with active TORC1 that metabolically resemble tumor cells, while CIT2 is a typical target induced in one subpopulation of starving lower (L) cells. The viability of the second L cell subpopulation is strictly dependent on RTG signaling. Additional co-activators of Rtg1p-Rtg3p specific to particular gene targets of each branch are required to regulate cell differentiation.
Koppenol-Raab, Marijke; Harwig, Megan Cleland; Posey, Ammon E.; Egner, John M.; MacKenzie, Kevin R.; Hill, R. Blake
2016-01-01
The tail-anchored protein Fis1 is implicated as a passive tether in yeast mitochondrial fission. We probed the functional role of Fis1 Glu-78, whose elevated side chain pKa suggests participation in protein interactions. Fis1 binds partners Mdv1 or Dnm1 tightly, but mutation E78A weakens Fis1 interaction with Mdv1, alters mitochondrial morphology, and abolishes fission in a growth assay. In fis1Δ rescue experiments, Fis1-E78A causes a novel localization pattern in which Dnm1 uniformly coats the mitochondria. By contrast, Fis1-E78A at lower expression levels recruits Dnm1 into mitochondrial punctate structures but fails to support normal fission. Thus, Fis1 makes multiple interactions that support Dnm1 puncta formation and may be essential after this step, supporting a revised model for assembly of the mitochondrial fission machinery. The insights gained by mutating a residue with a perturbed pKa suggest that side chain pKa values inferred from routine NMR sample pH optimization could provide useful leads for functional investigations. PMID:27496949
A promoter recognition mechanism common to yeast mitochondrial and phage t7 RNA polymerases.
Nayak, Dhananjaya; Guo, Qing; Sousa, Rui
2009-05-15
Yeast mitochondrial (YMt) and phage T7 RNA polymerases (RNAPs) are two divergent representatives of a large family of single subunit RNAPs that are also found in the mitochondria and chloroplasts of higher eukaryotes, mammalian nuclei, and many other bacteriophage. YMt and phage T7 promoters differ greatly in sequence and length, and the YMt RNAP uses an accessory factor for initiation, whereas T7 RNAP does not. We obtain evidence here that, despite these apparent differences, both the YMt and T7 RNAPs utilize a similar promoter recognition loop to bind their respective promoters. Mutations in this element in YMt RNAP specifically disrupt mitochondrial promoter utilization, and experiments with site-specifically tethered chemical nucleases indicate that this element binds the mitochondrial promoter almost identically to how the promoter recognition loop from the phage RNAP binds its promoter. Sequence comparisons reveal that the other members of the single subunit RNAP family display loops of variable sequence and size at a position corresponding to the YMt and T7 RNAP promoter recognition loops. We speculate that these elements may be involved in promoter recognition in most or all of these enzymes and that this element's structure allows it to accommodate significant sequence and length variation to provide a mechanism for rapid evolution of new promoter specificities in this RNAP family.
Regulation of yeast fatty acid desaturase in response to iron deficiency.
Romero, Antonia María; Jordá, Tania; Rozès, Nicolas; Martínez-Pastor, María Teresa; Puig, Sergi
2018-06-01
Unsaturated fatty acids (UFA) are essential components of phospholipids that greatly contribute to the biophysical properties of cellular membranes. Biosynthesis of UFAs relies on a conserved family of iron-dependent fatty acid desaturases, whose representative in the model yeast Saccharomyces cerevisiae is Ole1. OLE1 expression is tightly regulated to adapt UFA biosynthesis and lipid bilayer properties to changes in temperature, and in UFA or oxygen availability. Despite iron deficiency being the most extended nutritional disorder worldwide, very little is known about the mechanisms and the biological relevance of fatty acid desaturases regulation in response to iron starvation. In this report, we show that endoplasmic reticulum-anchored transcription factor Mga2 activates OLE1 transcription in response to nutritional and genetic iron deficiencies. Cells lacking MGA2 display low UFA levels and do not grow under iron-limited conditions, unless UFAs are supplemented or OLE1 is overexpressed. The proteasome, E3 ubiquitin ligase Rsp5 and the Cdc48 Npl4/Ufd1 complex are required for OLE1 activation during iron depletion. Interestingly, Mga2 also activates the transcription of its own mRNA in response to iron deficiency, hypoxia, low temperature and low UFAs. MGA2 up-regulation contributes to increase OLE1 expression in these situations. These results reveal the mechanism of OLE1 regulation when iron is scarce and identify the MGA2 auto-regulation as a potential activation strategy in multiple stresses. Copyright © 2018 Elsevier B.V. All rights reserved.
Andrade, María J; Rodríguez, Mar; Casado, Eva; Córdoba, Juan J
2010-03-01
The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)(4) and (GAC)(5) was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening. Copyright 2009 Elsevier Ltd. All rights reserved.
Biocavity laser spectroscopy of genetically altered yeast cells and isolated yeast mitochondria
NASA Astrophysics Data System (ADS)
Gourley, Paul L.; Hendricks, Judy K.; McDonald, Anthony E.; Copeland, R. Guild; Naviaux, Robert K.; Yaffe, Michael P.
2006-02-01
We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ +) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ -), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ + cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ - cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.
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.
Huang, Wei; Booth, David M; Cane, Matthew C; Chvanov, Michael; Javed, Muhammad A; Elliott, Victoria L; Armstrong, Jane A; Dingsdale, Hayley; Cash, Nicole; Li, Yan; Greenhalf, William; Mukherjee, Rajarshi; Kaphalia, Bhupendra S; Jaffar, Mohammed; Petersen, Ole H; Tepikin, Alexei V; Sutton, Robert; Criddle, David N
2014-01-01
Objective Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. Design Intracellular calcium ([Ca2+]C), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. Results Inhibition of OME with 4-MP converted predominantly transient [Ca2+]C rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. Conclusions A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation. PMID:24162590
Combined defects in oxidative phosphorylation and fatty acid β-oxidation in mitochondrial disease
Nsiah-Sefaa, Abena; McKenzie, Matthew
2016-01-01
Mitochondria provide the main source of energy to eukaryotic cells, oxidizing fats and sugars to generate ATP. Mitochondrial fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are two metabolic pathways which are central to this process. Defects in these pathways can result in diseases of the brain, skeletal muscle, heart and liver, affecting approximately 1 in 5000 live births. There are no effective therapies for these disorders, with quality of life severely reduced for most patients. The pathology underlying many aspects of these diseases is not well understood; for example, it is not clear why some patients with primary FAO deficiencies exhibit secondary OXPHOS defects. However, recent findings suggest that physical interactions exist between FAO and OXPHOS proteins, and that these interactions are critical for both FAO and OXPHOS function. Here, we review our current understanding of the interactions between FAO and OXPHOS proteins and how defects in these two metabolic pathways contribute to mitochondrial disease pathogenesis. PMID:26839416
Koves, Timothy R; Ussher, John R; Noland, Robert C; Slentz, Dorothy; Mosedale, Merrie; Ilkayeva, Olga; Bain, James; Stevens, Robert; Dyck, Jason R B; Newgard, Christopher B; Lopaschuk, Gary D; Muoio, Deborah M
2008-01-01
Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.
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.
Agrimi, Gennaro; Mena, Maria C; Izumi, Kazuki; Pisano, Isabella; Germinario, Lucrezia; Fukuzaki, Hisashi; Palmieri, Luigi; Blank, Lars M; Kitagaki, Hiroshi
2014-03-01
Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.
Kang, Min; Li, Sen; Zhong, Dejun; Yang, Zhimin; Li, Peng
2013-07-01
To investigate the role of hepatocyte apoptosis and mitochondrial permeability transition pore (MPTP) opening in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Thirty male SD rats were randomized into normal diet group and high-fat diet group. At 4, 8 and 12 week of feeding. The hepatocyte apoptosis index (AI) was measured using flow cytometry, and MPTP opening was evaluated with ultraviolet spectrophotometry. Immunohistochemistry was employed to detect hepatic expressions of Bcl-2 and Bax, and Western blotting was used to detect Bax protein expression changes. High-fat feeding resulted in significantly increased hepatocyte AI at 4-12 weeks and gradually increased MPTP opening. In the high-fat diet group, hepatic Bcl-2 expression was detected but the positive cell number remained stable, whereas Bax-positive cell number increased steadily with time with progressively increased intensity of Bax protein expression, resulting in gradually decreased Bcl-2/Bax ratio. Hepatocyte apoptosis occurs in the rat model of NAFLD in close correlation with mitochondrial damage. Increased MPTP opening as the result of increased Bax expression and aberrant Bcl-2/Bax ratio is an important mechanism of hepatocyte mitochondrial damage in NAFLD.
Zhao, Meng-Ge; Sheng, Xue-Ping; Huang, Ya-Ping; Wang, Yi-Ting; Jiang, Cui-Hua; Zhang, Jian; Yin, Zhi-Qi
2018-08-01
The effects of triterpenic acids-enriched fraction from Cyclocarya paliurus (CPT) on nonalcoholic fatty liver disease (NAFLD) were investigated using in vivo and in vitro models. In high fat diet-induced Wister rats, CPT significantly increased superoxide dismutase (SOD) activity and glutathione/oxidized glutathione (GSH/GSSG) ratio, reduced malondialdehyde (MDA) and protein carbonyl (PCO) levels. Moreover, CPT restored mitochondrial membrane potential dysfunction, decreased cytochrome P450 enzyme 2E1 (CYP2E1) activity, improved nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-mediated antioxidant enzyme heme oxygenase1 (HO-1) expression. In free fatty acids-induced HepG2 cells, CPT dramatically decreased ROS content, increased mitochondrial NADH dehydrogenase (Complex I) and mitochondrial cytochrome C oxidase (Complex IV) levels. Furthermore, CPT could upregulate HO-1, quinine oxidoreductase 1 (NQO1) expression, and increase Nrf2 translocation from cytoplasm-to-nucleus. The results indicated CPT could protect mitochondria function and improve oxidative stress by activating Nrf2. Therefore, it can be inferred that CPT may be a potential agent against NAFLD. Copyright © 2018 Elsevier Masson SAS. All rights reserved.
Advances in the Understanding and Treatment of Mitochondrial Fatty Acid Oxidation Disorders.
Goetzman, Eric S
2017-09-01
This review focuses on advances made in the past three years with regards to understanding the mitochondrial fatty acid oxidation (FAO) pathway, the pathophysiological ramifications of genetic lesions in FAO enzymes, and emerging therapies for FAO disorders. FAO has now been recognized to play a key energetic role in pulmonary surfactant synthesis, T-cell differentiation and memory, and the response of the proximal tubule to kidney injury. Patients with FAO disorders may face defects in these cellular systems as they age. Aspirin, statins, and nutritional supplements modulate the rate of FAO under normal conditions and could be risk factors for triggering symptoms in patients with FAO disorders. Patients have been identified with mutations in the ACAD9 and ECHS1 genes, which may represent new FAO disorders. New interventions for long-chain FAODs are in clinical trials. Finally, post-translational modifications that regulate fatty acid oxidation protein activities have been characterized that represent important new therapeutic targets. Recent research has led to a deeper understanding of FAO. New therapeutic avenues are being pursued that may ultimately cause a paradigm shift for patient care.
Smith, Stuart; Witkowski, Andrzej; Moghul, Ayesha; Yoshinaga, Yuko; Nefedov, Michael; de Jong, Pieter; Feng, Dejiang; Fong, Loren; Tu, Yiping; Hu, Yan; Young, Stephen G.; Pham, Thomas; Cheung, Carling; Katzman, Shana M.; Brand, Martin D.; Quinlan, Casey L.; Fens, Marcel; Kuypers, Frans; Misquitta, Stephanie; Griffey, Stephen M.; Tran, Son; Gharib, Afshin; Knudsen, Jens; Hannibal-Bach, Hans Kristian; Wang, Grace; Larkin, Sandra; Thweatt, Jennifer; Pasta, Saloni
2012-01-01
A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor. PMID:23077570
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
Gariani, Karim; Menzies, Keir J.; Ryu, Dongryeol; Wegner, Casey J.; Wang, Xu; Ropelle, Eduardo R.; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young‐Ki; Piersigilli, Alessandra; Pham, Tho X.; Yang, Yue; Ku, Chai Siah; Koo, Sung I.; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A.
2015-01-01
With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe −/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204) PMID:26404765
Van Kreijl, C F; Bos, J L
1977-01-01
The repeating nucleotide sequence of 68 base pairs in the mtDNA from an ethidium-induced cytoplasmic petite mutant of yeast has been determined. For sequence analysis specifically primed and terminated RNA copies, obtained by in vitro transcription of the separated strands, were use. The sequence consists of 66 consecutive AT base pairs flanked by two GC pairs and comprises nearly all of the mutant mitochondrial genome. The sequence, moreover, also represents the first part of wild-type mtDNA sequence so far. Images PMID:198740
Hill, J; McGraw, P; Tzagoloff, A
1985-03-25
The yeast nuclear gene CBP2 was previously proposed to code for a protein necessary for processing of the terminal intron in the cytochrome b pre-mRNA (McGraw, P., and Tzagoloff, A. (1983) J. Biol. Chem. 258, 9459-9468). In the present study we describe a mitochondrial mutation capable of suppressing the respiratory deficiency of cbp2 mutants. The mitochondrial suppressor mutation has been shown to be the result of a precise excision of the last intervening sequence from the cytochrome b gene. Strains with the altered mitochondrial DNA have normal levels of mature cytochrome b mRNA and of cytochrome b and exhibit wild type growth on glycerol. These results confirm that CBP2 codes for a protein specifically required for splicing of the cytochrome b intron and further suggest that absence of the intervening sequence does not noticeably affect the expression of respiratory function in mitochondria.
Malecki, Michal; Jedrzejczak, Robert; Stepien, Piotr P; Golik, Pawel
2007-09-07
The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.
Mitochondrial uncoupling proteins in unicellular eukaryotes.
Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E
2010-01-01
Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.
Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J
2017-03-06
Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used
Fatty alcohol production in Lipomyces starkeyi and Yarrowia lipolytica
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wang, Wei; Wei, Hui; Knoshaug, Eric
Current biological pathways to produce biofuel intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels are not cost effective. Previously, oleaginous yeasts have been investigated primarily for lipid production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. In addition, once recovered from the cells, lipids are difficult to blend directly with the existing fuels without upgrading. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels. In this study, we successfully demonstrate the production of fatty alcohols by the oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, throughmore » expression of the fatty acyl-CoA reductase gene from Marinobactor aquaeolei VT8. This strategy resulted in the production of 167 and 770 mg/L of fatty alcohols in shake flask from Y. lipolytica and L starkeyi, respectively. When using a dodecane overlay during fermentation, 92 and 99% of total fatty alcohols produced by Y. lipolytica and L. starkeyi, respectively, were extracted into the dodecane phase, which compares favorably to the 3 and 50% recovered, respectively, without the dodecane layer. In both oleaginous yeasts, long chain length, saturated fatty alcohols, i.e., hexadecanol (C16:0) and octadecanol (C18:0), were predominant and accounted for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Furthermore, this work demonstrates that the oleaginous yeasts, Y. lipolytica and L. starkeyi, can serve as platform organisms for the production of fatty acid-derived biofuels and bioproducts.« less
Fatty alcohol production in Lipomyces starkeyi and Yarrowia lipolytica
Wang, Wei; Wei, Hui; Knoshaug, Eric; ...
2016-10-24
Current biological pathways to produce biofuel intermediates amenable to separations and catalytic upgrading to hydrocarbon fuels are not cost effective. Previously, oleaginous yeasts have been investigated primarily for lipid production. However, yeasts store neutral lipids intracellularly making recovery difficult and expensive. In addition, once recovered from the cells, lipids are difficult to blend directly with the existing fuels without upgrading. We have, therefore, begun to investigate secreted fatty acid-derived products which can be easily recovered and upgraded to fuels. In this study, we successfully demonstrate the production of fatty alcohols by the oleaginous yeasts, Yarrowia lipolytica and Lipomyces starkeyi, throughmore » expression of the fatty acyl-CoA reductase gene from Marinobactor aquaeolei VT8. This strategy resulted in the production of 167 and 770 mg/L of fatty alcohols in shake flask from Y. lipolytica and L starkeyi, respectively. When using a dodecane overlay during fermentation, 92 and 99% of total fatty alcohols produced by Y. lipolytica and L. starkeyi, respectively, were extracted into the dodecane phase, which compares favorably to the 3 and 50% recovered, respectively, without the dodecane layer. In both oleaginous yeasts, long chain length, saturated fatty alcohols, i.e., hexadecanol (C16:0) and octadecanol (C18:0), were predominant and accounted for more than 85% of the total fatty alcohols produced. To the best of our knowledge, this is the first report of fatty alcohol production in L. starkeyi. Furthermore, this work demonstrates that the oleaginous yeasts, Y. lipolytica and L. starkeyi, can serve as platform organisms for the production of fatty acid-derived biofuels and bioproducts.« less
Shimazaki, Aiko; Sakamoto, Jin J; Furuta, Masakazu; Tsuchido, Tetsuaki
2016-01-01
The antifungal activities of diglycerin monoester of fatty acids (DGCs), which have been employed as food emulsifiers, were examined against three yeasts, Saccharomyces cerevisiae, Candida albicans and Candida utilis and were compared with those of sucrose monoester of palmitic acid (SC16) as another type of emulsifier and sodium benzoate (SB) as a weak acid food preservative. When the minimum growth inhibitory concentrations (MICs) of diglycerin monolaurate (DGC12) against these yeasts were determined 2 d after incubation in YM broth at pH5.0, they were relatively low, being 0.01% (w/v), for both S. cerevisiae and C. utilis, whereas was high, being 4.0% (w/v), for C. albicans. On the contrary, the MICs of sucrose monopalmitate (SC16) were high, being 3.0 and 4.0% (w/v), for the former two yeasts, respectively, but 0.6% (w/v) for the last yeast. In contrast to these emulsifiers, the MICs of sodium benzoate (SB) were similar independently upon the yeast strain, being in order 0.4, 0.3 and 0.5% (w/v), for the above yeasts, respectively. The anti-yeast activities of DGC12 and SC16 were gradually increased with a decrease in pH, in a manner similar to that of SB, except for the action of SC16 on C. albicans, for which the activity was more effective at pHs 5.0 and 6.0 than at pHs 4.0 and 7.0. Among DGCs tested having different fatty acid moieties in the molecule, lauroyl ester (DGC12) was more effective than myristoyl and palmitoyl esters against S. cerevisiae and C. utilis. The inhibitory effect of DGC12 on the yeast growth depended upon both the cell density and the strength of aeration during the treatment. Further, DGC12 was found to kill S. cerevisiae and C. utilis cells at a rather low concentration of 0.005% (w/v) in 50mM acetate buffer at pH5.0, although, against C. albicans cells, only slight fungicidal activity was demonstrated at a high concentration of 0.5% (w/v). The results obtained support the effectiveness of practical application of DGC12 to acidic
Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L.
2009-01-01
The assembly status of the cytochrome bc1 complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc1 subunits had been deleted. In all the yeast strains tested a bc1 sub-complex of about 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS-PAGE and immunodecoration, revealed the presence of the two catalytic subunits cytochrome b and cytochrome c1, associated with the non catalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Altogether these bc1 subunits build up the core structure of the cytochrome bc1 complex which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc1 core structure may represent a true assembly intermediate during the maturation of the bc1 complex, first because of its wide distribution in distinct yeast deletion strains and second for its characteristics of stability which resemble those of the intact homodimeric bc1 complex. Differently from this latter, however, the bc1 core structure is not able to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc1 complex provides a number of new elements for clarification of the molecular events leading to the maturation of the yeast cytochrome bc1 complex in the inner mitochondrial membrane. PMID:19236481
Kao, L R; Megraw, T L; Chae, C B
1993-06-15
The yeast mitochondrial histone protein HM is required for maintenance of the mitochondrial genome, and disruption of the gene encoding HM (HIM1/ABF2) results in formation of a respiration-deficient petite mutant phenotype. HM contains two homologous regions, which share sequence similarity with the eukaryotic nuclear nonhistone protein, HMG-1. Experiments with various deletion mutants of HM show that a single HMG domain of HM is functional and can restore respiration competency to cells that lack HM protein (him1 mutant cells). The gene encoding the putative yeast nuclear HMG-1 homolog, the NHP6A protein, can functionally complement the him1 mutation. These results suggest that the HMG domain is the basic unit for the function of HM in mitochondria and that the function of HMG-1 proteins in the nucleus and HM in the mitochondrion may be equivalent.
Vongsvivut, Jitraporn; Heraud, Philip; Gupta, Adarsha; Puri, Munish; McNaughton, Don; Barrow, Colin J
2013-10-21
The increase in polyunsaturated fatty acid (PUFA) consumption has prompted research into alternative resources other than fish oil. In this study, a new approach based on focal-plane-array Fourier transform infrared (FPA-FTIR) microspectroscopy and multivariate data analysis was developed for the characterisation of some marine microorganisms. Cell and lipid compositions in lipid-rich marine yeasts collected from the Australian coast were characterised in comparison to a commercially available PUFA-producing marine fungoid protist, thraustochytrid. Multivariate classification methods provided good discriminative accuracy evidenced from (i) separation of the yeasts from thraustochytrids and distinct spectral clusters among the yeasts that conformed well to their biological identities, and (ii) correct classification of yeasts from a totally independent set using cross-validation testing. The findings further indicated additional capability of the developed FPA-FTIR methodology, when combined with partial least squares regression (PLSR) analysis, for rapid monitoring of lipid production in one of the yeasts during the growth period, which was achieved at a high accuracy compared to the results obtained from the traditional lipid analysis based on gas chromatography. The developed FTIR-based approach when coupled to programmable withdrawal devices and a cytocentrifugation module would have strong potential as a novel online monitoring technology suited for bioprocessing applications and large-scale production.
Enlightenment of Yeast Mitochondrial Homoplasmy: Diversified Roles of Gene Conversion
Ling, Feng; Mikawa, Tsutomu; Shibata, Takehiko
2011-01-01
Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell (“homoplasmy”) during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3′ single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in immunoglobulin diversification in chicken. MtDNA can be regarded as a sequence family, in which the members tend to be diversified by a high frequency of spontaneous mutagenesis. Thus, it would be interesting to determine why and how double-stranded breakage and D-loop formation induce sequence homogenization in mitochondria and sequence diversification in nuclear DNA. We will review the mechanisms and roles of mtDNA homoplasmy, in contrast to nuclear gene conversion, which diversifies gene and genome sequences, to provide clues toward understanding how the common DNA processing pathway results in such divergent outcomes. PMID:24710143
Andrade, M J; Rodríguez, M; Casado, E M; Bermúdez, E; Córdoba, J J
2009-09-01
The efficiency of mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR and volatile compounds analysis to differentiate yeast biotypes involved in flavour development of dry-cured Iberian ham throughout the ripening process is evaluated. For this purpose, 86 yeasts isolated from Iberian hams in the main ripening stages at different industries of the four Protected Designations of Origin of this product, were used. The combination of mtDNA restriction analysis and RAPD-PCR using the primer (GACA)4 showed a higher variability in the yeast species detected than obtained using only mtDNA restriction analysis. Only two species, Debaryomyces hansenii and Candida zeylanoides, were identified throughout the whole ripening process and a wide diversity of biotypes was found in these two species, with those of D. hansenii predominating. Clear differences between biotypes were detected in the generation of volatile compounds, with the biotype C2-2 of D. hansenii showing the highest concentrations of volatiles. The combined use of mtDNA restriction analysis and RAPD-PCR distinguishes yeast biotypes with different production of volatile compounds. In addition, analysis of the production profile of volatile compounds is needed to differentiate yeast strains of the same biotype recovered at different stages of ripening. Thus, the combination of these three methods could be very useful to select or monitor yeasts as starter cultures in dry-cured meat products.
Gariani, Karim; Menzies, Keir J; Ryu, Dongryeol; Wegner, Casey J; Wang, Xu; Ropelle, Eduardo R; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young-Ki; Piersigilli, Alessandra; Pham, Tho X; Yang, Yue; Ku, Chai Siah; Koo, Sung I; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A; Lee, Ji-Young; Auwerx, Johan
2016-04-01
With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high-fat high-sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD(+) ) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD(+) repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD(+) biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1- and SIRT3-dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β-oxidation and mitochondrial complex content and activity. The cell-autonomous beneficial component of NR treatment was revealed in liver-specific Sirt1 knockout mice (Sirt1(hep-/-) ), whereas apolipoprotein E-deficient mice (Apoe(-/-) ) challenged with a high-fat high-cholesterol diet affirmed the use of NR in other independent models of NAFLD. Our data warrant the future evaluation of NAD(+) boosting strategies to manage the development or progression of NAFLD. © 2015 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver Diseases.
Wen, Yang-An; Xing, Xiaopeng; Harris, Jennifer W; Zaytseva, Yekaterina Y; Mitov, Mihail I; Napier, Dana L; Weiss, Heidi L; Mark Evers, B; Gao, Tianyan
2017-02-02
Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism by which adipocytes regulate the metabolism of colon cancer cells remains elusive. In this study, we showed that adipocytes isolated from adipose tissues of colon cancer patients have an important role in modulating cellular metabolism to support tumor growth and survival. Abundant adipocytes were found in close association with invasive tumor cells in colon cancer patients. Co-culture of adipocytes with colon cancer cells led to a transfer of free fatty acids that released from the adipocytes to the cancer cells. Uptake of fatty acids allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid β-oxidation. Mechanistically, co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize fatty acids and blocked the growth-promoting effect of adipocytes. In addition, we found that adipocytes stimulated the expression of genes associated with cancer stem cells and downregulated genes associated with intestinal epithelial cell differentiation in primary colon cancer cells and mouse tumor organoids. Importantly, the presence of adipocytes promoted the growth of xenograft tumors in vivo. Taken together, our results show that adipocytes in the tumor microenvironment serve as an energy provider and a metabolic regulator to promote the growth and survival of colon cancer cells.
Wen, Yang-An; Xing, Xiaopeng; Harris, Jennifer W; Zaytseva, Yekaterina Y; Mitov, Mihail I; Napier, Dana L; Weiss, Heidi L; Mark Evers, B; Gao, Tianyan
2017-01-01
Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism by which adipocytes regulate the metabolism of colon cancer cells remains elusive. In this study, we showed that adipocytes isolated from adipose tissues of colon cancer patients have an important role in modulating cellular metabolism to support tumor growth and survival. Abundant adipocytes were found in close association with invasive tumor cells in colon cancer patients. Co-culture of adipocytes with colon cancer cells led to a transfer of free fatty acids that released from the adipocytes to the cancer cells. Uptake of fatty acids allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid β-oxidation. Mechanistically, co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize fatty acids and blocked the growth-promoting effect of adipocytes. In addition, we found that adipocytes stimulated the expression of genes associated with cancer stem cells and downregulated genes associated with intestinal epithelial cell differentiation in primary colon cancer cells and mouse tumor organoids. Importantly, the presence of adipocytes promoted the growth of xenograft tumors in vivo. Taken together, our results show that adipocytes in the tumor microenvironment serve as an energy provider and a metabolic regulator to promote the growth and survival of colon cancer cells. PMID:28151470
Sembongi, Hiroshi; Di Re, Miriam; Bokori-Brown, Monika; Holt, Ian J
2007-10-01
Rearrangements of mitochondrial DNA (mtDNA) are a well-recognized cause of human disease; deletions are more frequent, but duplications are more readily transmitted to offspring. In theory, partial duplications of mtDNA can be resolved to partially deleted and wild-type (WT) molecules, via homologous recombination. Therefore, the yeast CCE1 gene, encoding a Holliday junction resolvase, was introduced into cells carrying partially duplicated or partially triplicated mtDNA. Some cell lines carrying the CCE1 gene had substantial amounts of WT mtDNA suggesting that the enzyme can mediate intramolecular recombination in human mitochondria. However, high levels of expression of CCE1 frequently led to mtDNA loss, and so it is necessary to strictly regulate the expression of CCE1 in human cells to ensure the selection and maintenance of WT mtDNA.
Hammond, Linda E.; Gallagher, Patricia A.; Wang, Shuli; Hiller, Sylvia; Kluckman, Kimberly D.; Posey-Marcos, Eugenia L.; Maeda, Nobuyo; Coleman, Rosalind A.
2002-01-01
Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT−/− mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT−/− liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT−/− liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production. PMID:12417724
Raza Shaikh, Saame; Brown, David A
2013-01-01
Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.
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.
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
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
Biogenesis of the yeast cytochrome bc1 complex.
Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L
2009-01-01
The mitochondrial respiratory chain is composed of four different protein complexes that cooperate in electron transfer and proton pumping across the inner mitochondrial membrane. The cytochrome bc1 complex, or complex III, is a component of the mitochondrial respiratory chain. This review will focus on the biogenesis of the bc1 complex in the mitochondria of the yeast Saccharomyces cerevisiae. In wild type yeast mitochondrial membranes the major part of the cytochrome bc1 complex was found in association with one or two copies of the cytochrome c oxidase complex. The analysis of several yeast mutant strains in which single genes or pairs of genes encoding bc1 subunits had been deleted revealed the presence of a common set of bc1 sub-complexes. These sub-complexes are represented by the central core of the bc1 complex, consisting of cytochrome b bound to subunit 7 and subunit 8, by the two core proteins associated with each other, by the Rieske protein associated with subunit 9, and by those deriving from the unexpected interaction of each of the two core proteins with cytochrome c1. Furthermore, a higher molecular mass sub-complex is that composed of cytochrome b, cytochrome c1, core protein 1 and 2, subunit 6, subunit 7 and subunit 8. The identification and characterization of all these sub-complexes may help in defining the steps and the molecular events leading to bc1 assembly in yeast mitochondria.
Zara, Vincenzo; Conte, Laura; Trumpower, Bernard L
2009-04-01
The assembly status of the cytochrome bc(1) complex has been analyzed in distinct yeast deletion strains in which genes for one or more of the bc(1) subunits were deleted. In all the yeast strains tested, a bc(1) sub-complex of approximately 500 kDa was found when the mitochondrial membranes were analyzed by blue native electrophoresis. The subsequent molecular characterization of this sub-complex, carried out in the second dimension by SDS/PAGE and immunodecoration, revealed the presence of the two catalytic subunits, cytochrome b and cytochrome c(1), associated with the noncatalytic subunits core protein 1, core protein 2, Qcr7p and Qcr8p. Together, these bc(1) subunits build up the core structure of the cytochrome bc(1) complex, which is then able to sequentially bind the remaining subunits, such as Qcr6p, Qcr9p, the Rieske iron-sulfur protein and Qcr10p. This bc(1) core structure may represent a true assembly intermediate during the maturation of the bc(1) complex; first, because of its wide distribution in distinct yeast deletion strains and, second, for its characteristics of stability, which resemble those of the intact homodimeric bc(1) complex. By contrast, the bc(1) core structure is unable to interact with the cytochrome c oxidase complex to form respiratory supercomplexes. The characterization of this novel core structure of the bc(1) complex provides a number of new elements clarifying the molecular events leading to the maturation of the yeast cytochrome bc(1) complex in the inner mitochondrial membrane.
Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L.
2011-01-01
CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM. PMID:21622568
Item, Flurin; Wueest, Stephan; Lemos, Vera; Stein, Sokrates; Lucchini, Fabrizio C; Denzler, Rémy; Fisser, Muriel C; Challa, Tenagne D; Pirinen, Eija; Kim, Youngsoo; Hemmi, Silvio; Gulbins, Erich; Gross, Atan; O'Reilly, Lorraine A; Stoffel, Markus; Auwerx, Johan; Konrad, Daniel
2017-09-07
Nonalcoholic fatty liver disease is one of the most prevalent metabolic disorders and it tightly associates with obesity, type 2 diabetes, and cardiovascular disease. Reduced mitochondrial lipid oxidation contributes to hepatic fatty acid accumulation. Here, we show that the Fas cell surface death receptor (Fas/CD95/Apo-1) regulates hepatic mitochondrial metabolism. Hepatic Fas overexpression in chow-fed mice compromises fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes promoting hepatic lipid accumulation and insulin resistance. In line, hepatocyte-specific ablation of Fas improves mitochondrial function and ameliorates high-fat-diet-induced hepatic steatosis, glucose tolerance, and insulin resistance. Mechanistically, Fas impairs fatty acid oxidation via the BH3 interacting-domain death agonist (BID). Mice with genetic or pharmacological inhibition of BID are protected from Fas-mediated impairment of mitochondrial oxidation and hepatic steatosis. We suggest Fas as a potential novel therapeutic target to treat obesity-associated fatty liver and insulin resistance.Hepatic steatosis is a common disease closely associated with metabolic syndrome and insulin resistance. Here Item et al. show that Fas, a member of the TNF receptor superfamily, contributes to mitochondrial dysfunction, steatosis development, and insulin resistance under high fat diet.
Malgoyre, Alexandra; Chabert, Clovis; Tonini, Julia; Koulmann, Nathalie; Bigard, Xavier; Sanchez, Hervé
2017-03-01
We investigated the effects of chronic hypoxia on the maximal use of and sensitivity of mitochondria to different substrates in rat slow-oxidative (soleus, SOL) and fast-glycolytic (extensor digitorum longus, EDL) muscles. We studied mitochondrial respiration in situ in permeabilized myofibers, using pyruvate, octanoate, palmitoyl-carnitine (PC), or palmitoyl-coenzyme A (PCoA). The hypophagia induced by hypoxia may also alter metabolism. Therefore, we used a group of pair-fed rats (reproducing the same caloric restriction, as observed in hypoxic animals), in addition to the normoxic control fed ad libitum. The resting respiratory exchange ratio decreased after 21 days of exposure to hypobaric hypoxia (simulated elevation of 5,500 m). The respiration supported by pyruvate and octanoate were unaffected. In contrast, the maximal oxidative respiratory rate for PCoA, the transport of which depends on carnitine palmitoyltransferase 1 (CPT-1), decreased in the rapid-glycolytic EDL and increased in the slow-oxidative SOL, although hypoxia improved affinity for this substrate in both muscle types. PC and PCoA were oxidized similarly in normoxic EDL, whereas chronic hypoxia limited transport at the CPT-1 step in this muscle. The effects of hypoxia were mediated by caloric restriction in the SOL and by hypoxia itself in the EDL. We conclude that improvements in mitochondrial affinity for PCoA, a physiological long-chain fatty acid, would facilitate fatty-acid use at rest after chronic hypoxia independently of quantitative alterations of mitochondria. Conversely, decreasing the maximal oxidation of PCoA in fast-glycolytic muscles would limit fatty-acid use during exercise. NEW & NOTEWORTHY Affinity for low concentrations of long-chain fatty acids (LCFA) in mitochondria skeletal muscles increases after chronic hypoxia. Combined with a lower respiratory exchange ratio, this suggests facility for fatty acid utilization at rest. This fuel preference is related to caloric
Chromosomal Aneuploidy Improves the Brewing Characteristics of Sake Yeast.
Kadowaki, Masafumi; Fujimaru, Yuki; Taguchi, Seiga; Ferdouse, Jannatul; Sawada, Kazutaka; Kimura, Yuta; Terasawa, Yohei; Agrimi, Gennaro; Anai, Toyoaki; Noguchi, Hideki; Toyoda, Atsushi; Fujiyama, Asao; Akao, Takeshi; Kitagaki, Hiroshi
2017-12-15
The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast ( Saccharomyces cerevisiae ) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their "petite" strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile. IMPORTANCE Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This study shows the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to α-acetolactate and, in turn, to high concentrations of diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic of
Chromosomal Aneuploidy Improves the Brewing Characteristics of Sake Yeast
Kadowaki, Masafumi; Fujimaru, Yuki; Taguchi, Seiga; Ferdouse, Jannatul; Sawada, Kazutaka; Kimura, Yuta; Terasawa, Yohei; Agrimi, Gennaro; Anai, Toyoaki; Noguchi, Hideki; Toyoda, Atsushi; Fujiyama, Asao; Akao, Takeshi
2017-01-01
ABSTRACT The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their “petite” strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile. IMPORTANCE Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This study shows the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to α-acetolactate and, in turn, to high concentrations of diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic
Kovaleva, Mariya V; Sukhanova, Evgeniya I; Trendeleva, Tatyana A; Zyl'kova, Marina V; Ural'skaya, Ludmila A; Popova, Kristina M; Saris, Nils-Erik L; Zvyagilskaya, Renata A
2009-06-01
In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca(2+) uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca(2+) transport system (Bazhenova et al. J Biol Chem 273:4372-4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96-100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352-1356, 2000; Deryabina et al. J Biol Chem 276:47801-47806, 2001) were very resistant to Ca(2+) overload. However, exposure of yeast mitochondria to 50-100 microM Ca(2+) in the presence of the Ca(2+) ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca(2+)/nH(+)-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca(2+)- ETH129-induced activation of the Ca(2+)/H(+)-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca(2+) overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319-331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37-51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools
Takakuwa, Naoya; Saito, Katsuichi
2010-01-01
Eighty-one yeast isolates from raw milk were surveyed for the production of fatty acid methyl esters (FAME). Only one species, identified as Cryptococcus curvatus, produced FAME at a detectable level. Cr. curvatus TYC-19 produced more FAME from beet molasses and cheese whey medium than other strains of the same species. In both media, the major FAME produced were linoleic and oleic acid methyl esters. Sequence analysis of the internal transcribed spacer region of ribosomal DNA indicated that TYC-19 diverged from the same species.
Fatty acids trigger mitochondrion-dependent necrosis.
Rockenfeller, Patrick; Ring, Julia; Muschett, Vera; Beranek, Andreas; Buettner, Sabrina; Carmona-Gutierrez, Didac; Eisenberg, Tobias; Khoury, Chamel; Rechberger, Gerald; Kohlwein, Sepp D; Kroemer, Guido; Madeo, Frank
2010-07-15
Obesity is characterised by lipid accumulation in non-adipose tissues, leading to organ degeneration and a wide range of diseases, including diabetes, heart attack and liver cirrhosis. Free fatty acids (FFA) are believed to be the principal toxic triggers mediating the adverse cellular effects of lipids. Here, we show that various cooking oils used in human nutrition cause cell death in yeast in the presence of a triacylglycerol lipase, mimicking the physiological microenvironment of the small intestine. Combining genetic and cell death assays, we demonstrate that elevated FFA concentrations lead to necrotic cell death, as evidenced by loss of membrane integrity and release of nuclear HMGB1. FFA-mediated necrosis depends on functional mitochondria and leads to the accumulation of reactive oxygen species. We conclude that lipotoxicity is executed via a mitochondrial necrotic pathway, challenging the dogma that the adverse effects of lipid stress are exclusively apoptotic.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chang, Soo-Ik; Hammes, G.G.
1989-11-01
Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chickenmore » and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The active sites of all of the enzymes are primarily in hydrophobic regions of the protein. This study suggests that the genes for the functional domains of fatty acid synthase were originally separated, and these genes were connected to each other by using different connecting nucleotide sequences in different species. An alternative explanation for the differences in rat and chicken is a common ancestry and mutations in the joining regions during evolution.« less
Mechanisms by Which Different Functional States of Mitochondria Define Yeast Longevity
Beach, Adam; Leonov, Anna; Arlia-Ciommo, Anthony; Svistkova, Veronika; Lutchman, Vicky; Titorenko, Vladimir I.
2015-01-01
Mitochondrial functionality is vital to organismal physiology. A body of evidence supports the notion that an age-related progressive decline in mitochondrial function is a hallmark of cellular and organismal aging in evolutionarily distant eukaryotes. Studies of the baker’s yeast Saccharomyces cerevisiae, a unicellular eukaryote, have led to discoveries of genes, signaling pathways and chemical compounds that modulate longevity-defining cellular processes in eukaryotic organisms across phyla. These studies have provided deep insights into mechanistic links that exist between different traits of mitochondrial functionality and cellular aging. The molecular mechanisms underlying the essential role of mitochondria as signaling organelles in yeast aging have begun to emerge. In this review, we discuss recent progress in understanding mechanisms by which different functional states of mitochondria define yeast longevity, outline the most important unanswered questions and suggest directions for future research. PMID:25768339
Cheng, Xin; Ivessa, Andreas S
2012-10-01
Translocation of mitochondrial DNA (mtDNA) fragments to the nucleus and insertion of those fragments into nuclear DNA has been observed in several organisms ranging from yeast to plants and mammals. Disruption of specific nuclear genes by de novo insertions of mtDNA fragments has even been linked to the initiation of several human diseases. Recently, we demonstrated that baker's yeast strains with high rates of mtDNA fragments migrating to the nucleus (yme1-1 mutant) exhibit short chronological life spans (CLS). The yeast CLS is determined by the survival of non-dividing cell populations. Here, we show that lack of the non-homologous-end-joining enzyme DNA ligase IV (DNL4) can rescue the short CLS of the yme1-1 mutant. In fission yeast, DNA ligase IV has been shown to be required for the capture of mtDNA fragments during the repair of double-stranded DNA breaks in nuclear DNA. In further analyses using pulse field gel and 2D gel electrophoresis we demonstrate that linear mtDNA fragments with likely nuclear localization accumulate in the yme1-1 mutant. The accumulation of the linear mtDNA fragments in the yme1-1 mutant is suppressed when Dnl4 is absent. We propose that the linear nuclear mtDNA fragments accelerate the aging process in the yme1-1 mutant cells by possibly affecting nuclear processes including DNA replication, recombination, and repair as well as transcription of nuclear genes. We speculate further that Dnl4 protein has besides its function as a ligase also a role in DNA protection. Dnl4 protein may stabilize the linear mtDNA fragments in the nucleus by binding to their physical ends. In the absence of Dnl4 protein the linear fragments are therefore unprotected and possibly degraded by nuclear nucleases. Copyright © 2012 Elsevier GmbH. All rights reserved.
Ling, Feng; Shibata, Takehiko
2004-01-01
Mitochondria carry many copies of mitochondrial DNA (mtDNA), but mt-alleles quickly segregate during mitotic growth through unknown mechanisms. Consequently, all mtDNA copies are often genetically homogeneous within each individual ("homoplasmic"). Our previous study suggested that tandem multimers ("concatemers") formed mainly by the Mhr1p (a yeast nuclear gene-encoded mtDNA-recombination protein)-dependent pathway are required for mtDNA partitioning into buds with concomitant monomerization. The transmission of a few randomly selected clones (as concatemers) of mtDNA into buds is a possible mechanism to establish homoplasmy. The current study provides evidence for this hypothesis as follows: the overexpression of MHR1 accelerates mt-allele-segregation in growing heteroplasmic zygotes, and mhr1-1 (recombination-deficient) causes its delay. The mt-allele-segregation rate correlates with the abundance of concatemers, which depends on Mhr1p. In G1-arrested cells, concatemeric mtDNA was labeled by [14C]thymidine at a much higher density than monomers, indicating concatemers as the immediate products of mtDNA replication, most likely in a rolling circle mode. After releasing the G1 arrest in the absence of [14C]thymidine, the monomers as the major species in growing buds of dividing cells bear a similar density of 14C as the concatemers in the mother cells, indicating that the concatemers in mother cells are the precursors of the monomers in buds.
Deryabina, Yulia; Isakova, Elena; Sekova, Varvara; Antipov, Alexey; Saris, Nils-Erik L
2014-12-01
In this study, we investigated the change in the antioxidant enzymes activity, cell respiration, reactive oxygen species (ROS), and impairment of membrane mitochondria permeability in the Endomyces magnusii yeasts during culture growth and aging. We showed that the transition into stationary phase is the key tool to understanding interaction of these processes. This growth stage is distinguished by two-fold increase in ROS production and respiration rate as compared to those in the logarithmic phase. It results in induction of alternative oxidase (AO) in the stationary phase, decline of the main antioxidant enzymes activities, ROS-production, and mitochondria membrane permeability. Significant increase in the share of mitochondrial isoform of superoxide dismutase (SOD2) occurred in the stationary phase from 51.8% (24 h of cultivation) to 68.6% (48 h of cultivation). Upon blocking the essential ROS-scavenging enzymes, SODs and catalases (CATs) some heterogeneity of cell population was observed: 80-90% of cells displayed evident signs of early apoptosis (such as disorientation of mitochondria cristae, mitochondrial fragmentation and deformation of nuclear chromatine). However, 10-20% of the population were definitely healthy. It allowed to draw the conclusion that a complete system of cell antioxidant protection underlies normal mitochondria functioning while the E. magnusii yeasts grow and age. Moreover, this system provides unimpaired cell physiology under oxidative stress during culture aging in the stationary phase. Failures in mitochondria functions due to inhibition of ROS-scavenging enzymes of CATs and SODs could lead to damage of the cells and some signs of early apoptosis.
Spiekerkoetter, Ute; Mueller, Martina; Cloppenburg, Eva; Motz, Reinald; Mayatepek, Ertan; Bueltmann, Burkhard; Korenke, Christoph
2008-08-01
Because of a switch in energy-producing substrate utilization from glucose in the fetal period to fatty acids postnatally, intrauterine morbidity of fatty acid oxidation defects has widely been denied. We report the intrauterine development of severe cardiomyopathy in a child with mitochondrial trifunctional protein deficiency after 27 weeks of gestation. The child was born at 31 weeks of gestation and died on day 3 of life. Severe cardiac mitochondrial proliferation was observed. Molecular analysis of both TFP genes was performed and confirmed a homozygous mutation in the TFP alpha-subunit introducing a stop codon at amino acid position 256 (g.871C>T, p.R256X). Despite severe intrauterine decompensation in our patient, no HELLP-syndrome or acute fatty liver of pregnancy was observed in the mother. In the pathogenesis of maternal HELLP-syndrome, toxic effects of accumulating long-chain hydroxy-acyl-CoAs or long-chain hydroxy-acylcarnitines are suspected. In our patient, acylcarnitine analysis on day 2 of life during severest metabolic decompensation did not reveal massive accumulation of long-chain hydroxy-acylcarnitines in blood, suggesting other pathogenic factors than toxic effects. The most important pathogenic mechanism for the development of intrauterine cardiomyopathy appears to be significant cardiac energy deficiency. In conclusion, our report implicates that fatty acid oxidation does play a significant role during intrauterine development with special regard to the heart. Severe cardiac mitochondrial proliferation in TFP deficiency suggests pathophysiologically relevant energy deficiency in this condition.
Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid-derived hydrocarbons.
Zhang, Yiming; Nielsen, Jens; Liu, Zihe
2018-06-05
Fatty acid-derived hydrocarbons attract increasing attention as biofuels due to their immiscibility with water, high-energy content, low freezing point, and high compatibility with existing refineries and end-user infrastructures. Yeast Saccharomyces cerevisiae has advantages for production of fatty acid-derived hydrocarbons as its native routes toward fatty acid synthesis involve only a few reactions that allow more efficient conversion of carbon substrates. Here we describe major biosynthetic pathways of fatty acid-derived hydrocarbons in yeast, and summarize key metabolic engineering strategies, including enhancing precursor supply, eliminating competing pathways, and expressing heterologous pathways. With recent advances in yeast production of fatty acid-derived hydrocarbons, our review identifies key research challenges and opportunities for future optimization, and concludes with perspectives and outlooks for further research directions. © 2018 Wiley Periodicals, Inc.
Montanari, Arianna; De Luca, Cristina; Di Micco, Patrizio; Morea, Veronica; Frontali, Laura; Francisci, Silvia
2011-01-01
Previous work has demonstrated the usefulness of the yeast model to investigate the molecular mechanisms underlying defects due to base substitutions in mitochondrial tRNA genes, and to identify suppressing molecules endowed with potential clinical relevance. The present paper extends these investigations to two human equivalent yeast mutations located at positions 32 and 33 in the anticodon loop of tRNAIle. Notwithstanding the proximity of the two T>C base substitutions, the effects of these mutations have been found to be quite different in yeast, as they are in human. The T32C substitution has a very severe effect in yeast, consisting in a complete inhibition of growth on nonfermentable substrates. Conversely, respiratory defects caused by the T33C mutation could only be observed in a defined genetic context. Analyses of available sequences and selected tRNA three-dimensional structures were performed to provide explanations for the different behavior of these adjacent mutations. Examination of the effects of previously identified suppressors demonstrated that overexpression of the TUF1 gene did not rescue the defective phenotypes determined by either mutation, possibly as a consequence of the lack of interactions between EF-Tu and the tRNA anticodon arm in known structures. On the contrary, both the cognate IleRS and the noncognate LeuRS and ValRS are endowed with suppressing activities toward both mutations. This allows us to extend to the tRNAIle mutants the cross-suppression activity of aminoacyl-tRNA synthetases previously demonstrated for tRNALeu and tRNAVal mutants. PMID:21914842
Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G.
2013-01-01
Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease. PMID:24161390
FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export
Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin
2015-01-01
Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734
Lalia, Antigoni Z; Dasari, Surendra; Robinson, Matthew M; Abid, Hinnah; Morse, Dawn M; Klaus, Katherine A; Lanza, Ian R
2017-04-01
Omega-3 polyunsaturated fatty acids (n3-PUFA) are recognized for their anti-inflammatory effects and may be beneficial in the context of sarcopenia. We determined the influence of n3-PUFA on muscle mitochondrial physiology and protein metabolism in older adults. Twelve young (18-35 years) and older (65-85 years) men and women were studied at baseline. Older adults were studied again following n3-PUFA supplementation (3.9g/day, 16 weeks). Muscle biopsies were used to evaluate respiratory capacity (high resolution respirometry) and oxidant emissions (spectrofluorometry) in isolated mitochondria. Maximal respiration was significantly lower in older compared to young. n3-PUFA did not change respiration, but significantly reduced oxidant emissions. Participants performed a single bout of resistance exercise, followed by biopsies at 15 and 18 hours post exercise. Several genes involved in muscle protein turnover were significantly altered in older adults at baseline and following exercise, yet muscle protein synthesis was similar between age groups under both conditions. Following n3-PUFA supplementation, mixed muscle, mitochondrial, and sarcoplasmic protein synthesis rates were increased in older adults before exercise. n3-PUFA increased post-exercise mitochondrial and myofibrillar protein synthesis in older adults. These results demonstrate that n3-PUFA reduce mitochondrial oxidant emissions, increase postabsorptive muscle protein synthesis, and enhance anabolic responses to exercise in older adults.
Lalia, Antigoni Z.; Dasari, Surendra; Robinson, Matthew M.; Abid, Hinnah; Morse, Dawn M.; Klaus, Katherine A.; Lanza, Ian R.
2017-01-01
Omega-3 polyunsaturated fatty acids (n3-PUFA) are recognized for their anti-inflammatory effects and may be beneficial in the context of sarcopenia. We determined the influence of n3-PUFA on muscle mitochondrial physiology and protein metabolism in older adults. Twelve young (18-35 years) and older (65-85 years) men and women were studied at baseline. Older adults were studied again following n3-PUFA supplementation (3.9g/day, 16 weeks). Muscle biopsies were used to evaluate respiratory capacity (high resolution respirometry) and oxidant emissions (spectrofluorometry) in isolated mitochondria. Maximal respiration was significantly lower in older compared to young. n3-PUFA did not change respiration, but significantly reduced oxidant emissions. Participants performed a single bout of resistance exercise, followed by biopsies at 15 and 18 hours post exercise. Several genes involved in muscle protein turnover were significantly altered in older adults at baseline and following exercise, yet muscle protein synthesis was similar between age groups under both conditions. Following n3-PUFA supplementation, mixed muscle, mitochondrial, and sarcoplasmic protein synthesis rates were increased in older adults before exercise. n3-PUFA increased post-exercise mitochondrial and myofibrillar protein synthesis in older adults. These results demonstrate that n3-PUFA reduce mitochondrial oxidant emissions, increase postabsorptive muscle protein synthesis, and enhance anabolic responses to exercise in older adults. PMID:28379838
Fricova, Dominika; Valach, Matus; Farkas, Zoltan; Pfeiffer, Ilona; Kucsera, Judit; Tomaska, Lubomir; Nosek, Jozef
2010-01-01
As a part of our initiative aimed at a large-scale comparative analysis of fungal mitochondrial genomes, we determined the complete DNA sequence of the mitochondrial genome of the yeast Candida subhashii and found that it exhibits a number of peculiar features. First, the mitochondrial genome is represented by linear dsDNA molecules of uniform length (29 795 bp), with an unusually high content of guanine and cytosine residues (52.7 %). Second, the coding sequences lack introns; thus, the genome has a relatively compact organization. Third, the termini of the linear molecules consist of long inverted repeats and seem to contain a protein covalently bound to terminal nucleotides at the 5′ ends. This architecture resembles the telomeres in a number of linear viral and plasmid DNA genomes classified as invertrons, in which the terminal proteins serve as specific primers for the initiation of DNA synthesis. Finally, although the mitochondrial genome of C. subhashii contains essentially the same set of genes as other closely related pathogenic Candida species, we identified additional ORFs encoding two homologues of the family B protein-priming DNA polymerases and an unknown protein. The terminal structures and the genes for DNA polymerases are reminiscent of linear mitochondrial plasmids, indicating that this genome architecture might have emerged from fortuitous recombination between an ancestral, presumably circular, mitochondrial genome and an invertron-like element. PMID:20395267
Rambold, Angelika S; Cohen, Sarah; Lippincott-Schwartz, Jennifer
2015-03-23
Fatty acids (FAs) provide cellular energy under starvation, yet how they mobilize and move into mitochondria in starved cells, driving oxidative respiration, is unclear. Here, we clarify this process by visualizing FA trafficking with a fluorescent FA probe. The labeled FA accumulated in lipid droplets (LDs) in well-fed cells but moved from LDs into mitochondria when cells were starved. Autophagy in starved cells replenished LDs with FAs, increasing LD number over time. Cytoplasmic lipases removed FAs from LDs, enabling their transfer into mitochondria. This required mitochondria to be highly fused and localized near LDs. When mitochondrial fusion was prevented in starved cells, FAs neither homogeneously distributed within mitochondria nor became efficiently metabolized. Instead, FAs reassociated with LDs and fluxed into neighboring cells. Thus, FAs engage in complex trafficking itineraries regulated by cytoplasmic lipases, autophagy, and mitochondrial fusion dynamics, ensuring maximum oxidative metabolism and avoidance of FA toxicity in starved cells. Copyright © 2015 Elsevier Inc. All rights reserved.
Ragnini, A; Fukuhara, H
1989-01-01
In the yeast Kluyveromyces lactis, mutations affecting mitochondrial functions are often highly unstable. In order to understand the basis of this genetic instability, we examined the case of an oligomycin resistant mutant. When the mutant was grown in the absence of the drug, the resistance was rapidly lost. This character showed a typical cytoplasmic inheritance. The unstable resistance was found to be associated with the presence of a repetitive DNA in which the repeating unit was a specific segment of the mitochondrial DNA. The amplified molecules were co-replicating with the wild type genome in the mutant cells. The spontaneous loss of the drug resistance was accompanied by the disappearance of the amplified DNA. The repetitive sequence came from a 405 base-pair segment immediately downstream of a cluster of two transfer RNA genes (threonyl 2 and glutamyl). Modified processing of these tRNAs was detected in the mutant. A possible mechanism by which these events could lead to drug resistance is discussed. Images PMID:2780315
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
Gerhold, Joachim M.; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan
2014-01-01
Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. PMID:24951592
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
English, Jane A.; Harauma, Akiko; Föcking, Melanie; Wynne, Kieran; Scaife, Caitriona; Cagney, Gerard; Moriguchi, Toru; Cotter, David R.
2013-01-01
Omega-3 fatty acid (n-3 FA) deficiency is an environmental risk factor for schizophrenia, yet characterization of the consequences of deficiency at the protein level in the brain is limited. We aimed to identify the protein pathways disrupted as a consequence of chronic n-3 deficiency in the hippocampus of mice. Fatty acid analysis of the hippocampus following chronic dietary deficiency revealed a 3-fold decrease (p < 0.001) in n-3 FA levels. Label free LC-MS/MS analysis identified and profiled 1008 proteins, of which 114 were observed to be differentially expressed between n-3 deficient and control groups (n = 8 per group). The cellular processes that were most implicated were neuritogenesis, endocytosis, and exocytosis, while specific protein pathways that were most significantly dysregulated were mitochondrial dysfunction and clathrin mediated endocytosis (CME). In order to characterize whether these processes and pathways are ones influenced by antipsychotic medication, we used LC-MS/MS to test the differential expression of these 114 proteins in the hippocampus of mice chronically treated with the antipsychotic agent haloperidol. We observed 23 of the 114 proteins to be differentially expressed, 17 of which were altered in the opposite direction to that observed following n-3 deficiency. Overall, our findings point to disturbed synaptic function, neuritogenesis, and mitochondrial function as a consequence of dietary deficiency in n-3 FA. This study greatly aids our understanding of the molecular mechanism by which n-3 deficiency impairs normal brain function, and provides clues as to how n-3 FA exert their therapeutic effect in early psychosis. PMID:24194745
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
Hu, Jiamiao; Kyrou, Ioannis; Tan, Bee K; Dimitriadis, Georgios K; Ramanjaneya, Manjunath; Tripathi, Gyanendra; Patel, Vanlata; James, Sean; Kawan, Mohamed; Chen, Jing; Randeva, Harpal S
2016-05-01
Short-chain fatty acids play crucial roles in a range of physiological functions. However, the effects of short-chain fatty acids on brown adipose tissue have not been fully investigated. We examined the role of acetate, a short-chain fatty acid formed by fermentation in the gut, in the regulation of brown adipocyte metabolism. Our results show that acetate up-regulates adipocyte protein 2, peroxisomal proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1 expression and affects the morphological changes of brown adipocytes during adipogenesis. Moreover, an increase in mitochondrial biogenesis was observed after acetate treatment. Acetate also elicited the activation of ERK and cAMP response element-binding protein, and these responses were sensitive to G(i/o)-type G protein inactivator, Gβγ-subunit inhibitor, phospholipase C inhibitor, and MAPK kinase inhibitor, indicating a role for the G(i/o)βγ/phospholipase C/protein kinase C/MAPK kinase signaling pathway in these responses. These effects of acetate were mimicked by treatment with 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide, a synthetic G protein-coupled receptor 43 (GPR43) agonist and were impaired in GPR43 knockdown cells. Taken together, our results indicate that acetate may have important physiological roles in brown adipocytes through the activation of GPR43.
de Velasco, Patricia C; Chicaybam, Gustavo; Ramos-Filho, Dionizio M; Dos Santos, Raísa M A R; Mairink, Caroline; Sardinha, Fátima L C; El-Bacha, Tatiana; Galina, Antonio; Tavares-do-Carmo, Maria das Graças
2017-07-01
The quality of dietary lipids in the maternal diet can programme the offspring to diseases in later life. We investigated whether the maternal intake of palm oil or interesterified fat, substitutes for trans-unsaturated fatty acids (FA), induces metabolic changes in the adult offspring. During pregnancy and lactation, C57BL/6 female mice received normolipidic diets containing partially hydrogenated vegetable fat rich in trans-unsaturated fatty acids (TG), palm oil (PG), interesterified fat (IG) or soyabean oil (CG). After weaning, male offspring from all groups received the control diet until day 110. Plasma glucose and TAG and liver FA profiles were ascertained. Liver mitochondrial function was accessed with high-resolution respirometry by measuring VO2, fluorimetry for detection of hydrogen peroxide (H2O2) production and mitochondrial Ca2+ uptake. The results showed that the IG offspring presented a 20 % increase in plasma glucose and both the IG and TG offspring presented a 2- and 1·9-fold increase in TAG, respectively, when compared with CG offspring. Liver MUFA and PUFA contents decreased in the TG and IG offspring when compared with CG offspring. Liver MUFA content also decreased in the PG offspring. These modifications in FA composition possibly affected liver mitochondrial function, as respiration was impaired in the TG offspring and H2O2 production was higher in the IG offspring. In addition, mitochondrial Ca2+ retention capacity was reduced by approximately 40 and 55 % in the TG and IG offspring, respectively. In conclusion, maternal consumption of trans-unsaturated and interesterified fat affected offspring health by compromising mitochondrial bioenergetics and lipid metabolism in the liver.
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
Evaluation of the Microbial Identification System for identification of clinically isolated yeasts.
Crist, A E; Johnson, L M; Burke, P J
1996-01-01
The Microbial Identification System (MIS; Microbial ID, Inc., Newark, Del.) was evaluated for the identification of 550 clinically isolated yeasts. The organisms evaluated were fresh clinical isolates identified by methods routinely used in our laboratory (API 20C and conventional methods) and included Candida albicans (n = 294), C. glabrata (n = 145), C. tropicalis (n = 58), C. parapsilosis (n = 33), and other yeasts (n = 20). In preparation for fatty acid analysis, yeasts were inoculated onto Sabouraud dextrose agar and incubated at 28 degrees C for 24 h. Yeasts were harvested, saponified, derivatized, and extracted, and fatty acid analysis was performed according to the manufacturer's instructions. Fatty acid profiles were analyzed, and computer identifications were made with the Yeast Clinical Library (database version 3.8). Of the 550 isolates tested, 374 (68.0%) were correctly identified to the species level, with 87 (15.8%) being incorrectly identified and 89 (16.2%) giving no identification. Repeat testing of isolates giving no identification resulted in an additional 18 isolates being correctly identified. This gave the MIS an overall identification rate of 71.3%. The most frequently misidentified yeast was C. glabrata, which was identified as Saccharomyces cerevisiae 32.4% of the time. On the basis of these results, the MIS, with its current database, does not appear suitable for the routine identification of clinically important yeasts. PMID:8880489
Singh, Purnima; Singh, Shiv M; Tsuji, Masaharu; Prasad, Gandham S; Hoshino, Tamotsu
2014-02-01
A psychrophilic yeast species was isolated from glacier cryoconite holes of Svalbard. Nucleotide sequences of the strains were studied using D1/D2 domain, ITS region and partial sequences of mitochondrial cytochrome b gene. The strains belonged to a clade of psychrophilic yeasts, but showed marked differences from related species in the D1/D2 domain and biochemical characters. Effects of temperature, salt and media on growth of the cultures were also studied. Screening of the cultures for amylase, cellulase, protease, lipase, urease and catalase activities was carried out. The strains expressed high amylase and lipase activities. Freeze tolerance ability of the isolates indicated the formation of unique hexagonal ice crystal structures due to presence of 'antifreeze proteins' (AFPs). FAME analysis of cultures showed a unique trend of increase in unsaturated fatty acids with decrease in temperature. The major fatty acids recorded were oleic acid, linoleic acid, linolenic acid, palmitic acid, stearic acid, myristic acid and pentadecanoic acid. Based on sequence data and, physiological and morphological properties of the strains, we propose a novel species, Rhodotorula svalbardensis and designate strains MLB-I (CCP-II) and CRY-YB-1 (CBS 12863, JCM 19699, JCM 19700, MTCC 10952) as its type strains (Etymology: sval.bar.den'sis. N.L. fem. adj. svalbardensis pertaining to Svalbard). Copyright © 2014 Elsevier Inc. All rights reserved.
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.
Lipoic acid metabolism and mitochondrial redox regulation.
Solmonson, Ashley D; DeBerardinis, Ralph J
2017-11-30
Lipoic acid is an essential cofactor for mitochondrial metabolism and is synthesized de novo using intermediates from mitochondrial fatty acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a critical role in stabilizing and regulating these multi-enzyme complexes. Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety. Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity and coordination of fuel metabolism. Lipoic acid is an essential cofactor for mitochondrial metabolism and is synthesized de novo using intermediates from mitochondrial fatty acid synthesis type II, S-adenosylmethionine and iron-sulfur clusters. This cofactor is required for catalysis by multiple mitochondrial 2-ketoacid dehydrogenase complexes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase. Lipoic acid also plays a critical role in stabilizing and regulating these multi-enzyme complexes. Many of these dehydrogenases are regulated by reactive oxygen species, mediated through the disulfide bond of the prosthetic lipoyl moiety. Collectively, its functions explain why lipoic acid is required for cell growth, mitochondrial activity and coordination of fuel metabolism. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.
DNA polymerase γ and disease: what we have learned from yeast
Lodi, Tiziana; Dallabona, Cristina; Nolli, Cecilia; Goffrini, Paola; Donnini, Claudia; Baruffini, Enrico
2015-01-01
Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1. PMID:25852747
Kitagaki, Hiroshi; Kitamoto, Katsuhiko
2013-01-01
Sake is an alcoholic beverage of Japan, with a tradition lasting more than 1,300 years; it is produced from rice and water by fermenting with the koji mold Aspergillus oryzae and sake yeast Saccharomyces cerevisiae. Breeding research on sake yeasts was originally developed in Japan by incorporating microbiological and genetic research methodologies adopted in other scientific areas. Since the advent of a genetic paradigm, isolation of yeast mutants has been a dominant approach for the breeding of favorable sake yeasts. These sake yeasts include (a) those that do not form foams (produced by isolating a mutant that does not stick to foams, thus decreasing the cost of sake production); (b) those that do not produce urea, which leads to the formation of ethyl carbamate, a possible carcinogen (isolated by positive selection in a canavanine-, arginine-, and ornithine-containing medium); (c) those that produce an increased amount of ethyl caproate, an apple-like flavor (produced by isolating a mutant resistant to cerulenin, an inhibitor of fatty-acid synthesis); and (d) those that produce a decreased amount of pyruvate (produced by isolating a mutant resistant to an inhibitor of mitochondrial transport, thus decreasing the amount of diacetyl). Given that sake yeasts perform sexual reproduction, sporulation and mating are potent approaches for their breeding. Recently, the genome sequences of sake yeasts have been determined and made publicly accessible. By utilizing this information, the quantitative trait loci (QTLs) for the brewing characteristics of sake yeasts have been identified, which paves a way to DNA marker-assisted selection of the mated strains. Genetic engineering technologies for experimental yeast strains have recently been established by academic groups, and these technologies have also been applied to the breeding of sake yeasts. Sake yeasts whose genomes have been modified with these technologies correspond to genetically modified organisms (GMOs
Tesson, Christelle; Nawara, Magdalena; Salih, Mustafa A.M.; Rossignol, Rodrigue; Zaki, Maha S.; Al Balwi, Mohammed; Schule, Rebecca; Mignot, Cyril; Obre, Emilie; Bouhouche, Ahmed; Santorelli, Filippo M.; Durand, Christelle M.; Oteyza, Andrés Caballero; El-Hachimi, Khalid H.; Al Drees, Abdulmajeed; Bouslam, Naima; Lamari, Foudil; Elmalik, Salah A.; Kabiraj, Mohammad M.; Seidahmed, Mohammed Z.; Esteves, Typhaine; Gaussen, Marion; Monin, Marie-Lorraine; Gyapay, Gabor; Lechner, Doris; Gonzalez, Michael; Depienne, Christel; Mochel, Fanny; Lavie, Julie; Schols, Ludger; Lacombe, Didier; Yahyaoui, Mohamed; Al Abdulkareem, Ibrahim; Zuchner, Stephan; Yamashita, Atsushi; Benomar, Ali; Goizet, Cyril; Durr, Alexandra; Gleeson, Joseph G.; Darios, Frederic; Brice, Alexis; Stevanin, Giovanni
2012-01-01
Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function. PMID:23176821
Gerhold, Joachim M; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan
2014-08-15
Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Ubiquitination of specific mitochondrial matrix proteins
DOE Office of Scientific and Technical Information (OSTI.GOV)
Lehmann, Gilad; Ziv, Tamar; Braten, Ori
2016-06-17
Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinatedmore » proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.« less
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
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.
2014-01-01
Background Inborn enzyme defects of mitochondrial fatty acid beta-oxidation (FAO) form a large group of genetic disorders associated to variable clinical presentations ranging from life-threatening pediatric manifestations up to milder late onset phenotypes, including myopathy. Very few candidate drugs have been identified in this group of disorders. Resveratrol (RSV) is a natural polyphenol with anti-oxidant and anti-inflammatory effects, recently shown to have beneficial metabolic properties in mice models. Our study explores its possible effects on FAO and mitochondrial energy metabolism in human cells, which are still very little documented. Methods Using cells from controls and from patients with Carnitine Palmitoyl Transferase 2 (CPT2) or Very Long Chain AcylCoA Dehydrogenase (VLCAD) deficiency we characterized the metabolic effects of RSV, RSV metabolites, and other stilbenes. We also focused on analysis of RSV uptake, and on the effects of low RSV concentrations, considering the limited bioavailability of RSV in vivo. Results Time course of RSV accumulation in fibroblasts over 48 h of treatment were consistent with the resulting stimulation or correction of FAO capacities. At 48 h, half maximal and maximal FAO stimulations were respectively achieved for 37,5 microM (EC50) and 75 microM RSV, but we found that serum content of culture medium negatively modulated RSV uptake and FAO induction. Indeed, decreasing serum from 12% to 3% led to shift EC50 from 37,5 to 13 microM, and a 2.6-3.6-fold FAO stimulation was reached with 20 microM RSV at 3% serum, that was absent at 12% serum. Two other stilbenes often found associated with RSV, i.e. cis- RSV and piceid, also triggered significant FAO up-regulation. Resveratrol glucuro- or sulfo- conjugates had modest or no effects. In contrast, dihydro-RSV, one of the most abundant circulating RSV metabolites in human significantly stimulated FAO (1.3-2.3-fold). Conclusions This study provides the first compared data on
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.
Guo, Yongzheng; Wang, Zhen; Qin, Xinghua; Xu, Jie; Hou, Zuoxu; Yang, Hongyan; Mao, Xuechao; Xing, Wenjuan; Li, Xiaoliang; Zhang, Xing; Gao, Feng
2018-06-01
Heart failure (HF) is characterized by reduced fatty acid (FA) utilization associated with mitochondrial dysfunction. Recent evidence has shown that enhancing FA utilization may provide cardioprotection against HF. Our aim was to investigate the effects and the underlying mechanisms of cardiac FA utilization on cardiac function in response to pressure overload. Transverse aortic constriction (TAC) was used in C57 mice to establish pressure overload-induced HF. TAC mice fed on a high fat diet (HFD) exhibited increased cardiac FA utilization and improved cardiac function and survival compared with those on control diet. Such cardioprotection could also be provided by cardiac-specific overexpression of CD36. Notably, both HFD and CD36 overexpression attenuated mitochondrial fragmentation and improved mitochondrial function in the failing heart. Pressure overload decreased ATP-dependent metalloprotease (YME1L) expression and induced the proteolytic cleavage of the dynamin-like guanosine triphosphatase OPA1 as a result of suppressed FA utilization. Enhancing FA utilization upregulated YME1L expression and subsequently rebalanced OPA1 processing, resulting in restoration of mitochondrial morphology in the failing heart. In addition, cardiac-specific overexpression of YME1L exerted similar cardioprotective effects against HF to those provided by HFD or CD36 overexpression. These findings demonstrate that enhancing FA utilization ameliorates mitochondrial fragmentation and cardiac dysfunction via rebalancing OPA1 processing in pressure overload-induced HF, suggesting a unique metabolic intervention approach to improving cardiac functions in HF.
Oleaginous yeasts: Promising platforms for the production of oleochemicals and biofuels.
Adrio, José L
2017-09-01
Oleaginous yeasts have a unique physiology that makes them the best suited hosts for the production of lipids, oleochemicals, and diesel-like fuels. Their high lipogenesis, capability of growing on many different carbon sources (including lignocellulosic sugars), easy large-scale cultivation, and an increasing number of genetic tools are some of the advantages that have encouraged their use to develop sustainable processes. This mini-review summarizes the metabolic engineering strategies developed in oleaginous yeasts within the last 2 years to improve process metrics (titer, yield, and productivity) for the production of lipids, free fatty acids, fatty acid-based chemicals (e.g., fatty alcohols, fatty acid ethyl esters), and alkanes. During this short period of time, tremendous progress has been made in Yarrowia lipolytica, the model oleaginous yeast, which has been engineered to improve lipid production by different strategies including increasing lipogenic pathway flux and biosynthetic precursors, and blocking degradation pathways. Moreover, remarkable advances have also been reported in Rhodosporidium toruloides and Lipomyces starkey despite the limited genetic tools available for these two very promising hosts. Biotechnol. Bioeng. 2017;114: 1915-1920. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
Dynamin-Related Protein 1 and Mitochondrial Fragmentation in Neurodegenerative Diseases
Reddy, P. Hemachandra; Reddy, Tejaswini P.; Manczak, Maria; Calkins, Marcus J.; Shirendeb, Ulziibat; Mao, Peizhong
2010-01-01
The purpose of this article is to review the recent developments of abnormal mitochondrial dynamics, mitochondrial fragmentation, and neuronal damage in neurodegenerative diseases, including Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. The GTPase family of proteins, including fission proteins, dynamin related protein 1 (Drp1), mitochondrial fission 1 (Fis1), and fusion proteins (Mfn1, Mfn2 and Opa1) are essential to maintain mitochondrial fission and fusion balance, and to provide necessary adenosine triphosphate to neurons. Among these, Drp1 is involved in several important aspects of mitochondria, including shape, size, distribution, remodeling, and maintenance of X in mammalian cells. In addition, recent advancements in molecular, cellular, electron microscopy, and confocal imaging studies revealed that Drp1 is associated with several cellular functions, including mitochondrial and peroxisomal fragmentation, phosphorylation, SUMOylation, ubiquitination, and cell death. In the last two decades, tremendous progress has been made in researching mitochondrial dynamics, in yeast, worms, and mammalian cells; and this research has provided evidence linking Drp1 to neurodegenerative diseases. Researchers in the neurodegenerative disease field are beginning to recognize the possible involvement of Drp1 in causing mitochondrial fragmentation and abnormal mitochondrial dynamics in neurodegenerative diseases. This article summarizes research findings relating Drp1 to mitochondrial fission and fusion, in yeast, worms, and mammals. Based on findings from the Reddy laboratory and others’, we propose that mutant proteins of neurodegenerative diseases, including AD, PD, HD, and ALS, interact with Drp1, activate mitochondrial fission machinery, fragment mitochondria excessively, and impair mitochondrial transport and mitochondrial dynamics, ultimately causing mitochondrial dysfunction and neuronal damage. PMID:21145355
DOE Office of Scientific and Technical Information (OSTI.GOV)
Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi
2010-11-26
Research highlights: {yields} POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. {yields} Deletion of POR1 caused growth defects on fatty acids. {yields} {Delta}por1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y.more » lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in {beta}-oxidation and peroxisome proliferation by oleate was distinctly diminished in the {Delta}por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.« less
Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission
Koirala, Sajjan; Guo, Qian; Kalia, Raghav; Bui, Huyen T.; Eckert, Debra M.; Frost, Adam; Shaw, Janet M.
2013-01-01
Mitochondrial fission is mediated by the dynamin-related GTPases Dnm1/Drp1 (yeast/mammals), which form spirals around constricted sites on mitochondria. Additional membrane-associated adaptor proteins (Fis1, Mdv1, Mff, and MiDs) are required to recruit these GTPases from the cytoplasm to the mitochondrial surface. Whether these adaptors participate in both GTPase recruitment and membrane scission is not known. Here we use a yeast strain lacking all fission proteins to identify the minimal combinations of GTPases and adaptors sufficient for mitochondrial fission. Although Fis1 is dispensable for fission, membrane-anchored Mdv1, Mff, or MiDs paired individually with their respective GTPases are sufficient to divide mitochondria. In addition to their role in Drp1 membrane recruitment, MiDs coassemble with Drp1 in vitro. The resulting heteropolymer adopts a dramatically different structure with a narrower diameter than Drp1 homopolymers assembled in isolation. This result demonstrates that an adaptor protein alters the architecture of a mitochondrial dynamin GTPase polymer in a manner that could facilitate membrane constriction and severing activity. PMID:23530241
Preferential inclusion of extrachromosomal genetic elements in yeast meiotic spores.
Brewer, B J; Fangman, W L
1980-09-01
During meiosis and sporulation in the yeast Saccharomyces cerevisiae, extrachromosomal traits are efficiently transmitted to haploid spores. Although the pattern of inheritance of chromosomal traits reflects the mechanism of regular chromosomal segregation in meiosis, it is not known what processes are reflected by the efficient inheritance of extrachromosomal traits. Because extrachromosomal genetic elements in yeast are present in multiple copies, perpetuation of an extrachromosomal trait could occur by the passive envelopment of a subset of copies or by an active sequestering of all or a subset of copies within the four spores. We show that only subsets of the four extrachromosomal nucleic acids commonly found in yeast are transmitted through meiosis--55% of mitochondrial DNA copies, 82% of the 2-micron DNA plasmids, and about 70% of the L and M double-stranded RNAs. However, electron micrographs of serial sections through yeast asci indicate that the four spore enclose only 30% of the total ascus material. Thus these extrachromosomal elements are preferentially included within the spores, indicating that their inheritance is not a random process. Transmission of mitochondrial DNA can be accounted for by the observed enclosure of 52% of the mitochondrial volume within the spores. The high transmission frequencies of the double-stranded RNAs (which exist as virus-like particles in the cytoplasm) and 2-micron DNA must indicate that either these nucleic acids are actively recruited from the cytoplasm by some mechanism or they are associated in some way with the nucleus during meiosis.
Francis, Brian R; White, Karen H; Thorsness, Peter E
2007-04-01
ATP1-111, a suppressor of the slow-growth phenotype of yme1Delta lacking mitochondrial DNA is due to the substitution of phenylalanine for valine at position 111 of the alpha-subunit of mitochondrial ATP synthase (Atp1p in yeast). The suppressing activity of ATP1-111 requires intact beta (Atp2p) and gamma (Atp3p) subunits of mitochondrial ATP synthase, but not the stator stalk subunits b (Atp4p) and OSCP (Atp5p). ATP1-111 and other similarly suppressing mutations in ATP1 and ATP3 increase the growth rate of wild-type strains lacking mitochondrial DNA. These suppressing mutations decrease the growth rate of yeast containing an intact mitochondrial chromosome on media requiring oxidative phosphorylation, but not when grown on fermentable media. Measurement of chronological aging of yeast in culture reveals that ATP1 and ATP3 suppressor alleles in strains that contain mitochondrial DNA are longer lived than the isogenic wild-type strain. In contrast, the chronological life span of yeast cells lacking mitochondrial DNA and containing these mutations is shorter than that of the isogenic wild-type strain. Spore viability of strains bearing ATP1-111 is reduced compared to wild type, although ATP1-111 enhances the survival of spores that lacked mitochondrial DNA.
Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Alvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A
2012-11-01
MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. According to our results, supplementation with riboflavin or coenzyme Q(10) effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
Garrido-Maraver, Juan; Cordero, Mario D; Moñino, Irene Domínguez; Pereira-Arenas, Sheila; Lechuga-Vieco, Ana V; Cotán, David; De la Mata, Mario; Oropesa-Ávila, Manuel; De Miguel, Manuel; Bautista Lorite, Juan; Rivas Infante, Eloy; Álvarez-Dolado, Manuel; Navas, Plácido; Jackson, Sandra; Francisci, Silvia; Sánchez-Alcázar, José A
2012-01-01
BACKGROUND AND PURPOSE MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors. EXPERIMENTAL APPROACH We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease. KEY RESULTS According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models. CONCLUSIONS AND IMPLICATIONS Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment. PMID:22747838
Rodríguez-Enríquez, Sara; Hernández-Esquivel, Luz; Marín-Hernández, Alvaro; El Hafidi, Mohammed; Gallardo-Pérez, Juan Carlos; Hernández-Reséndiz, Ileana; Rodríguez-Zavala, José S; Pacheco-Velázquez, Silvia C; Moreno-Sánchez, Rafael
2015-08-01
Oxidative phosphorylation (OxPhos) is functional and sustains tumor proliferation in several cancer cell types. To establish whether mitochondrial β-oxidation of free fatty acids (FFAs) contributes to cancer OxPhos functioning, its protein contents and enzyme activities, as well as respiratory rates and electrical membrane potential (ΔΨm) driven by FFA oxidation were assessed in rat AS-30D hepatoma and liver (RLM) mitochondria. Higher protein contents (1.4-3 times) of β-oxidation (CPT1, SCAD) as well as proteins and enzyme activities (1.7-13-times) of Krebs cycle (KC: ICD, 2OGDH, PDH, ME, GA), and respiratory chain (RC: COX) were determined in hepatoma mitochondria vs. RLM. Although increased cholesterol content (9-times vs. RLM) was determined in the hepatoma mitochondrial membranes, FFAs and other NAD-linked substrates were oxidized faster (1.6-6.6 times) by hepatoma mitochondria than RLM, maintaining similar ΔΨm values. The contents of β-oxidation, KC and RC enzymes were also assessed in cells. The mitochondrial enzyme levels in human cervix cancer HeLa and AS-30D cells were higher than those observed in rat hepatocytes whereas in human breast cancer biopsies, CPT1 and SCAD contents were lower than in human breast normal tissue. The presence of CPT1 and SCAD in AS-30D mitochondria and HeLa cells correlated with an active FFA utilization in HeLa cells. Furthermore, the β-oxidation inhibitor perhexiline blocked FFA utilization, OxPhos and proliferation in HeLa and other cancer cells. In conclusion, functional mitochondria supported by FFA β-oxidation are essential for the accelerated cancer cell proliferation and hence anti-β-oxidation therapeutics appears as an alternative promising approach to deter malignant tumor growth. Copyright © 2015 Elsevier Ltd. All rights reserved.
Mitochondrial lipids in Bufo arenarum full-grown oocytes.
Gili, Valeria; Alonso, Telma S
2004-05-01
Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.
van Weeghel, Michel; te Brinke, Heleen; van Lenthe, Henk; Kulik, Wim; Minkler, Paul E; Stoll, Maria S K; Sass, Jörn Oliver; Janssen, Uwe; Stoffel, Wilhelm; Schwab, K Otfried; Wanders, Ronald J A; Hoppel, Charles L; Houten, Sander M
2012-10-01
Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood β-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 μM vs. KO 0.09 μM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 μM vs. KO 0.04 μM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.
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.
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.
Feldkamp, Thorsten; Weinberg, Joel M.; Hörbelt, Markus; Von Kropff, Christina; Witzke, Oliver; Nürnberger, Jens; Kribben, Andreas
2009-01-01
Background. Proximal tubules subjected to hypoxia in vitro under conditions relevant to ischaemia in vivo develop an energetic deficit that is not corrected even after full reoxygenation. We have provided evidence that accumulation of nonesterified fatty acids (NEFA) is the primary reason for this energetic deficit. In this study, we have further investigated the mechanism for the NEFA-induced energetic deficit. Methods. Mitochondrial membrane potential (Δψ) was measured in digitonin-permeabilized, freshly isolated proximal tubules by safranin O uptake. Addition of the potassium/proton exchanger nigericin enables the determination of the mitochondrial proton motive force (Δp) and the proton gradient (ΔpH). ATP was measured luminometrically and NEFA colorimetrically. Results. Tubule ATP content was depleted after hypoxia and recovered incompletely, even after full reoxygenation. Mitochondrial safranin O uptake was decreased in proximal tubules after hypoxia and reoxygenation (H/R). This decrease was attenuated by delipidated bovine serum albumin (dBSA) or citrate. Addition of nigericin increased safranin O uptake of mitochondria in normoxic proximal tubules, but not in proximal tubules after H/R. Addition of dBSA restored the effect of nigericin to increase mitochondrial safranin O uptake. Addition of the NEFA oleate had the same impact on mitochondrial safranin O uptake as subjecting proximal tubules to H/R. Conclusion. The mechanism of the NEFA-induced energetic deficit in freshly isolated rat proximal tubules induced by H/R is characterized by impaired ATP production after full reoxygenation, impaired recovery of Δψ and Δp, abrogation of ΔpH and sensitivity to citrate, consistent with involvement of the tricarboxylate carrier. The data support the concept that protonophoric uncoupling by NEFA movement on anion carriers plays a critical role in proximal tubule mitochochondrial dysfunction after H/R. PMID:18678559
Vergnano, Marta; Wan, Chris
2017-01-01
ABSTRACT We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. PMID:28743817
Signal presequences increase mitochondrial permeability and open the multiple conductance channel.
Kushnareva, Y E; Campo, M L; Kinnally, K W; Sokolove, P M
1999-06-01
We have reported that the signal presequence of cytochrome oxidase subunit IV from Neurospora crassa increases the permeability of isolated rat liver mitochondria [P. M. Sokolove and K. W. Kinnally (1996) Arch. Biochem. Biophys. 336, 69] and regulates the behavior of the mutiple conductance channel (MCC) of yeast inner mitochondrial membrane [T. A. Lohret and K. W. Kinnally (1995) J. Biol. Chem. 270, 15950]. Here we examine in greater detail the action of a number of mitochondrial presequences from various sources and of several control peptides on the permeability of isolated rat liver mitochondria and on MCC activity monitored via patch-clamp techniques in both mammalian mitoplasts and a reconstituted yeast system. The data indicate that the ability to alter mitochondrial permeability is a property of most, but not all, signal peptides. Furthermore, it is clear that, although signal peptides are characterized by positive charge and the ability to form amphiphilic alpha helices, these two characteristics are not sufficient to guarantee mitochondrial effects. Finally, the results reveal a strong correlation between peptide effects on the permeability of isolated mitochondria and on MCC activity: peptides that induced swelling of mouse and rat mitochondria also activated the quiescent MCC of mouse mitoplasts and induced flickering of active MCC reconstituted from yeast mitochondrial membranes. Moreover, relative peptide efficacies were very similar for mitochondrial swelling and both types of patch-clamp experiments. We propose that patch-clamp recordings of MCC activity and the high-amplitude swelling induced by signal peptides reflect the opening of a single channel. Based on the selective responsiveness of that channel to signal peptides and the dependence of its opening in isolated mitochondria on membrane potential, we further suggest that the channel is involved in the mitochondrial protein import process. Copyright 1999 Academic Press.
Role of 3-Hydroxy Fatty Acid-Induced Hepatic Lipotoxicity in Acute Fatty Liver of Pregnancy
Ibdah, Jamal A.
2018-01-01
Acute fatty liver of pregnancy (AFLP), a catastrophic illness for both the mother and the unborn offspring, develops in the last trimester of pregnancy with significant maternal and perinatal mortality. AFLP is also recognized as an obstetric and medical emergency. Maternal AFLP is highly associated with a fetal homozygous mutation (1528G>C) in the gene that encodes for mitochondrial long-chain hydroxy acyl-CoA dehydrogenase (LCHAD). The mutation in LCHAD results in the accumulation of 3-hydroxy fatty acids, such as 3-hydroxy myristic acid, 3-hydroxy palmitic acid and 3-hydroxy dicarboxylic acid in the placenta, which are then shunted to the maternal circulation leading to the development of acute liver injury observed in patients with AFLP. In this review, we will discuss the mechanistic role of increased 3-hydroxy fatty acid in causing lipotoxicity to the liver and in inducing oxidative stress, mitochondrial dysfunction and hepatocyte lipoapoptosis. Further, we also review the role of 3-hydroxy fatty acids in causing placental damage, pancreatic islet β-cell glucolipotoxicity, brain damage, and retinal epithelial cells lipoapoptosis in patients with LCHAD deficiency. PMID:29361796
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.
Sarkar, Jaya; Poruri, Kiranmai; Boniecki, Michal T.; McTavish, Katherine K.; Martinis, Susan A.
2012-01-01
The yeast mitochondrial leucyl-tRNA synthetase (ymLeuRS) performs dual essential roles in group I intron splicing and protein synthesis. A specific LeuRS domain called CP1 is responsible for clearing noncognate amino acids that are misactivated during aminoacylation. The ymLeuRS CP1 domain also plays a critical role in splicing. Herein, the ymLeuRS CP1 domain was isolated from the full-length enzyme and was active in RNA splicing in vitro. Unlike its Escherichia coli LeuRS CP1 domain counterpart, it failed to significantly hydrolyze misaminoacylated tRNALeu. In addition and in stark contrast to the yeast domain, the editing-active E. coli LeuRS CP1 domain failed to recapitulate the splicing activity of the full-length E. coli enzyme. Although LeuRS-dependent splicing activity is rooted in an ancient adaptation for its aminoacylation activity, these results suggest that the ymLeuRS has functionally diverged to confer a robust splicing activity. This adaptation could have come at some expense to the protein's housekeeping role in aminoacylation and editing. PMID:22383526
Řezanka, Tomáš; Matoulková, Dagmar; Kolouchová, Irena; Masák, Jan; Viden, Ivan; Sigler, Karel
2015-05-01
The methods of preparation of fatty acids from brewer's yeast and its use in production of biofuels and in different branches of industry are described. Isolation of fatty acids from cell lipids includes cell disintegration (e.g., with liquid nitrogen, KOH, NaOH, petroleum ether, nitrogenous basic compounds, etc.) and subsequent processing of extracted lipids, including analysis of fatty acid and computing of biodiesel properties such as viscosity, density, cloud point, and cetane number. Methyl esters obtained from brewer's waste yeast are well suited for the production of biodiesel. All 49 samples (7 breweries and 7 methods) meet the requirements for biodiesel quality in both the composition of fatty acids and the properties of the biofuel required by the US and EU standards.
Ruggles, Kelly V.; Garbarino, Jeanne; Liu, Ying; Moon, James; Schneider, Kerry; Henneberry, Annette; Billheimer, Jeff; Millar, John S.; Marchadier, Dawn; Valasek, Mark A.; Joblin-Mills, Aidan; Gulati, Sonia; Munkacsi, Andrew B.; Repa, Joyce J.; Rader, Dan; Sturley, Stephen L.
2014-01-01
The toxic subcellular accumulation of lipids predisposes several human metabolic syndromes, including obesity, type 2 diabetes, and some forms of neurodegeneration. To identify pathways that prevent lipid-induced cell death, we performed a genome-wide fatty acid sensitivity screen in Saccharomyces cerevisiae. We identified 167 yeast mutants as sensitive to 0.5 mm palmitoleate, 45% of which define pathways that were conserved in humans. 63 lesions also impacted the status of the lipid droplet; however, this was not correlated to the degree of fatty acid sensitivity. The most liposensitive yeast strain arose due to deletion of the “ARE2 required for viability” (ARV1) gene, encoding an evolutionarily conserved, potential lipid transporter that localizes to the endoplasmic reticulum membrane. Down-regulation of mammalian ARV1 in MIN6 pancreatic β-cells or HEK293 cells resulted in decreased neutral lipid synthesis, increased fatty acid sensitivity, and lipoapoptosis. Conversely, elevated expression of human ARV1 in HEK293 cells or mouse liver significantly increased triglyceride mass and lipid droplet number. The ARV1-induced hepatic triglyceride accumulation was accompanied by up-regulation of DGAT1, a triglyceride synthesis gene, and the fatty acid transporter, CD36. Furthermore, ARV1 was identified as a transcriptional of the protein peroxisome proliferator-activated receptor α (PPARα), a key regulator of lipid homeostasis whose transcriptional targets include DGAT1 and CD36. These results implicate ARV1 as a protective factor in lipotoxic diseases due to modulation of fatty acid metabolism. In conclusion, a lipotoxicity-based genetic screen in a model microorganism has identified 75 human genes that may play key roles in neutral lipid metabolism and disease. PMID:24273168
Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T
2009-01-01
The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766
Live-Cell Imaging of Mitochondria and the Actin Cytoskeleton in Budding Yeast.
Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A
2016-01-01
Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images.
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
Yamagoshi, Ryohei; Yamamoto, Takenori; Hashimoto, Mitsuru; Sugahara, Ryohei; Shiotsuki, Takahiro; Miyoshi, Hideto; Terada, Hiroshi; Shinohara, Yasuo
2017-01-01
The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate "revertants" viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
Inflexibility of AMPK-mediated metabolic reprogramming in mitochondrial disease
Lin, Dar-Shong; Kao, Shu-Huei; Ho, Che-Sheng; Wei, Yau-Huei; Hung, Pi-Lien; Hsu, Mei-Hsin; Wu, Tsu-Yen; Wang, Tuan-Jen; Jian, Yuan-Ren; Lee, Tsung-Han; Chiang, Ming-Fu
2017-01-01
Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is most commonly caused by the A3243G mutation of mitochondrial DNA. The capacity to utilize fatty acid or glucose as a fuel source and how such dynamic switches of metabolic fuel preferences and transcriptional modulation of adaptive mechanism in response to energy deficiency in MELAS syndrome have not been fully elucidated. The fibroblasts from patients with MELAS syndrome demonstrated a remarkable deficiency of electron transport chain complexes I and IV, an impaired cellular biogenesis under glucose deprivation, and a decreased ATP synthesis. In situ analysis of the bioenergetic properties of MELAS cells demonstrated an attenuated fatty acid oxidation that concomitantly occurred with impaired mitochondrial respiration, while energy production was mostly dependent on glycolysis. Furthermore, the transcriptional modulation was mediated by the AMP-activated protein kinase (AMPK) signaling pathway, which activated its downstream modulators leading to a subsequent increase in glycolytic flux through activation of pyruvate dehydrogenase. In contrast, the activities of carnitine palmitoyltransferase for fatty acid oxidation and acetyl-CoA carboxylase-1 for fatty acid synthesis were reduced and transcriptional regulation factors for biogenesis were not altered. These results provide novel information that MELAS cells lack the adaptive mechanism to switch fuel source from glucose to fatty acid, as glycolysis rates increase in response to energy deficiency. The aberrant secondary cellular responses to disrupted metabolic homeostasis mediated by AMPK signaling pathway may contribute to the development of the clinical phenotype. PMID:29088732
Hesketh, Andy; Vergnano, Marta; Wan, Chris; Oliver, Stephen G
2017-07-25
We have engineered Saccharomyces cerevisiae to inducibly synthesize the prokaryotic signaling nucleotides cyclic di-GMP (cdiGMP), cdiAMP, and ppGpp in order to characterize the range of effects these nucleotides exert on eukaryotic cell function during bacterial pathogenesis. Synthetic genetic array (SGA) and transcriptome analyses indicated that, while these compounds elicit some common reactions in yeast, there are also complex and distinctive responses to each of the three nucleotides. All three are capable of inhibiting eukaryotic cell growth, with the guanine nucleotides exhibiting stronger effects than cdiAMP. Mutations compromising mitochondrial function and chromatin remodeling show negative epistatic interactions with all three nucleotides. In contrast, certain mutations that cause defects in chromatin modification and ribosomal protein function show positive epistasis, alleviating growth inhibition by at least two of the three nucleotides. Uniquely, cdiGMP is lethal both to cells growing by respiration on acetate and to obligately fermentative petite mutants. cdiGMP is also synthetically lethal with the ribonucleotide reductase (RNR) inhibitor hydroxyurea. Heterologous expression of the human ppGpp hydrolase Mesh1p prevented the accumulation of ppGpp in the engineered yeast and restored cell growth. Extensive in vivo interactions between bacterial signaling molecules and eukaryotic gene function occur, resulting in outcomes ranging from growth inhibition to death. cdiGMP functions through a mechanism that must be compensated by unhindered RNR activity or by functionally competent mitochondria. Mesh1p may be required for abrogating the damaging effects of ppGpp in human cells subjected to bacterial infection. IMPORTANCE During infections, pathogenic bacteria can release nucleotides into the cells of their eukaryotic hosts. These nucleotides are recognized as signals that contribute to the initiation of defensive immune responses that help the infected
Cecatto, Cristiane; Godoy, Kálita Dos Santos; da Silva, Janaína Camacho; Amaral, Alexandre Umpierrez; Wajner, Moacir
2016-10-01
The pathogenesis of the muscular symptoms and recurrent rhabdomyolysis that are commonly manifested in patients with mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiencies is still unknown. In this study we investigated the effects of the major long-chain monocarboxylic 3-hydroxylated fatty acids (LCHFA) accumulating in these disorders, namely 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, on important mitochondrial functions in rat skeletal muscle mitochondria. 3HTA and 3HPA markedly increased resting (state 4) and decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration. 3HPA provoked similar effects in permeabilized skeletal muscle fibers, validating the results obtained in purified mitochondria. Furthermore, 3HTA and 3HPA markedly diminished mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded mitochondria. Mitochondrial permeability transition (mPT) induction probably underlie these effects since they were totally prevented by cyclosporin A and ADP. In contrast, the dicarboxylic analogue of 3HTA did not alter the tested parameters. Our data strongly indicate that 3HTA and 3HPA behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers in skeletal muscle. It is proposed that these pathomechanisms disrupting mitochondrial homeostasis may be involved in the muscle alterations characteristic of MTP and LCHAD deficiencies. Copyright © 2016 Elsevier Ltd. All rights reserved.
Draft genome sequence of the oleaginous yeast Cryptococcus curvatus ATCC 20509
DOE Office of Scientific and Technical Information (OSTI.GOV)
Close, Dan; Ojumu, John O.
Cryptococcus curvatus ATCC 20509 is a commonly used nonmodel oleaginous yeast capable of converting a variety of carbon sources into fatty acids. In addition, we present the draft genome sequence of this popular organism to provide a means for more in-depth studies of its fatty acid production potential.
Draft genome sequence of the oleaginous yeast Cryptococcus curvatus ATCC 20509
Close, Dan; Ojumu, John O.
2016-11-03
Cryptococcus curvatus ATCC 20509 is a commonly used nonmodel oleaginous yeast capable of converting a variety of carbon sources into fatty acids. In addition, we present the draft genome sequence of this popular organism to provide a means for more in-depth studies of its fatty acid production potential.
Barbosa, Marina R; Sampaio, Igor H; Teodoro, Bruno G; Sousa, Thais A; Zoppi, Claudio C; Queiroz, André L; Passos, Madla A; Alberici, Luciane C; Teixeira, Felipe R; Manfiolli, Adriana O; Batista, Thiago M; Cappelli, Ana Paula Gameiro; Reis, Rosana I; Frasson, Danúbia; Kettelhut, Isis C; Parreiras-e-Silva, Lucas T; Costa-Neto, Claudio M; Carneiro, Everardo M; Curi, Rui; Silveira, Leonardo R
2013-10-01
The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids. Copyright © 2013 Elsevier B.V. All rights reserved.
Hulshof, Martijn F. M.; van den Berg, Sjoerd A. A.; Schaart, Gert; van Dijk, Ko Willems; Smit, Egbert; Mariman, Edwin C. M.
2011-01-01
Background Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance. Methodology C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR. Principal Findings At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks. Conclusions/Interpretation Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in
A mitochondria-dependent pathway mediates the apoptosis of GSE-induced yeast.
Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun
2012-01-01
Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency.
A Mitochondria-Dependent Pathway Mediates the Apoptosis of GSE-Induced Yeast
Cao, Sishuo; Xu, Wentao; Zhang, Nan; Wang, Yan; Luo, YunBo; He, Xiaoyun; Huang, Kunlun
2012-01-01
Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase–mediated dUTP Nick End Labeling (TUNEL) and 4,6′-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency. PMID:22403727
Peng, Huadong; Moghaddam, Lalehvash; Brinin, Anthony; Williams, Brett; Mundree, Sagadevan; Haritos, Victoria S
2018-03-01
As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks. © 2017 International Union of Biochemistry and Molecular Biology, Inc.
Weight loss by Ppc-1, a novel small molecule mitochondrial uncoupler derived from slime mold.
Suzuki, Toshiyuki; Kikuchi, Haruhisa; Ogura, Masato; Homma, Miwako K; Oshima, Yoshiteru; Homma, Yoshimi
2015-01-01
Mitochondria play a key role in diverse processes including ATP synthesis and apoptosis. Mitochondrial function can be studied using inhibitors of respiration, and new agents are valuable for discovering novel mechanisms involved in mitochondrial regulation. Here, we screened small molecules derived from slime molds and other microorganisms for their effects on mitochondrial oxygen consumption. We identified Ppc-1 as a novel molecule which stimulates oxygen consumption without adverse effects on ATP production. The kinetic behavior of Ppc-1 suggests its function as a mitochondrial uncoupler. Serial administration of Ppc-1 into mice suppressed weight gain with no abnormal effects on liver or kidney tissues, and no evidence of tumor formation. Serum fatty acid levels were significantly elevated in mice treated with Ppc-1, while body fat content remained low. After a single administration, Ppc-1 distributes into various tissues of individual animals at low levels. Ppc-1 stimulates adipocytes in culture to release fatty acids, which might explain the elevated serum fatty acids in Ppc-1-treated mice. The results suggest that Ppc-1 is a unique mitochondrial regulator which will be a valuable tool for mitochondrial research as well as the development of new drugs to treat obesity.
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.
Network Thermodynamic Curation of Human and Yeast Genome-Scale Metabolic Models
Martínez, Verónica S.; Quek, Lake-Ee; Nielsen, Lars K.
2014-01-01
Genome-scale models are used for an ever-widening range of applications. Although there has been much focus on specifying the stoichiometric matrix, the predictive power of genome-scale models equally depends on reaction directions. Two-thirds of reactions in the two eukaryotic reconstructions Homo sapiens Recon 1 and Yeast 5 are specified as irreversible. However, these specifications are mainly based on biochemical textbooks or on their similarity to other organisms and are rarely underpinned by detailed thermodynamic analysis. In this study, a to our knowledge new workflow combining network-embedded thermodynamic and flux variability analysis was used to evaluate existing irreversibility constraints in Recon 1 and Yeast 5 and to identify new ones. A total of 27 and 16 new irreversible reactions were identified in Recon 1 and Yeast 5, respectively, whereas only four reactions were found with directions incorrectly specified against thermodynamics (three in Yeast 5 and one in Recon 1). The workflow further identified for both models several isolated internal loops that require further curation. The framework also highlighted the need for substrate channeling (in human) and ATP hydrolysis (in yeast) for the essential reaction catalyzed by phosphoribosylaminoimidazole carboxylase in purine metabolism. Finally, the framework highlighted differences in proline metabolism between yeast (cytosolic anabolism and mitochondrial catabolism) and humans (exclusively mitochondrial metabolism). We conclude that network-embedded thermodynamics facilitates the specification and validation of irreversibility constraints in compartmentalized metabolic models, at the same time providing further insight into network properties. PMID:25028891
Skeletal muscle mitochondrial energetics in obesity and type 2 diabetes mellitus: endocrine aspects.
Aguer, Céline; Harper, Mary-Ellen
2012-12-01
During the development of type 2 diabetes mellitus, skeletal muscle is a major site of insulin resistance. The latter has been linked to mitochondrial dysfunction and impaired fatty acid oxidation. Some hormones like insulin, thyroid hormones and adipokines (e.g., leptin, adiponectin) have positive effects on muscle mitochondrial bioenergetics through their direct or indirect effects on mitochondrial biogenesis, mitochondrial protein expression, mitochondrial enzyme activities and/or AMPK pathway activation--all of which can improve fatty acid oxidation. It is therefore not surprising that treatment with these hormones has been proposed to improve muscle and whole body insulin sensitivity. However, treatment of diabetic patients with leptin and adiponectin has no effect on muscle mitochondrial bioenergetics showing resistance to these hormones during type 2 diabetes. Furthermore, treatment with most thyroid hormones has unexpectedly revealed negative effects on muscle insulin sensitivity. Future research should focus on development of agents that improve metabolic dysfunction downstream of hormone receptors. Copyright © 2012 Elsevier Ltd. All rights reserved.
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
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.
McFarlan, Jay T.; Yoshida, Yuko; Jain, Swati S.; Han, Xioa-Xia; Snook, Laelie A.; Lally, James; Smith, Brennan K.; Glatz, Jan F. C.; Luiken, Joost J. F. P.; Sayer, Ryan A.; Tupling, A. Russell; Chabowski, Adrian; Holloway, Graham P.; Bonen, Arend
2012-01-01
For ∼40 years it has been widely accepted that (i) the exercise-induced increase in muscle fatty acid oxidation (FAO) is dependent on the increased delivery of circulating fatty acids, and (ii) exercise training-induced FAO up-regulation is largely attributable to muscle mitochondrial biogenesis. These long standing concepts were developed prior to the recent recognition that fatty acid entry into muscle occurs via a regulatable sarcolemmal CD36-mediated mechanism. We examined the role of CD36 in muscle fuel selection under basal conditions, during a metabolic challenge (exercise), and after exercise training. We also investigated whether CD36 overexpression, independent of mitochondrial changes, mimicked exercise training-induced FAO up-regulation. Under basal conditions CD36-KO versus WT mice displayed reduced fatty acid transport (−21%) and oxidation (−25%), intramuscular lipids (less than or equal to −31%), and hepatic glycogen (−20%); but muscle glycogen, VO2max, and mitochondrial content and enzymes did not differ. In acutely exercised (78% VO2max) CD36-KO mice, fatty acid transport (−41%), oxidation (−37%), and exercise duration (−44%) were reduced, whereas muscle and hepatic glycogen depletions were accelerated by 27–55%, revealing 2-fold greater carbohydrate use. Exercise training increased mtDNA and β-hydroxyacyl-CoA dehydrogenase similarly in WT and CD36-KO muscles, but FAO was increased only in WT muscle (+90%). Comparable CD36 increases, induced by exercise training (+44%) or by CD36 overexpression (+41%), increased FAO similarly (84–90%), either when mitochondrial biogenesis and FAO enzymes were up-regulated (exercise training) or when these were unaltered (CD36 overexpression). Thus, sarcolemmal CD36 has a key role in muscle fuel selection, exercise performance, and training-induced muscle FAO adaptation, challenging long held views of mechanisms involved in acute and adaptive regulation of muscle FAO. PMID:22584574
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.
The Regulation of Coenzyme Q Biosynthesis in Eukaryotic Cells: All That Yeast Can Tell Us
González-Mariscal, Isabel; García-Testón, Elena; Padilla, Sergio; Martín-Montalvo, Alejandro; Pomares Viciana, Teresa; Vazquez-Fonseca, Luis; Gandolfo Domínguez, Pablo; Santos-Ocaña, Carlos
2014-01-01
Coenzyme Q (CoQ) is a mitochondrial lipid, which functions mainly as an electron carrier from complex I or II to complex III at the mitochondrial inner membrane, and also as antioxidant in cell membranes. CoQ is needed as electron acceptor in β-oxidation of fatty acids and pyridine nucleotide biosynthesis, and it is responsible for opening the mitochondrial permeability transition pore. The yeast model has been very useful to analyze the synthesis of CoQ, and therefore, most of the knowledge about its regulation was obtained from the Saccharomyces cerevisiae model. CoQ biosynthesis is regulated to support 2 processes: the bioenergetic metabolism and the antioxidant defense. Alterations of the carbon source in yeast, or in nutrient availability in yeasts or mammalian cells, upregulate genes encoding proteins involved in CoQ synthesis. Oxidative stress, generated by chemical or physical agents or by serum deprivation, modifies specifically the expression of some COQ genes by means of stress transcription factors such as Msn2/4p, Yap1p or Hsf1p. In general, the induction of COQ gene expression produced by metabolic changes or stress is modulated downstream by other regulatory mechanisms such as the protein import to mitochondria, the assembly of a multi-enzymatic complex composed by Coq proteins and also the existence of a phosphorylation cycle that regulates the last steps of CoQ biosynthesis. The CoQ biosynthetic complex assembly starts with the production of a nucleating lipid such as HHB by the action of the Coq2 protein. Then, the Coq4 protein recognizes the precursor HHB acting as the nucleus of the complex. The activity of Coq8p, probably as kinase, allows the formation of an initial pre-complex containing all Coq proteins with the exception of Coq7p. This pre-complex leads to the synthesis of 5-demethoxy-Q6 (DMQ6), the Coq7p substrate. When de novo CoQ biosynthesis is required, Coq7p becomes dephosphorylated by the action of Ptc7p increasing the synthesis
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.
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.
Weight Loss by Ppc-1, a Novel Small Molecule Mitochondrial Uncoupler Derived from Slime Mold
Suzuki, Toshiyuki; Kikuchi, Haruhisa; Ogura, Masato; Homma, Miwako K.; Oshima, Yoshiteru; Homma, Yoshimi
2015-01-01
Mitochondria play a key role in diverse processes including ATP synthesis and apoptosis. Mitochondrial function can be studied using inhibitors of respiration, and new agents are valuable for discovering novel mechanisms involved in mitochondrial regulation. Here, we screened small molecules derived from slime molds and other microorganisms for their effects on mitochondrial oxygen consumption. We identified Ppc-1 as a novel molecule which stimulates oxygen consumption without adverse effects on ATP production. The kinetic behavior of Ppc-1 suggests its function as a mitochondrial uncoupler. Serial administration of Ppc-1 into mice suppressed weight gain with no abnormal effects on liver or kidney tissues, and no evidence of tumor formation. Serum fatty acid levels were significantly elevated in mice treated with Ppc-1, while body fat content remained low. After a single administration, Ppc-1 distributes into various tissues of individual animals at low levels. Ppc-1 stimulates adipocytes in culture to release fatty acids, which might explain the elevated serum fatty acids in Ppc-1-treated mice. The results suggest that Ppc-1 is a unique mitochondrial regulator which will be a valuable tool for mitochondrial research as well as the development of new drugs to treat obesity. PMID:25668511
Wang, Shaoxiao; Zhang, Siyuan; Xu, Chuan; Barron, Addie; Galiano, Floyd; Patel, Dhaval; Lee, Yong Joo; Caldwell, Guy A.; Caldwell, Kim A.
2016-01-01
We have been investigating the role that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) content plays in modulating the solubility of the Parkinson’s disease protein alpha-synuclein (α-syn) using Saccharomyces cerevisiae and Caenorhabditis elegans. One enzyme that synthesizes PE is the conserved enzyme phosphatidylserine decarboxylase (Psd1/yeast; PSD-1/worms), which is lodged in the inner mitochondrial membrane. We previously found that decreasing the level of PE due to knockdown of Psd1/psd-1 affects the homeostasis of α-syn in vivo. In S. cerevisiae, the co-occurrence of low PE and α-syn in psd1Δ cells triggers mitochondrial defects, stress in the endoplasmic reticulum, misprocessing of glycosylphosphatidylinositol-anchored proteins, and a 3-fold increase in the level of α-syn. The goal of this study was to identify drugs that rescue this phenotype. We screened the Prestwick library of 1121 Food and Drug Administration-approved drugs using psd1Δ + α-syn cells and identified cyclosporin A, meclofenoxate hydrochloride, and sulfaphenazole as putative protective compounds. The protective activity of these drugs was corroborated using C. elegans in which α-syn is expressed specifically in the dopaminergic neurons, with psd-1 depleted by RNAi. Worm populations were examined for dopaminergic neuron survival following psd-1 knockdown. Exposure to cyclosporine, meclofenoxate, and sulfaphenazole significantly enhanced survival at day 7 in α-syn-expressing worm populations whereby 50–55% of the populations displayed normal neurons, compared to only 10–15% of untreated animals. We also found that all three drugs rescued worms expressing α-syn in dopaminergic neurons that were deficient in the phospholipid cardiolipin following cardiolipin synthase (crls-1) depletion by RNAi. We discuss how these drugs might block α-syn pathology in dopaminergic neurons. PMID:27736935
Study the oxidative injury of yeast cells by NADH autofluorescence
NASA Astrophysics Data System (ADS)
Liang, Ju; Wu, Wen-Lan; Liu, Zhi-Hong; Mei, Yun-Jun; Cai, Ru-Xiu; Shen, Ping
2007-06-01
Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H 2O 2 and ONOO - have been recorded in detail in this work. In the presence of different amounts of H 2O 2 and ONOO -, necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.
Genetic and Biochemical Analysis of High Iron Toxicity in Yeast
Lin, Huilan; Li, Liangtao; Jia, Xuan; Ward, Diane McVey; Kaplan, Jerry
2011-01-01
Iron storage in yeast requires the activity of the vacuolar iron transporter Ccc1. Yeast with an intact CCC1 are resistant to iron toxicity, but deletion of CCC1 renders yeast susceptible to iron toxicity. We used genetic and biochemical analysis to identify suppressors of high iron toxicity in Δccc1 cells to probe the mechanism of high iron toxicity. All genes identified as suppressors of high iron toxicity in aerobically grown Δccc1 cells encode organelle iron transporters including mitochondrial iron transporters MRS3, MRS4, and RIM2. Overexpression of MRS3 suppressed high iron toxicity by decreasing cytosolic iron through mitochondrial iron accumulation. Under anaerobic conditions, Δccc1 cells were still sensitive to high iron toxicity, but overexpression of MRS3 did not suppress iron toxicity and did not result in mitochondrial iron accumulation. We conclude that Mrs3/Mrs4 can sequester iron within mitochondria under aerobic conditions but not anaerobic conditions. We show that iron toxicity in Δccc1 cells occurred under both aerobic and anaerobic conditions. Microarray analysis showed no evidence of oxidative damage under anaerobic conditions, suggesting that iron toxicity may not be solely due to oxidative damage. Deletion of TSA1, which encodes a peroxiredoxin, exacerbated iron toxicity in Δccc1 cells under both aerobic and anaerobic conditions, suggesting a unique role for Tsa1 in iron toxicity. PMID:21115478
Dual Fatty Acid Elongase Complex Interactions in Arabidopsis
Morineau, Céline; Gissot, Lionel; Bellec, Yannick; Hematy, Kian; Tellier, Frédérique; Renne, Charlotte; Haslam, Richard; Beaudoin, Frédéric; Napier, Johnathan; Faure, Jean-Denis
2016-01-01
Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting. PMID:27583779
Dual Fatty Acid Elongase Complex Interactions in Arabidopsis.
Morineau, Céline; Gissot, Lionel; Bellec, Yannick; Hematy, Kian; Tellier, Frédérique; Renne, Charlotte; Haslam, Richard; Beaudoin, Frédéric; Napier, Johnathan; Faure, Jean-Denis
2016-01-01
Very long chain fatty acids (VLCFAs) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFAs in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFAs, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase 3 hydroxyacyl-CoA dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFAs elongation but not the plant pas2-1 defects. PTPLA interacted with elongase subunits in the Endoplasmic Reticulum (ER) and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function increased VLCFA levels, an effect that was dependent on the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted to the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes based on PAS2 or PTPLA dehydratase that are functionally interacting.
Network thermodynamic curation of human and yeast genome-scale metabolic models.
Martínez, Verónica S; Quek, Lake-Ee; Nielsen, Lars K
2014-07-15
Genome-scale models are used for an ever-widening range of applications. Although there has been much focus on specifying the stoichiometric matrix, the predictive power of genome-scale models equally depends on reaction directions. Two-thirds of reactions in the two eukaryotic reconstructions Homo sapiens Recon 1 and Yeast 5 are specified as irreversible. However, these specifications are mainly based on biochemical textbooks or on their similarity to other organisms and are rarely underpinned by detailed thermodynamic analysis. In this study, a to our knowledge new workflow combining network-embedded thermodynamic and flux variability analysis was used to evaluate existing irreversibility constraints in Recon 1 and Yeast 5 and to identify new ones. A total of 27 and 16 new irreversible reactions were identified in Recon 1 and Yeast 5, respectively, whereas only four reactions were found with directions incorrectly specified against thermodynamics (three in Yeast 5 and one in Recon 1). The workflow further identified for both models several isolated internal loops that require further curation. The framework also highlighted the need for substrate channeling (in human) and ATP hydrolysis (in yeast) for the essential reaction catalyzed by phosphoribosylaminoimidazole carboxylase in purine metabolism. Finally, the framework highlighted differences in proline metabolism between yeast (cytosolic anabolism and mitochondrial catabolism) and humans (exclusively mitochondrial metabolism). We conclude that network-embedded thermodynamics facilitates the specification and validation of irreversibility constraints in compartmentalized metabolic models, at the same time providing further insight into network properties. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Marcovina, Santica M; Sirtori, Cesare; Peracino, Andrea; Gheorghiade, Mihai; Borum, Peggy; Remuzzi, Giuseppe; Ardehali, Hossein
2013-02-01
Mitochondria play important roles in human physiological processes, and therefore, their dysfunction can lead to a constellation of metabolic and nonmetabolic abnormalities such as a defect in mitochondrial gene expression, imbalance in fuel and energy homeostasis, impairment in oxidative phosphorylation, enhancement of insulin resistance, and abnormalities in fatty acid metabolism. As a consequence, mitochondrial dysfunction contributes to the pathophysiology of insulin resistance, obesity, diabetes, vascular disease, and chronic heart failure. The increased knowledge on mitochondria and their role in cellular metabolism is providing new evidence that these disorders may benefit from mitochondrial-targeted therapies. We review the current knowledge of the contribution of mitochondrial dysfunction to chronic diseases, the outcomes of experimental studies on mitochondrial-targeted therapies, and explore the potential of metabolic modulators in the treatment of selected chronic conditions. As an example of such modulators, we evaluate the efficacy of the administration of L-carnitine and its analogues acetyl and propionyl L-carnitine in several chronic diseases. L-carnitine is intrinsically involved in mitochondrial metabolism and function as it plays a key role in fatty acid oxidation and energy metabolism. In addition to the transportation of free fatty acids across the inner mitochondrial membrane, L-carnitine modulates their oxidation rate and is involved in the regulation of vital cellular functions such as apoptosis. Thus, L-carnitine and its derivatives show promise in the treatment of chronic conditions and diseases associated with mitochondrial dysfunction but further translational studies are needed to fully explore their potential. Copyright © 2013 Mosby, Inc. All rights reserved.
Antioxidant dietary approach in treatment of fatty liver: New insights and updates
Ferramosca, Alessandra; Di Giacomo, Mariangela; Zara, Vincenzo
2017-01-01
Non-alcoholic fatty liver disease (NAFLD) is a common clinicopathological condition, encompassing a range of conditions caused by lipid deposition within liver cells. To date, no approved drugs are available for the treatment of NAFLD, despite the fact that it represents a serious and growing clinical problem in the Western world. Identification of the molecular mechanisms leading to NAFLD-related fat accumulation, mitochondrial dysfunction and oxidative balance impairment facilitates the development of specific interventions aimed at preventing the progression of hepatic steatosis. In this review, we focus our attention on the role of dysfunctions in mitochondrial bioenergetics in the pathogenesis of fatty liver. Major data from the literature about the mitochondrial targeting of some antioxidant molecules as a potential treatment for hepatic steatosis are described and critically analysed. There is ample evidence of the positive effects of several classes of antioxidants, such as polyphenols (i.e., resveratrol, quercetin, coumestrol, anthocyanins, epigallocatechin gallate and curcumin), carotenoids (i.e., lycopene, astaxanthin and fucoxanthin) and glucosinolates (i.e., glucoraphanin, sulforaphane, sinigrin and allyl-isothiocyanate), on the reversion of fatty liver. Although the mechanism of action is not yet fully elucidated, in some cases an indirect interaction with mitochondrial metabolism is expected. We believe that such knowledge will eventually translate into the development of novel therapeutic approaches for fatty liver. PMID:28694655
Antioxidant dietary approach in treatment of fatty liver: New insights and updates.
Ferramosca, Alessandra; Di Giacomo, Mariangela; Zara, Vincenzo
2017-06-21
Non-alcoholic fatty liver disease (NAFLD) is a common clinicopathological condition, encompassing a range of conditions caused by lipid deposition within liver cells. To date, no approved drugs are available for the treatment of NAFLD, despite the fact that it represents a serious and growing clinical problem in the Western world. Identification of the molecular mechanisms leading to NAFLD-related fat accumulation, mitochondrial dysfunction and oxidative balance impairment facilitates the development of specific interventions aimed at preventing the progression of hepatic steatosis. In this review, we focus our attention on the role of dysfunctions in mitochondrial bioenergetics in the pathogenesis of fatty liver. Major data from the literature about the mitochondrial targeting of some antioxidant molecules as a potential treatment for hepatic steatosis are described and critically analysed. There is ample evidence of the positive effects of several classes of antioxidants, such as polyphenols ( i.e ., resveratrol, quercetin, coumestrol, anthocyanins, epigallocatechin gallate and curcumin), carotenoids ( i.e ., lycopene, astaxanthin and fucoxanthin) and glucosinolates ( i.e ., glucoraphanin, sulforaphane, sinigrin and allyl-isothiocyanate), on the reversion of fatty liver. Although the mechanism of action is not yet fully elucidated, in some cases an indirect interaction with mitochondrial metabolism is expected. We believe that such knowledge will eventually translate into the development of novel therapeutic approaches for fatty liver.
Navarro, Claudia D C; Figueira, Tiago R; Francisco, Annelise; Dal'Bó, Genoefa A; Ronchi, Juliana A; Rovani, Juliana C; Escanhoela, Cecilia A F; Oliveira, Helena C F; Castilho, Roger F; Vercesi, Anibal E
2017-12-01
The mechanisms by which a high fat diet (HFD) promotes non-alcoholic fatty liver disease (NAFLD) appear to involve liver mitochondrial dysfunctions and redox imbalance. We hypothesized that a HFD would increase mitochondrial reliance on NAD(P)-transhydrogenase (NNT) as the source of NADPH for antioxidant systems that counteract NAFLD development. Therefore, we studied HFD-induced liver mitochondrial dysfunctions and NAFLD in C57Unib.B6 congenic mice with (Nnt +/+ ) or without (Nnt -/- ) NNT activity; the spontaneously mutated allele (Nnt -/- ) was inherited from the C57BL/6J mouse substrain. After 20 weeks on a HFD, Nnt -/- mice exhibited a higher prevalence of steatohepatitis and content of liver triglycerides compared to Nnt +/+ mice on an identical diet. Under a HFD, the aggravated NAFLD phenotype in the Nnt -/- mice was accompanied by an increased H 2 O 2 release rate from mitochondria, decreased aconitase activity (a redox-sensitive mitochondrial enzyme) and higher susceptibility to Ca 2+ -induced mitochondrial permeability transition. In addition, HFD led to the phosphorylation (inhibition) of pyruvate dehydrogenase (PDH) and markedly reduced the ability of liver mitochondria to remove peroxide in Nnt -/- mice. Bypass or pharmacological reactivation of PDH by dichloroacetate restored the peroxide removal capability of mitochondria from Nnt -/- mice on a HFD. Noteworthy, compared to mice that were chow-fed, the HFD did not impair peroxide removal nor elicit redox imbalance in mitochondria from Nnt +/+ mice. Therefore, HFD interacted with Nnt mutation to generate PDH inhibition and further suppression of peroxide removal. We conclude that NNT plays a critical role in counteracting mitochondrial redox imbalance, PDH inhibition and advancement of NAFLD in mice fed a HFD. The present study provide seminal experimental evidence that redox imbalance in liver mitochondria potentiates the progression from simple steatosis to steatohepatitis following a HFD. Copyright
Wang, Zhe; Shen, Yan
2017-03-01
The fast growing evidences have indicated that the natural product osthole is a promising drug candidate for fighting several serious human diseases, for example, cancer and inflammation. However, the mode-of-action (MoA) of osthole remains largely incomplete. In this study, we investigated the growth inhibition activity of osthole using fission yeast as a model, with the goal of understanding the osthole's mechanism of action, especially from the molecular level. Microarray analysis indicated that osthole has significant impacts on gene transcription levels (In total, 214 genes are up-regulated, and 97 genes are down-regulated). Gene set enrichment analysis (GSEA) indicated that 11 genes belong to the "Respiration module" category, especially including the components of complex III and V of mitochondrial respiration chain. Based on GSEA and network analysis, we also found that 54 up-regulated genes belong to the "Core Environmental Stress Responses" category, particularly including many transporter genes, which suggests that the rapidly activated nutrient exchange between cell and environment is part of the MoA of osthole. In summary, osthole can greatly impact on fission yeast transcriptome, and it primarily represses the expression levels of the genes in respiration chain, which next causes the inefficiency of ATP production and thus largely explains osthole's growth inhibition activity in Schizosaccharomyces pombe (S. pombe). The complexity of the osthole's MoA shown in previous studies and our current research demonstrates that the omics approach and bioinformatics tools should be applied together to acquire the complete landscape of osthole's growth inhibition activity.
Retrograde Signaling as a Mechanism of Yeast Adaptation to Unfavorable Factors.
Trendeleva, T A; Zvyagilskaya, R A
2018-02-01
Mitochondria perform many essential functions in eukaryotic cells. Being the main producers of ATP and the site of many catabolic and anabolic reactions, they participate in intracellular signaling, proliferation, aging, and formation of reactive oxygen species. Mitochondrial dysfunction is the cause of many diseases and even cell death. The functioning of mitochondria in vivo is impossible without interaction with other cellular compartments. Mitochondrial retrograde signaling is a signaling pathway connecting mitochondria and the nucleus. The major signal transducers in the yeast retrograde response are Rtg1p, Rtg2p, and Rtg3p proteins, as well as four additional negative regulatory factors - Mks1p, Lst8p, and two 14-3-3 proteins (Bmh1/2p). In this review, we analyze current information on the retrograde signaling in yeast that is regarded as a stress or homeostatic response mechanism to changes in various metabolic and biosynthetic activities that occur upon mitochondrial dysfunction. We also discuss relations between retrograde signaling and other signaling pathways in the cell.
Zoladz, Jerzy A; Koziel, Agnieszka; Broniarek, Izabela; Woyda-Ploszczyca, Andrzej M; Ogrodna, Karolina; Majerczak, Joanna; Celichowski, Jan; Szkutnik, Zbigniew; Jarmuszkiewicz, Wieslawa
2017-01-01
We studied the effects of various assay temperatures, representing hypothermia (25°C), normothermia (35°C), and hyperthermia (42°C), on the oxidation of lipid-derived fuels in rat skeletal muscle mitochondria of untrained and endurance-trained rats. Adult 4-month-old male Wistar rats were assigned to a training group (rats trained on a treadmill for 8 weeks) or a sedentary control group. In skeletal muscle mitochondria of both control and trained rats, an increase in the assay temperature from 25°C to 42°C was accompanied by a consistent increase in the oxidation of palmitoylcarnitine and glycerol-3-phosphate. Moreover, endurance training increased mitochondrial capacity to oxidize the lipid-derived fuels at all studied temperatures. The endurance training-induced increase in mitochondrial capacity to oxidize fatty acids was accompanied by an enhancement of mitochondrial biogenesis, as shown by the elevated expression levels of Nrf2, PGC1α, and mitochondrial marker and by the elevated expression levels of mitochondrial proteins involved in fatty acid metabolism, such as fatty acid transporter CD36, carnitine palmitoyltransferase 1A (CPT1A), and acyl-CoA dehydrogenase (ACADS). We conclude that hyperthermia enhances but hypothermia attenuates the rate of the oxidation of fatty acids and glycerol-3-phosphate in rat skeletal muscle mitochondria isolated from both untrained and trained rats. Moreover, our results indicate that endurance training up-regulates mitochondrial biogenesis markers, lipid-sustained oxidative capacity, and CD36 and CPT1A proteins involved in fatty acid transport, possibly via PGC1α and Nrf2 signaling pathways.
Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome
Garone, Caterina; D’Souza, Aaron R; Dallabona, Cristina; Lodi, Tiziana; Rebelo-Guiomar, Pedro; Rorbach, Joanna; Donati, Maria Alice; Procopio, Elena; Montomoli, Martino; Guerrini, Renzo; Zeviani, Massimo; Calvo, Sarah E; Mootha, Vamsi K; DiMauro, Salvatore; Ferrero, Ileana; Minczuk, Michal
2017-01-01
Abstract Defects in nuclear-encoded proteins of the mitochondrial translation machinery cause early-onset and tissue-specific deficiency of one or more OXPHOS complexes. Here, we report a 7-year-old Italian boy with childhood-onset rapidly progressive encephalomyopathy and stroke-like episodes. Multiple OXPHOS defects and decreased mtDNA copy number (40%) were detected in muscle homogenate. Clinical features combined with low level of plasma citrulline were highly suggestive of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was excluded. Targeted exome sequencing of genes encoding the mitochondrial proteome identified a damaging mutation, c.567 G > A, affecting a highly conserved amino acid residue (p.Gly189Arg) of the MRM2 protein. MRM2 has never before been linked to a human disease and encodes an enzyme responsible for 2’-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA. We generated a knockout yeast model for the orthologous gene that showed a defect in respiration and the reduction of the 2’-O-methyl modification at the equivalent position (U2791) in the yeast mitochondrial 21S rRNA. Complementation with the mrm2 allele carrying the equivalent yeast mutation failed to rescue the respiratory phenotype, which was instead completely rescued by expressing the wild-type allele. Our findings establish that defective MRM2 causes a MELAS-like phenotype, and suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation. PMID:28973171
Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome.
Garone, Caterina; D'Souza, Aaron R; Dallabona, Cristina; Lodi, Tiziana; Rebelo-Guiomar, Pedro; Rorbach, Joanna; Donati, Maria Alice; Procopio, Elena; Montomoli, Martino; Guerrini, Renzo; Zeviani, Massimo; Calvo, Sarah E; Mootha, Vamsi K; DiMauro, Salvatore; Ferrero, Ileana; Minczuk, Michal
2017-11-01
Defects in nuclear-encoded proteins of the mitochondrial translation machinery cause early-onset and tissue-specific deficiency of one or more OXPHOS complexes. Here, we report a 7-year-old Italian boy with childhood-onset rapidly progressive encephalomyopathy and stroke-like episodes. Multiple OXPHOS defects and decreased mtDNA copy number (40%) were detected in muscle homogenate. Clinical features combined with low level of plasma citrulline were highly suggestive of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was excluded. Targeted exome sequencing of genes encoding the mitochondrial proteome identified a damaging mutation, c.567 G > A, affecting a highly conserved amino acid residue (p.Gly189Arg) of the MRM2 protein. MRM2 has never before been linked to a human disease and encodes an enzyme responsible for 2'-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA. We generated a knockout yeast model for the orthologous gene that showed a defect in respiration and the reduction of the 2'-O-methyl modification at the equivalent position (U2791) in the yeast mitochondrial 21S rRNA. Complementation with the mrm2 allele carrying the equivalent yeast mutation failed to rescue the respiratory phenotype, which was instead completely rescued by expressing the wild-type allele. Our findings establish that defective MRM2 causes a MELAS-like phenotype, and suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation. © The Author 2017. Published by Oxford University Press.
Hess, Kenneth C.; Liu, Jingjing; Manfredi, Giovanni; Mühlschlegel, Fritz A.; Buck, Jochen; Levin, Lonny R.; Barrientos, Antoni
2014-01-01
Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.—Hess, K. C., Liu, J., Manfredi, G., Mühlschlegel, F. A., Buck, J., Levin, L. R., Barrientos, A. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity. PMID:25002117
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.
Draft genome sequence of the D-Xylose-Fermenting yeast Spathaspora xylofermentans UFMG-HMD23.3
USDA-ARS?s Scientific Manuscript database
Here, we report the draft genome sequence of the yeast Spathaspora xylofermentans UFMG-HMD23.3 (CBMAI 1427=CBS 12681), a D-xylose fermenting yeast isolated from the Amazonian forest. The genome consists of 298 contigs, with a total size of 15.1 Mb, including the mitochondrial genome, and 5,948 predi...
Lemeshko, Victor V
2017-11-01
Inhibition of cell respiration by high concentrations of glucose (glucose repression), known as "Crabtree effect", has been demonstrated for various cancerous strains, highly proliferating cells and yeast lines. Although significant progress in understanding metabolic events associated with the glucose repression of cell respiration has been achieved, it is not yet clear whether the Crabtree effect is the result of a limited activity of the respiratory chain, or of some glucose-mediated regulation of mitochondrial metabolic state. In this work we propose an electrical mechanism of glucose repression of the yeast S. cerevisiae, resulting from generation of the mitochondrial outer membrane potential (OMP) coupled to the direct oxidation of cytosolic NADH in mitochondria. This yeast-type mechanism of OMP generation is different from the earlier proposed VDAC-hexokinase-mediated voltage generation of cancer-type, associated with the mitochondrial outer membrane. The model was developed assuming that VDAC is more permeable to NADH than to NAD + . Thermodynamic estimations of OMP, generated as a result of NADH(2-)/NAD + (1-) turnover through the outer membrane, demonstrated that the values of calculated negative OMP match the known range of VDAC voltage sensitivity, thus suggesting a possibility of OMP-dependent VDAC-mediated regulation of cell energy metabolism. According to the proposed mechanism, we suggest that the yeast-type Crabtree effect is the result of a fast VDAC-mediated electrical repression of mitochondria due to a decrease in the outer membrane permeability to charged metabolites and owing their redistribution between the mitochondrial intermembrane space and the cytosol, both controlled by metabolically-derived OMP. Copyright © 2017 Elsevier B.V. All rights reserved.
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
Aung, Hnin W.; Henry, Susan A.
2013-01-01
Abstract Genome-scale metabolic models are built using information from an organism's annotated genome and, correspondingly, information on reactions catalyzed by the set of metabolic enzymes encoded by the genome. These models have been successfully applied to guide metabolic engineering to increase production of metabolites of industrial interest. Congruity between simulated and experimental metabolic behavior is influenced by the accuracy of the representation of the metabolic network in the model. In the interest of applying the consensus model of Saccharomyces cerevisiae metabolism for increased productivity of triglycerides, we manually evaluated the representation of fatty acid, glycerophospholipid, and glycerolipid metabolism in the consensus model (Yeast v6.0). These areas of metabolism were chosen due to their tightly interconnected nature to triglyceride synthesis. Manual curation was facilitated by custom MATLAB functions that return information contained in the model for reactions associated with genes and metabolites within the stated areas of metabolism. Through manual curation, we have identified inconsistencies between information contained in the model and literature knowledge. These inconsistencies include incorrect gene-reaction associations, improper definition of substrates/products in reactions, inappropriate assignments of reaction directionality, nonfunctional β-oxidation pathways, and missing reactions relevant to the synthesis and degradation of triglycerides. Suggestions to amend these inconsistencies in the Yeast v6.0 model can be implemented through a MATLAB script provided in the Supplementary Materials, Supplementary Data S1 (Supplementary Data are available online at www.liebertpub.com/ind). PMID:24678285
Mitochondrial pleomorphy in plant cells is driven by contiguous ER dynamics
Jaipargas, Erica-Ashley; Barton, Kiah A.; Mathur, Neeta; Mathur, Jaideep
2015-01-01
Mitochondria are pleomorphic, double membrane-bound organelles involved in cellular energetics in all eukaryotes. Mitochondria in animal and yeast cells are typically tubular-reticulate structures and several micro-meters long but in green plants they are predominantly observed as 0.2–1.5 μm punctae. While fission and fusion, through the coordinated activity of several conserved proteins, shapes mitochondria, the endoplasmic reticulum (ER) has recently been identified as an additional player in this process in yeast and mammalian cells. The mitochondria-ER relationship in plant cells remains largely uncharacterized. Here, through live-imaging of the entire range of mitochondria pleomorphy we uncover the underlying basis for the predominantly punctate mitochondrial form in plants. We demonstrate that mitochondrial morphology changes in response to light and cytosolic sugar levels in an ER mediated manner. Whereas, large ER polygons and low dynamics under dark conditions favor mitochondrial fusion and elongation, small ER polygons result in increased fission and predominantly small mitochondria. Hypoxia also reduces ER dynamics and increases mitochondrial fusion to produce giant mitochondria. By observing elongated mitochondria in normal plants and fission-impaired Arabidopsis nmt1-2 and drp3a mutants we also establish that thin extensions called matrixules and a beads-on-a-string mitochondrial phenotype are direct consequences of mitochondria-ER interactions. PMID:26442089
Martin, Nicolas; Kraffe, Edouard; Le Grand, Fabienne; Marty, Yanic; Bureau, Dominique P; Guderley, Helga
2015-01-01
Although dietary lipid quality markedly affects fatty acid (FA) composition of mitochondrial membranes from rainbow trout red muscle (Oncorhynchus mykiss), mitochondrial processes are relatively unchanged. As certain classes of phospholipids interact more intimately with membrane proteins than others, we examined whether specific phospholipid classes from these muscle mitochondria were more affected by dietary FA composition than others. To test this hypothesis, we fed trout with two diets differing only in their FA composition: Diet 1 had higher levels of 18:1n-9 and 18:2n-6 than Diet 2, while 22:6n-3 and 22:5n-6 were virtually absent from Diet 1 and high in Diet 2. After 5 months, trout fed Diet 2 had higher proportions of phosphatidylcholine (PC) and less phosphatidylethanolamine (PE) in mitochondrial membranes than those fed Diet 1. The FA composition of PC, PE and cardiolipin (CL) showed clear evidence of regulated incorporation of dietary FA. For trout fed Diet 2, 22:6n-3 was the most abundant FA in PC, PE and CL. The n-6 FA were consistently higher in all phospholipid classes of trout fed Diet 1, with shorter n-6 FA being favoured in CL than in PC and PE. Despite these marked changes in individual FA levels with diet, general characteristics such as total polyunsaturated FA, total monounsaturated FA and total saturated FA were conserved in PE and CL, confirming differential regulation of the FA composition of PC, PE and CL. The regulated changes of phospholipid classes presumably maintain critical membrane characteristics despite varying nutritional quality. We postulate that these changes aim to protect mitochondrial function. © 2014 Wiley Periodicals, Inc.
Lipase production in lipolytic yeast from Wonorejo mangrove area
NASA Astrophysics Data System (ADS)
Alami, Nur Hidayatul; Nasihah, Liziyatin; Umar, Rurin Luswidya Artaty; Kuswytasari, Nengah Dwianita; Zulaika, Enny; Shovitri, Maya
2017-06-01
Lipase is an enzyme that is often used in industry and become a commercial enzyme. One group of microorganisms capable of producing lipase is a yeast. This study aims to screen yeast from Wonorejo mangrove that potential to produce lipase and to optimize the production of these enzymes. Screening test include the measurement of lipolytic index and value of fatty acid. Yeast with the best value of fatty acid will be continued to the measurement of lipase activity. It is affected by several environmental factors, such as pH, temperature, and incubation time. This research was conducted to observe the optimization variation on environmental factors combination to produce lipase. Lipase activity was tested by using p-Nitrophenyl Palmitate (pNPP). Absorbency was measured by spectrofotometer on wavelength of 410 nm. Measurement of the enzyme activity was done by interpolating the absorbance values on the p-nitrophenol standard curve then calculated by the formula. All data were analyzed by using descriptive quantitative method. The results show that the highest lypolityc index was 2.08. The highest value of fatty acid was 0.49 that was reached on 168 hours of incubation. Candida W3.8 expressed the highest lypolylitic potential. The optimum environment to produce lipase by Candida W 3.8 was on 120 hours of incubation time, in temperature range of 27°C - 45°C and pH range of 4,5 - 7.
Kim, Dong-Gyu; Yoo, Jae Cheal; Kim, Eunju; Lee, Young-Sun; Yarishkin, Oleg V; Lee, Da Yong; Lee, Kun Ho; Hong, Seong-Geun; Hwang, Eun Mi; Park, Jae-Yong
2014-06-01
Mitochondrial trans-2-enoyl-CoA reductase (MECR) is involved in mitochondrial synthesis of fatty acids and is highly expressed in mitochondria. MECR is also known as nuclear receptor binding factor-1, which was originally reported with yeast two-hybrid screening as a binding protein of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα). However, MECR and PPARα are localized at different compartment, mitochondria, and the nucleus, respectively. Therefore, the presence of a cytosolic or nuclear isoform of MECR is necessary for functional interaction between MECR and PPARα. To identify the expression pattern of MECR and the cytosolic form of MECR (cMECR), we performed reverse transcription polymerase chain reaction (RT-PCR) with various tissue samples from Sprague-Dawley rats. To confirm the interaction between cMECR and PPARα, we performed several binding assays such as yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation. To observe subcellular localization of these proteins, immunocytochemistry was performed. A luciferase assay was used to measure PPARα activity. We provide evidence of an alternatively spliced variant of the rat MECR gene that yields cMECR. The cMECR lacks the N-terminal 76 amino acids of MECR and shows uniform distribution in the cytoplasm and nucleus of HeLa cells. cMECR directly bound PPARα in the nucleus and increased PPARα-dependent luciferase activity in HeLa cells. We found the cytosolic form of MECR (cMECR) was expressed in the cytosolic and/or nuclear region, directly binds with PPARα, and enhances PPARα activity.
Hadwiger, Lee A; Polashock, James
2013-01-01
Previous reports on the model nonhost resistance interaction between Fusarium solani f. sp. phaseoli and pea endocarp tissue have described the disease resistance-signaling role of a fungal DNase1-like protein. The response resulted in no further growth beyond spore germination. This F. solani f. sp. phaseoli DNase gene, constructed with a pathogenesis-related (PR) gene promoter, when transferred to tobacco, generated resistance against Pseudomonas syringe pv. tabaci. The current analytical/theoretical article proposes similar roles for the additional nuclear and mitochondrial nucleases, the coding regions for which are identified in newly available fungal genome sequences. The amino acid sequence homologies within functional domains are conserved within a wide array of fungi. The potato pathogen Verticillium dahliae nuclease was divergent from that of the saprophyte, yeast; however, the purified DNase from yeast also elicited nonhost defense responses in pea, including pisatin accumulation, PR gene induction, and resistance against a true pea pathogen. The yeast mitochondrial DNase gene (open reading frame) predictably codes for a signal peptide providing the mechanism for secretion. Mitochondrial DNase genes appear to provide an unlimited source of components for developing transgenic resistance in all transformable plants.
Leighton, F; Bergseth, S; Rørtveit, T; Christiansen, E N; Bremer, J
1989-06-25
The fate of the acetyl-CoA units released during peroxisomal fatty acid oxidation was studied in isolated hepatocytes from normal and peroxisome-proliferated rats. Ketogenesis and hydrogen peroxide generation were employed as indicators of mitochondrial and peroxisomal fatty acid oxidation, respectively. Butyric and hexanoic acids were employed as mitochondrial substrates, 1, omega-dicarboxylic acids as predominantly peroxisomal substrates, and lauric acid as a substrate for both mitochondria and peroxisomes. Ketogenesis from dicarboxylic acids was either absent or very low in normal and peroxisome-proliferated hepatocytes, but free acetate release was detected at rates that could account for all the acetyl-CoA produced in peroxisomes by dicarboxylic and also by monocarboxylic acids. Mitochondrial fatty acid oxidation also led to free acetate generation but at low rates relative to ketogenesis. The origin of the acetate released was confirmed employing [1-14C]dodecanedioic acid. Thus, the activity of peroxisomes might contribute significantly to the free acetate generation known to occur during fatty acid oxidation in rats and possibly also in humans.
Nanolaser Spectroscopy of Genetically Engineered Yeast: New Tool for a Better Brew?
NASA Astrophysics Data System (ADS)
Gourley, Paul L.; Hendricks, Judy K.; Naviaux, Robert K.; Yaffe, Michael P.
2006-03-01
A basic function of the cell membrane is to selectively uptake ions or molecules from its environment to concentrate them into the interior. This concentration difference results in an osmostic pressure difference across the membrane. Ultimately, this pressure and its fluctuation from cell to cell will be limited by the availability and fluctuations of the solute concentrations in solution, the extent of inter-cell communication, and the state of respiring intracellular mitochondria that fuel the process. To measure these fluctuations, we have employed a high-speed nanolaser technique that samples the osmotic pressure in individual yeast cells and isolated mitochondria. We analyzed 2 yeast cell strains, normal baker’s yeast and a genetically-altered version, that differ only by the presence of mitochondrial DNA. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes. These cells have mitochondria, but the mitochondria lack most normal respiratory chain complexes. The frequency distributions in the nanolaser spectra produced by wild-type and modified cells and mitochondria show a striking shift from Gaussian to Poissonian distributions, revealing a powerful novel method for studying statistical physics of yeast.
Trendeleva, Tatiana A; Sukhanova, Evgenia I; Rogov, Anton G; Zvyagilskaya, Renata A; Seveina, Inna I; Ilyasova, Tatiana M; Cherepanov, Dmitry A; Skulachev, Vladimir P
2013-09-01
The effects of the mitochondria-targeted lipophilic cation dodecyltriphenylphosphonium (C12TPP, the charge is delocalized and screened by bulky hydrophobic residues) and those of lipophilic cations decyltriethylammonium bromide and cetyltrimethylammonium bromide (C10TEA and C16TMA, the charges are localized and screened by less bulky residues) on bilayer planar phospholipid membranes and tightly-coupled mitochondria from the yeast Yarrowia lipolytica have been compared. In planar membranes, C12TPP was found to generate a diffusion potential as if it easily penetrates these membranes. In the presence of palmitate, C12TPP induced H(+) permeability like plastoquinonyl decyltriphenilphosphonium that facilitates transfer of fatty acid anions (Severin et al., PNAS, 2010, 107, 663-668). C12TPP was shown to stimulate State 4 respiration of mitochondria and caused a mitochondrial membrane depolarization with a half-maximal effect at 6μM. Besides, C12TPP profoundly potentiated the uncoupling effect of endogenous or added fatty acids. C10TEA and C16TMA inhibited State 4 respiration and decreased the membrane potential, though at much higher concentrations than C12TPP, and they did not promote the uncoupling action of fatty acids. These relationships were modeled by molecular dynamics. They can be explained by different membrane permeabilities for studied cations, which in turn are due to different availabilities of the positive charge in these cations to water dipoles. Copyright © 2012 Elsevier B.V. All rights reserved.
USDA-ARS?s Scientific Manuscript database
Oleaginous yeasts can accumulate up to 70% of cell biomass as lipids, predominantly as triacylglycerols. Yeast lipid fatty acid profiles have been reported to be similar to that of vegetable oils and consist primarily of oleic, palmitic, stearic, and linoleic acids. This capability provides the oppo...
Heeren, Gino; Rinnerthaler, Mark; Laun, Peter; von Seyerl, Phyllis; Kössler, Sonja; Klinger, Harald; Hager, Matthias; Bogengruber, Edith; Jarolim, Stefanie; Simon-Nobbe, Birgit; Schüller, Christoph; Carmona-Gutierrez, Didac; Breitenbach-Koller, Lore; Mück, Christoph; Jansen-Dürr, Pidder; Criollo, Alfredo; Kroemer, Guido; Madeo, Frank; Breitenbach, Michael
2009-07-13
Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.
Ghosh, Alok; Trivedi, Prachi P; Timbalia, Shrishiv A; Griffin, Aaron T; Rahn, Jennifer J; Chan, Sherine S L; Gohil, Vishal M
2014-07-01
Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx₉CxnCx₁₀C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx₉CxnCx₁₀C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Yoshihisa, Tohru; Yunoki-Esaki, Kaori; Ohshima, Chie; Tanaka, Nobuyuki; Endo, Toshiya
2003-08-01
Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.
Noland, Robert C; Koves, Timothy R; Seiler, Sarah E; Lum, Helen; Lust, Robert M; Ilkayeva, Olga; Stevens, Robert D; Hegardt, Fausto G; Muoio, Deborah M
2009-08-21
In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine diminution was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete beta-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.
Noland, Robert C.; Koves, Timothy R.; Seiler, Sarah E.; Lum, Helen; Lust, Robert M.; Ilkayeva, Olga; Stevens, Robert D.; Hegardt, Fausto G.; Muoio, Deborah M.
2009-01-01
In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine dimunition was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete β-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome. PMID:19553674
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
Methods to study the biogenesis of membrane proteins in yeast mitochondria.
Weckbecker, Daniel; Herrmann, Johannes M
2013-01-01
The biogenesis of mitochondrial membrane proteins is an intricate process that relies on the import and submitochondrial sorting of nuclear-encoded preproteins and on the synthesis of mitochondrial translation products in the matrix. Subsequently, these polypeptides need to be inserted into the outer and the inner membranes of the organelle where many of them assemble into multisubunit complexes. In this chapter we provide established protocols to study these different processes experimentally using mitochondria of budding yeast. In particular, methods are described in detail to purify mitochondria, to study mitochondrial protein synthesis, to follow the import of radiolabeled preproteins into isolated mitochondria, and to assess membrane association and the aggregation of mitochondrial proteins by fractionation. These protocols and a list of dos and don'ts shall enable beginners and experienced scientists to address the targeting and assembly of mitochondrial membrane proteins.
Fatty acyl-CoA reductases of birds
2011-01-01
Background Birds clean and lubricate their feathers with waxes that are produced in the uropygial gland, a holocrine gland located on their back above the tail. The type and the composition of the secreted wax esters are dependent on the bird species, for instance the wax ester secretion of goose contains branched-chain fatty acids and unbranched fatty alcohols, whereas that of barn owl contains fatty acids and alcohols both of which are branched. Alcohol-forming fatty acyl-CoA reductases (FAR) catalyze the reduction of activated acyl groups to fatty alcohols that can be esterified with acyl-CoA thioesters forming wax esters. Results cDNA sequences encoding fatty acyl-CoA reductases were cloned from the uropygial glands of barn owl (Tyto alba), domestic chicken (Gallus gallus domesticus) and domestic goose (Anser anser domesticus). Heterologous expression in Saccharomyces cerevisiae showed that they encode membrane associated enzymes which catalyze a NADPH dependent reduction of acyl-CoA thioesters to fatty alcohols. By feeding studies of transgenic yeast cultures and in vitro enzyme assays with membrane fractions of transgenic yeast cells two groups of isozymes with different properties were identified, termed FAR1 and FAR2. The FAR1 group mainly synthesized 1-hexadecanol and accepted substrates in the range between 14 and 18 carbon atoms, whereas the FAR2 group preferred stearoyl-CoA and accepted substrates between 16 and 20 carbon atoms. Expression studies with tissues of domestic chicken indicated that FAR transcripts were not restricted to the uropygial gland. Conclusion The data of our study suggest that the identified and characterized avian FAR isozymes, FAR1 and FAR2, can be involved in wax ester biosynthesis and in other pathways like ether lipid synthesis. PMID:22151413
Close, Dan; Ojumu, John O.; Zhang, Gui X.
2016-11-03
Cryptococcus terricola JCM 24523 has recently been identified as an oleaginous yeast capable of converting starch into fatty acids. Here, this draft genome sequence provides a platform for elucidating its fatty acid production potential and supporting comparisons with other oleaginous species.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Close, Dan; Ojumu, John O.; Zhang, Gui X.
Cryptococcus terricola JCM 24523 has recently been identified as an oleaginous yeast capable of converting starch into fatty acids. Here, this draft genome sequence provides a platform for elucidating its fatty acid production potential and supporting comparisons with other oleaginous species.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yu X. H.; Shanklin J.; Rawat, R.
Cyclopropane fatty acids (CPA) have been found in certain gymnosperms, Malvales, Litchi and other Sapindales. The presence of their unique strained ring structures confers physical and chemical properties characteristic of unsaturated fatty acids with the oxidative stability displayed by saturated fatty acids making them of considerable industrial interest. While cyclopropenoid fatty acids (CPE) are well-known inhibitors of fatty acid desaturation in animals, CPE can also inhibit the stearoyl-CoA desaturase and interfere with the maturation and reproduction of some insect species suggesting that in addition to their traditional role as storage lipids, CPE can contribute to the protection of plants frommore » herbivory. Three genes encoding cyclopropane synthase homologues GhCPS1, GhCPS2 and GhCPS3 were identified in cotton. Determination of gene transcript abundance revealed differences among the expression of GhCPS1, 2 and 3 showing high, intermediate and low levels, respectively, of transcripts in roots and stems; whereas GhCPS1 and 2 are both expressed at low levels in seeds. Analyses of fatty acid composition in different tissues indicate that the expression patterns of GhCPS1 and 2 correlate with cyclic fatty acid (CFA) distribution. Deletion of the N-terminal oxidase domain lowered GhCPS's ability to produce cyclopropane fatty acid by approximately 70%. GhCPS1 and 2, but not 3 resulted in the production of cyclopropane fatty acids upon heterologous expression in yeast, tobacco BY2 cell and Arabidopsis seed. In cotton GhCPS1 and 2 gene expression correlates with the total CFA content in roots, stems and seeds. That GhCPS1 and 2 are expressed at a similar level in seed suggests both of them can be considered potential targets for gene silencing to reduce undesirable seed CPE accumulation. Because GhCPS1 is more active in yeast than the published Sterculia CPS and shows similar activity when expressed in model plant systems, it represents a strong candidate
Effect of Selenium on Lipid and Amino Acid Metabolism in Yeast Cells.
Kieliszek, Marek; Błażejak, Stanisław; Bzducha-Wróbel, Anna; Kot, Anna M
2018-04-19
This article discusses the effect of selenium in aqueous solutions on aspects of lipid and amino acid metabolism in the cell biomass of Saccharomyces cerevisiae MYA-2200 and Candida utilis ATCC 9950 yeasts. The yeast biomass was obtained by using waste products (potato wastewater and glycerol). Selenium, at a dose of 20 mg/L of aqueous solution, affected the differentiation of cellular morphology. Yeast enriched with selenium was characterized by a large functional diversity in terms of protein and amino acid content. The protein content in the biomass of S. cerevisiae enriched with selenium (42.6%) decreased slightly as compared to that in the control sample without additional selenium supplementation (48.4%). Moreover, yeasts of both strains enriched with selenium contained a large amount of glutamic acid, aspartic acid, lysine, and leucine. Analysis of fatty acid profiles in S. cerevisiae yeast supplemented with selenium showed an increase in the unsaturated fatty acid content (e.g., C18:1). The presence of margaric acid (C17:0) and hexadecanoic acid (C17:1) was found in the C. utilis biomass enriched with selenium, in contrast to that of S. cerevisiae. These results indicate that selenium may induce lipid peroxidation, which consequently affects the loss of integrity of the cytoplasmic membrane. Yeast enriched with selenium with optimal amino acid and lipid composition can be used to prepare a novel formula of dietary supplements, which can be applied directly to various diets for both humans and animals.
Javed, Muhammad Ahsan; Wen, Li; Awais, Muhammad; Latawiec, Diane; Huang, Wei; Chvanov, Michael; Schaller, Sophie; Bordet, Thierry; Michaud, Magali; Pruss, Rebecca; Tepikin, Alexei; Criddle, David; Sutton, Robert
2018-01-01
Objectives Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP). Methods Changes in mitochondrial membrane potential (Δψm), cytosolic Ca2+ ([Ca2+]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology. Results TRO40303 prevented loss of Δψm and necrosis induced by 100 μM palmitoleic acid ethyl ester or 500 μM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP. Conclusions TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP. PMID:29200128
Arlia-Ciommo, Anthony; Leonov, Anna; Beach, Adam; Richard, Vincent R.; Bourque, Simon D.; Burstein, Michelle T.; Kyryakov, Pavlo; Gomez-Perez, Alejandra; Koupaki, Olivia; Feldman, Rachel; Titorenko, Vladimir I.
2018-01-01
A dietary regimen of caloric restriction delays aging in evolutionarily distant eukaryotes, including the budding yeast Saccharomyces cerevisiae. Here, we assessed how caloric restriction influences morphological, biochemical and cell biological properties of chronologically aging yeast advancing through different stages of the aging process. Our findings revealed that this low-calorie diet slows yeast chronological aging by mechanisms that coordinate the spatiotemporal dynamics of various cellular processes before entry into a non-proliferative state and after such entry. Caloric restriction causes a stepwise establishment of an aging-delaying cellular pattern by tuning a network that assimilates the following: 1) pathways of carbohydrate and lipid metabolism; 2) communications between the endoplasmic reticulum, lipid droplets, peroxisomes, mitochondria and the cytosol; and 3) a balance between the processes of mitochondrial fusion and fission. Through different phases of the aging process, the caloric restriction-dependent remodeling of this intricate network 1) postpones the age-related onsets of apoptotic and liponecrotic modes of regulated cell death; and 2) actively increases the chance of cell survival by supporting the maintenance of cellular proteostasis. Because caloric restriction decreases the risk of cell death and actively increases the chance of cell survival throughout chronological lifespan, this dietary intervention extends longevity of chronologically aging yeast. PMID:29662634
The role of the mitochondrial pyruvate carrier in substrate regulation
Vacanti, Nathaniel M.; Divakaruni, Ajit S.; Green, Courtney R.; Parker, Seth J.; Henry, Robert R.; Ciaraldi, Theodore P.; Murphy, Anne N.; Metallo, Christian M.
2014-01-01
SUMMARY Pyruvate lies at a central biochemical node connecting carbohydrate, amino acid, and fatty acid metabolism, and the regulation of pyruvate flux into mitochondria represents a critical step in intermediary metabolism impacting numerous diseases. To characterize changes in mitochondrial substrate utilization in the context of compromised mitochondrial pyruvate transport, we applied 13C metabolic flux analysis (MFA) to cells after transcriptional or pharmacological inhibition of the mitochondrial pyruvate carrier (MPC). Despite profound suppression of both glucose and pyruvate oxidation, cell growth, oxygen consumption, and tricarboxylic acid (TCA) metabolism were surprisingly maintained. Oxidative TCA flux was achieved through enhanced reliance on glutaminolysis through malic enzyme and pyruvate dehydrogenase (PDH) as well as fatty acid and branched chain amino acid oxidation. Thus, in contrast to inhibition of complex I or PDH, suppression of pyruvate transport induces a form of metabolic flexibility associated with use of lipids and amino acids as catabolic and anabolic fuels. PMID:25458843
Weekes, M. P.; Antrobus, R.; Rorbach, J.; van Haute, L.; Umrania, Y.; Smith, D. L.; Minczuk, M.; Lehner, P. J.; Sinclair, J. H.
2016-01-01
ABSTRACT Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. PMID:27025248
The rice mitochondrial iron transporter is essential for plant growth
Bashir, Khurram; Ishimaru, Yasuhiro; Shimo, Hugo; Nagasaka, Seiji; Fujimoto, Masaru; Takanashi, Hideki; Tsutsumi, Nobuhiro; An, Gynheung; Nakanishi, Hiromi; Nishizawa, Naoko K.
2011-01-01
In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Δmrs3Δmrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development. PMID:21610725
Uric Acid Induces Hepatic Steatosis by Generation of Mitochondrial Oxidative Stress
Lanaspa, Miguel A.; Sanchez-Lozada, Laura G.; Choi, Yea-Jin; Cicerchi, Christina; Kanbay, Mehmet; Roncal-Jimenez, Carlos A.; Ishimoto, Takuji; Li, Nanxing; Marek, George; Duranay, Murat; Schreiner, George; Rodriguez-Iturbe, Bernardo; Nakagawa, Takahiko; Kang, Duk-Hee; Sautin, Yuri Y.; Johnson, Richard J.
2012-01-01
Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states. PMID:23035112
Unraveling Biochemical Pathways Affected by Mitochondrial Dysfunctions Using Metabolomic Approaches
Demine, Stéphane; Reddy, Nagabushana; Renard, Patricia; Raes, Martine; Arnould, Thierry
2014-01-01
Mitochondrial dysfunction(s) (MDs) can be defined as alterations in the mitochondria, including mitochondrial uncoupling, mitochondrial depolarization, inhibition of the mitochondrial respiratory chain, mitochondrial network fragmentation, mitochondrial or nuclear DNA mutations and the mitochondrial accumulation of protein aggregates. All these MDs are known to alter the capacity of ATP production and are observed in several pathological states/diseases, including cancer, obesity, muscle and neurological disorders. The induction of MDs can also alter the secretion of several metabolites, reactive oxygen species production and modify several cell-signalling pathways to resolve the mitochondrial dysfunction or ultimately trigger cell death. Many metabolites, such as fatty acids and derived compounds, could be secreted into the blood stream by cells suffering from mitochondrial alterations. In this review, we summarize how a mitochondrial uncoupling can modify metabolites, the signalling pathways and transcription factors involved in this process. We describe how to identify the causes or consequences of mitochondrial dysfunction using metabolomics (liquid and gas chromatography associated with mass spectrometry analysis, NMR spectroscopy) in the obesity and insulin resistance thematic. PMID:25257998
Mitochondrial DNA repair and damage tolerance.
Stein, Alexis; Sia, Elaine A
2017-01-01
The accurate maintenance of mitochondrial DNA (mtDNA) is required in order for eukaryotic cells to assemble a functional electron transport chain. This independently-maintained genome relies on nuclear-encoded proteins that are imported into the mitochondria to carry out replication and repair processes. Decades of research has made clear that mitochondria employ robust and varied mtDNA repair and damage tolerance mechanisms in order to ensure the proper maintenance of the mitochondrial genome. This review focuses on our current understanding of mtDNA repair and damage tolerance pathways including base excision repair, mismatch repair, homologous recombination, non-homologous end joining, translesion synthesis and mtDNA degradation in both yeast and mammalian systems.
Förster, M E; Staib, W
1992-07-01
1. The kinetics of mitochondrial mammalian pyruvate dehydrogenase multienzyme complex (PDHC) is studied by the formation of CO2 using tracer amounts of [1-14C]pyruvate. It is found that the Hill plot results in a (pseudo-)cooperativity with a transition of n-1----3 at a pyruvate concentration about Ks. 2. Addition of L-carnitine, octanoate, palmitoyl-CoA or palmitate + L-carnitine + fatty acid-binding protein results in a Hill coefficient of n = 2 following the kinetics of pyruvate oxidation. 3. Addition of fatty acid-binding protein to an assay system oxidizing palmitate in presence of L-carnitine alters the pattern of the kinetics in the Hill plot so that an apparently lower level of L-carnitine is necessary for the reaction course of beta-degradation. 4. It is concluded that beta-degradation is a coordinated, multienzyme-complex based mechanism tightly linked to citric acid cycle and it is proposed that L-carnitine is actively involved into the reaction and not only functioning as carrier-molecule for transmembrane transport.
Transport of pyruvate and lactate in yeast mitochondria.
Briquet, M
1977-02-07
Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented. 1. The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited. 2. The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate. 3. The [14C]pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate. 4. Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate. These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA.
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.
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.
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.
Cappello, M S; Poltronieri, P; Blaiotta, G; Zacheo, G
2010-11-15
The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO₂ and temperature) useful for industrial application. Copyright © 2010 Elsevier B.V. All rights reserved.
Effects of a spoilage yeast from silage on in vitro ruminal fermentation.
Santos, M C; Lock, A L; Mechor, G D; Kung, L
2015-04-01
Feeding silages with high concentrations of yeasts from aerobic spoilage is often implicated as a cause of poor animal performance on dairies. Our objective was to determine if a commonly found spoilage yeast, isolated from silage, had the potential to alter in vitro ruminal fermentations. A single colony of Issatchenkia orientalis, isolated from high-moisture corn, was grown in selective medium. The yeast culture was purified and added to in vitro culture tubes containing a total mixed ration (43% concentrate, 43% corn silage, 11% alfalfa haylage, and 3% alfalfa hay on a dry matter basis), buffer, and ruminal fluid to achieve added theoretical final concentrations of 0 (CTR), 4.40 (low yeast; LY), 6.40 (medium yeast; MY), and 8.40 (high yeast; HY) log10 cfu of yeast/mL of in vitro fluid. Seven separate tubes were prepared for each treatment and each time point and incubated for 12 and 24h at 39 °C. At the end of the incubation period, samples were analyzed for pH, yeast number, neutral detergent fiber (NDF) digestibility, volatile fatty acids (VFA), and fatty acids (FA). We found that total viable yeast counts decreased for all treatments in in vitro incubations but were still relatively high (5.3 log10 cfu of yeasts/mL) for HY after 24h of incubation. Addition of HY resulted in a lower pH and higher concentration of total VFA in culture fluid compared with other treatments. Moreover, additions of MY and HY decreased in vitro NDF digestibility compared with CTR, and the effect was greatest for HY. Overall, the biohydrogenation of dietary unsaturated FA was not altered by addition of I. orientalis and decreased over time with an increase in the accumulation of saturated FA, especially palmitic and stearic acids. We conclude that addition of I. orientalis, especially at high levels, has the potential to reduce in vitro NDF digestion and alter other aspects of ruminal fermentations. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All
Modification of the feeding behavior of dairy cows through live yeast supplementation.
DeVries, T J; Chevaux, E
2014-10-01
The objective of this study was to determine if the feeding behavior of dairy cows is modified through live yeast supplementation. Twelve lactating Holstein dairy cows (2 primiparous and 10 multiparous) were individually exposed, in a replicated crossover design, to each of 2 treatment diets (over 35-d periods): (1) a control TMR and (2) a control TMR plus 1 × 10(10) cfu/head per day of live yeast (Saccharomyces cerevisiae CNCM I-1077; Levucell SC20; Lallemand Animal Nutrition, Montreal, QC, Canada). Milk production, feeding, and rumination behavior were electronically monitored for each animal for the last 7 d of each treatment period. Milk samples were collected for the last 6 d of each period for milk component analysis. Dry matter intake (28.3 kg/d), eating time (229.3 min/d), and rate (0.14 kg of dry matter/min) were similar between treatments. With yeast supplementation, meal criteria (minimum intermeal interval) were shorter (20.0 vs. 25.8 min), translating to cows tending to have more meals (9.0 vs. 7.8 meals/d), which tended to be smaller in size (3.4 vs. 3.8 kg/meal). Yeast-supplemented cows also tended to ruminate longer (570.3 vs. 544.9 min/d). Milk yield (45.8 kg/d) and efficiency of production (1.64 kg of milk/kg of dry matter intake) were similar between treatments. A tendency for higher milk fat percent (3.71 vs. 3.55%) and yield (1.70 vs. 1.63 kg/d) was observed when cows were supplemented with yeast. No differences in milk fatty acid composition were observed, with the exception of a tendency for a greater concentration of 18:2 cis-9,cis-12 fatty acid (2.71 vs. 2.48% of total fatty acids) with yeast supplementation. Yeast-supplemented cows had lower mean ruminal temperature (38.4 vs. 38.5 °C) and spent less time with rumen temperature above 39.0 °C (353.1 vs. 366.9 min/d), potentially indicating improved rumen pH conditions. Overall, the results show that live yeast supplementation tended to improve meal patterns and rumination, rumen
Echinococcus granulosus fatty acid binding proteins subcellular localization.
Alvite, Gabriela; Esteves, Adriana
2016-05-01
Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.
Rajvanshi, Praveen Kumar; Arya, Madhuri; Rajasekharan, Ram
2017-11-10
The transcription factors Msn2 and Msn4 (multicopy suppressor of SNF1 mutation proteins 2 and 4) bind the stress-response element in gene promoters in the yeast Saccharomyces cerevisiae However, the roles of Msn2/4 in primary metabolic pathways such as fatty acid β-oxidation are unclear. Here, in silico analysis revealed that the promoters of most genes involved in the biogenesis, function, and regulation of the peroxisome contain Msn2/4-binding sites. We also found that transcript levels of MSN2/MSN4 are increased in glucose-depletion conditions and that during growth in nonpreferred carbon sources, Msn2 is constantly localized to the nucleus in wild-type cells. Of note, the double mutant msn2 Δ msn4 Δ exhibited a severe growth defect when grown with oleic acid as the sole carbon source and had reduced transcript levels of major β-oxidation genes. ChIP indicated that Msn2 has increased occupancy on the promoters of β-oxidation genes in glucose-depleted conditions, and in vivo reporter gene analysis indicated reduced expression of these genes in msn2 Δ msn4 Δ cells. Moreover, mobility shift assays revealed that Msn4 binds β-oxidation gene promoters. Immunofluorescence microscopy with anti-peroxisome membrane protein antibodies disclosed that the msn2 Δ msn4 Δ strain had fewer peroxisomes than the wild type, and lipid analysis indicated that the msn2 Δ msn4 Δ strain had increased triacylglycerol and steryl ester levels. Collectively, our data suggest that Msn2/Msn4 transcription factors activate expression of the genes involved in fatty acid oxidation. Because glucose sensing, signaling, and fatty acid β-oxidation pathways are evolutionarily conserved throughout eukaryotes, the msn2 Δ msn4 Δ strain could therefore be a good model system for further study of these critical processes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
USDA-ARS?s Scientific Manuscript database
Oleaginous yeasts can accumulate up to 70% of cell biomass as lipid, predominantly as triacylglycerols. Yeast lipid fatty acid profiles have been reported to be similar to that of vegetable oils and consist primarily of oleic, palmitic, stearic, and linoleic acids. This capability provides the oppor...
NASA Technical Reports Server (NTRS)
Hong, Mee Young; Chapkin, Robert S.; Barhoumi, Rola; Burghardt, Robert C.; Turner, Nancy D.; Henderson, Cara E.; Sanders, Lisa M.; Fan, Yang-Yi; Davidson, Laurie A.; Murphy, Mary E.;
2002-01-01
We have shown that a combination of fish oil (high in n-3 fatty acids) with the butyrate-producing fiber pectin, upregulates apoptosis in colon cells exposed to the carcinogen azoxymethane, protecting against colon tumor development. We now hypothesize that n-3 fatty acids prime the colonocytes such that butyrate can initiate apoptosis. To test this, 30 Sprague-Dawley rats were provided with diets differing in the fatty acid composition (corn oil, fish oil or a purified fatty acid ethyl ester diet). Intact colon crypts were exposed ex vivo to butyrate, and analyzed for reactive oxygen species (ROS), mitochondrial membrane potential (MMP), translocation of cytochrome C to the cytosol, and caspase-3 activity (early events in apoptosis). The fatty acid composition of the three major mitochondrial phospholipids was also determined, and an unsaturation index calculated. The unsaturation index in cardiolipin was correlated with ROS levels (R = 0.99; P = 0.02). When colon crypts from fish oil and FAEE-fed rats were exposed to butyrate, MMP decreased (P = 0.041); and translocation of cytochrome C to the cytosol (P = 0.037) and caspase-3 activation increased (P = 0.032). The data suggest that fish oil may prime the colonocytes for butyrate-induced apoptosis by enhancing the unsaturation of mitochondrial phospholipids, especially cardiolipin, resulting in an increase in ROS and initiating apoptotic cascade.
Bienholz, Anja; Al-Taweel, Ahmad; Roeser, Nancy F; Kribben, Andreas; Feldkamp, Thorsten; Weinberg, Joel M
2014-01-01
Kidney proximal tubules subjected to hypoxia/reoxygenation develop a nonesterified fatty acid-induced energetic deficit characterized by persistent partial mitochondrial deenergization that can be prevented and reversed by citric acid cycle substrates. To further assess the role of competition between fatty acids and substrates on inner membrane substrate carriers in the deenergization and the contribution to deenergization of fatty acid effects on respiratory function, digitonin-permeabilized rabbit and mouse tubules were studied using either addition of exogenous oleate after control normoxic incubation or increases of endogenous fatty acids produced by hypoxia/reoxygenation. The results demonstrated major effects of matrix oxaloacetate accumulation on succinate-supported energization and respiration and their modification by fatty acids. Improvements of energization in the presence of fatty acids by glutamate were shown to result predominantly from lowering matrix oxaloacetate rather than from amelioration of transmembrane cycling of fatty acids and uncoupling. Mouse tubules had 2.5 fold higher rates of succinate utilization, which resulted in stronger effects of oxaloacetate accumulation than rabbit tubules. Hypoxia/reoxygenation induced respiratory inhibition that was more severe for complex I-dependent substrates. Fatty acids themselves did not acutely contribute to this respiratory inhibition, but lowering them during 60 min. reoxygenation to allow recovery of ATP during that period alleviated it. These data clarify the basis for the nonesterified fatty acid-induced mitochondrial energetic deficit in kidney proximal tubules that impairs structural and functional recovery and provide insight into interactions that need to be considered in the design of substrate-based interventions to improve mitochondrial function.
Noninvasive probes of mitochondrial molecular motors
NASA Astrophysics Data System (ADS)
Nawarathna, Dharmakeerthna; Claycomb, James
2005-03-01
We report on a noninvasive method of probing mitochondrial molecular motors using nonlinear dielectric spectroscopy. It has been found previously that enzymes in the plasma membrane, particularly H+ ATPase, result in a strong low frequency (less than 100 Hz) nonlinear harmonic response. In this study, we find evidence that molecular motors located in the inner membranes of mitochondria cause the generation of harmonics at relatively high frequencies (1 - 30 kHz). In particular, we find that potassium cyanide (KCN), a respiratory inhibitor that binds to cytochrome c oxidase and thus prevents transport of protons across the mitochondrial inner membrane, suppresses the harmonic response. We observe this behavior in yeast (S. cerevisiae), a eucaryote that typically contains about 300 mitochondria, and B. indicas, a procaryote believed to be related to the ancient ancestor of mitochondria. Our current modeling efforts are focusing on a Brownian ratchet model of the F0 unit of ATP synthase, a remarkable molecular turbine driven by the proton gradient across the mitochondrial inner membrane.
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.
Yeast aconitase binds and provides metabolically coupled protection to mitochondrial DNA.
Chen, Xin Jie; Wang, Xiaowen; Butow, Ronald A
2007-08-21
Aconitase (Aco1p) is a multifunctional protein: It is an enzyme of the tricarboxylic acid cycle. In animal cells, Aco1p also is a cytosolic protein binding to mRNAs to regulate iron metabolism. In yeast, Aco1p was identified as a component of mtDNA nucleoids. Here we show that yeast Aco1p protects mtDNA from excessive accumulation of point mutations and ssDNA breaks and suppresses reductive recombination of mtDNA. Aconitase binds to both ds- and ssDNA, with a preference for GC-containing sequences. Therefore, mitochondria are opportunistic organelles that seize proteins, such as metabolic enzymes, for construction of the nucleoid, an mtDNA maintenance/segregation apparatus.
Tucci, Sara; Mingirulli, Nadja; Wehbe, Zeinab; Dumit, Verónica I; Kirschner, Janbernd; Spiekerkoetter, Ute
2018-01-01
The white skeletal muscle of very long-chain acyl-CoA-dehydrogenase-deficient (VLCAD -/- ) mice undergoes metabolic modification to compensate for defective β-oxidation in a progressive and time-dependent manner by upregulating glucose oxidation. This metabolic regulation seems to be accompanied by morphologic adaptation of muscle fibers toward the glycolytic fiber type II with the concomitant upregulation of mitochondrial fatty acid biosynthesis (mFASII) and lipoic acid biosynthesis. Dietary supplementation of VLCAD -/- mice with different medium-chain triglycerides over 1 year revealed that odd-chain species has no effect on muscle fiber switch, whereas even-chain species inhibit progressive metabolic adaptation. Our study shows that muscle may undergo adaptive mechanisms that are modulated by dietary supplementation. We describe for the first time a concomitant change of mFASII in this muscular adaptation process. © 2017 Federation of European Biochemical Societies.
Kolondra, Adam; Labedzka-Dmoch, Karolina; Wenda, Joanna M; Drzewicka, Katarzyna; Golik, Pawel
2015-10-21
Yeasts show remarkable variation in the organization of their mitochondrial genomes, yet there is little experimental data on organellar gene expression outside few model species. Candida albicans is interesting as a human pathogen, and as a representative of a clade that is distant from the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Unlike them, it encodes seven Complex I subunits in its mtDNA. No experimental data regarding organellar expression were available prior to this study. We used high-throughput RNA sequencing and traditional RNA biology techniques to study the mitochondrial transcriptome of C. albicans strains BWP17 and SN148. The 14 protein-coding genes, two ribosomal RNA genes, and 24 tRNA genes are expressed as eight primary polycistronic transcription units. We also found transcriptional activity in the noncoding regions, and antisense transcripts that could be a part of a regulatory mechanism. The promoter sequence is a variant of the nonanucleotide identified in other yeast mtDNAs, but some of the active promoters show significant departures from the consensus. The primary transcripts are processed by a tRNA punctuation mechanism into the monocistronic and bicistronic mature RNAs. The steady state levels of various mature transcripts exhibit large differences that are a result of posttranscriptional regulation. Transcriptome analysis allowed to precisely annotate the positions of introns in the RNL (2), COB (2) and COX1 (4) genes, as well as to refine the annotation of tRNAs and rRNAs. Comparative study of the mitochondrial genome organization in various Candida species indicates that they undergo shuffling in blocks usually containing 2-3 genes, and that their arrangement in primary transcripts is not conserved. tRNA genes with their associated promoters, as well as GC-rich sequence elements play an important role in these evolutionary events. The main evolutionary force shaping the mitochondrial genomes of yeasts is the
Khot, Mahesh; Ghosh, Debashish
2017-04-01
This study analyzes the single cell oil (SCO), fatty acid profile, and biodiesel fuel properties of the yeast Rhodotorula mucilaginosa IIPL32 grown on the pentose fraction of acid pre-treated sugarcane bagasse as a carbon source. The yeast biomass from nitrogen limiting culture conditions (15.3 g L -1 ) was able to give the SCO yield of 0.17 g g -1 of xylose consumed. Acid digestion, cryo-pulverization, direct in situ transesterification, and microwave assisted techniques were evaluated in comparison to the Soxhlet extraction for the total intracellular yeast lipid recovery. The significant differences were observed among the SCO yield of different methods and the in situ transesterification stood out most for effective yeast lipid recovery generating 97.23 mg lipid as FAME per gram dry biomass. The method was fast and consumed lesser solvent with greater FAME yield while accessing most cellular fatty acids present. The yeast lipids showed the major presence of monounsaturated fatty esters (35-55%; 18:1, 16:1) suitable for better ignition quality, oxidative stability, and cold-flow properties of the biodiesel. Analyzed fuel properties (density, kinematic viscosity, cetane number) of the yeast oil were in good agreement with international biodiesel standards. The sugarcane bagasse-derived xylose and the consolidated comparative assessment of lab scale SCO recovery methods highlight the necessity for careful substrate choice and validation of analytical method in yeast oil research. The use of less toxic co-solvents together with solvent recovery and recycling would help improve process economics for sustainable production of biodiesel from the hemicellulosic fraction of cheap renewable sources. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe
Duhig, Trevor; Nam, Miyoung; Palmer, Georgia; Han, Sangjo; Jeffery, Linda; Baek, Seung-Tae; Lee, Hyemi; Shim, Young Sam; Lee, Minho; Kim, Lila; Heo, Kyung-Sun; Noh, Eun Joo; Lee, Ah-Reum; Jang, Young-Joo; Chung, Kyung-Sook; Choi, Shin-Jung; Park, Jo-Young; Park, Youngwoo; Kim, Hwan Mook; Park, Song-Kyu; Park, Hae-Joon; Kang, Eun-Jung; Kim, Hyong Bai; Kang, Hyun-Sam; Park, Hee-Moon; Kim, Kyunghoon; Song, Kiwon; Song, Kyung Bin; Nurse, Paul; Hoe, Kwang-Lae
2014-01-01
SUMMARY We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome. This resource provides a powerful tool for biotechnological and eukaryotic cell biology research. Comprehensive gene dispensability comparisons with budding yeast, the first time such studies have been possible between two eukaryotes, revealed that 83% of single copy orthologues in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than non-essential genes to be single copy, broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth. PMID:20473289
Van Hoeck, V; Leroy, J L M R; Arias Alvarez, M; Rizos, D; Gutierrez-Adan, A; Schnorbusch, K; Bols, P E J; Leese, H J; Sturmey, R G
2013-01-01
Elevated plasma nonesterified fatty acid (NEFA) concentrations are associated with negative energy balance and metabolic disorders such as obesity and type II diabetes. Such increased plasma NEFA concentrations induce changes in the microenvironment of the ovarian follicle, which can compromise oocyte competence. Exposing oocytes to elevated NEFA concentrations during maturation affects the gene expression and phenotype of the subsequent embryo, notably prompting a disrupted oxidative metabolism. We hypothesized that these changes in the embryo are a consequence of modified energy metabolism in the oocyte. To investigate this, bovine cumulus oocyte complexes were matured under elevated NEFA conditions, and energy metabolism-related gene expression, mitochondrial function, and ultrastructure evaluated. It was found that expression of genes related to REDOX maintenance was modified in NEFA-exposed oocytes, cumulus cells, and resultant blastocysts. Moreover, the expression of genes related to fatty acid synthesis in embryos that developed from NEFA-exposed oocytes was upregulated. From a functional perspective, inhibition of fatty acid β-oxidation in maturing oocytes exposed to elevated NEFA concentrations restored developmental competence. There were no clear differences in mitochondrial morphology or oxygen consumption between treatments, although there was a trend for a higher mitochondrial membrane potential in zygotes derived from NEFA-exposed oocytes. These data show that the degree of mitochondrial fatty acid β-oxidation has a decisive impact on the development of NEFA-exposed oocytes. Furthermore, the gene expression data suggest that the resulting embryos adapt through altered metabolic strategies, which might explain the aberrant energy metabolism previously observed in these embryos originating from NEFA-exposed maturing oocytes.
A Functional Genomics Approach to Identify Novel Breast Cancer Gene Targets in Yeast
2005-05-01
Chaleff DT, Valent B, Fink GR. Mutations affecting Ty-mediated expression of the HIS4 gene of Saccharomyces cerevisiae. Genetics 1984; 107(2): 179-97... mutations , and are synthetically lethal with rotl mutations ROX3 YBL093C Repressor Of hypoXic genes : RNA polymerase I1 holcenzyme component 3,3 SSS...mitochondrial gene products; mutation causes an elevated rate of mitochondrial turnover; 3 MOD after 60 generations, MOD on NaCI YNDI YER005W Yeast Nucleoside
Murakawa, Tomokazu; Yamaguchi, Osamu; Hashimoto, Ayako; Hikoso, Shungo; Takeda, Toshihiro; Oka, Takafumi; Yasui, Hiroki; Ueda, Hiromichi; Akazawa, Yasuhiro; Nakayama, Hiroyuki; Taneike, Manabu; Misaka, Tomofumi; Omiya, Shigemiki; Shah, Ajay M.; Yamamoto, Akitsugu; Nishida, Kazuhiko; Ohsumi, Yoshinori; Okamoto, Koji; Sakata, Yasushi; Otsu, Kinya
2015-01-01
Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells. PMID:26146385
Kochan, Kamila; Peng, Huadong; Wood, Bayden R; Haritos, Victoria S
2018-01-01
Biodiesel is a valuable renewable fuel made from derivatized fatty acids produced in plants, animals, and oleaginous microbes. Of the latter, yeasts are of special interest due to their wide use in biotechnology, ability to synthesize fatty acids and store large amounts of triacylglycerols while utilizing non-food carbon sources. While yeast efficiently produce lipids, genetic modification and indeed, lipid pathway metabolic engineering, is usually required for cost-effective production. Traditionally, gas chromatography (GC) is used to measure fatty acid production and to track the success of a metabolic engineering strategy in a microbial culture; here we have employed vibrational spectroscopy approaches at population and single cell level of engineered yeast while simultaneously investigating metabolite levels in subcellular structures. Firstly, a strong correlation ( r 2 > 0.99) was established between Fourier transform infrared (FTIR) lipid in intact cells and GC analysis of fatty acid methyl esters in the differently engineered strains. Confocal Raman spectroscopy of individual cells carrying genetic modifications to enhance fatty acid synthesis and lipid accumulation revealed changes to the lipid body (LB), the storage organelle for lipids in yeast, with their number increasing markedly (up to tenfold higher); LB size was almost double in the strain that also expressed a LB stabilizing gene but considerable variation was also noted between cells. Raman spectroscopy revealed a clear trend toward reduced unsaturated fatty acid content in lipids of cells carrying more complex metabolic engineering. Atomic force microscopy-infrared spectroscopy (AFM-IR) analysis of individual cells indicated large differences in subcellular constituents between strains: cells of the most highly engineered strain had elevated lipid and much reduced carbohydrate in their cytoplasm compared with unmodified cells. Vibrational spectroscopy analysis allowed the simultaneous
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.
Zhang, Lin; Zhou, Ying; Wu, Wangjun; Hou, Liming; Chen, Hongxing; Zuo, Bo; Xiong, Yuanzhu; Yang, Jinzeng
2017-01-01
Individual skeletal muscles in the animal body are heterogeneous, as each is comprised of different fiber types. Type I muscle fibers are rich with mitochondria, and have high oxidative metabolisms while type IIB fibers have few mitochondria and high glycolytic metabolic capacity. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a transcriptional co-activator that regulates mitochondrial biogenesis and respiratory function, is implicated in muscle fiber-type switching. Over-expression of PGC-1α in transgenic mice increased the proportion of red/oxidative type I fiber. During pig muscle growth, an increased number of type I fibers can give meat more red color. To explore the roles of PGC-1α in regulation of muscle fiber type conversion, we generated skeletal muscle-specific PGC-1α transgenic mice and pig. Ectopic over-expression of PGC-1α was detected in both fast and slow muscle fibers. The transgenic animals displayed a remarkable amount of red/oxidative muscle fibers in major skeletal muscle tissues. Skeletal muscles from transgenic mice and pigs have increased expression levels of oxidative fiber markers such as MHC1, MHC2x, myoglobin and Tnni1, and decreased expressions of glycolytic fiber genes (MHC2a, MHC2b, CASQ-1 and Tnni2). The genes responsible for the TCA cycle and oxidative phosphorylation, cytochrome coxidase 2 and 4, and citrate synthase were also increased in the transgenic mice and pigs. These results suggested that transgenic over-expressed PGC-1α significantly increased muscle mitochondrial biogenesis, resulting in qualitative changes from glycolytic to oxidative energy generation. The transgenic animals also had elevated levels of PDK4 and PPARγ proteins in muscle tissue, which can lead to increased glycogen deposition and fatty acid oxidation. Therefore, the results support a significant role of PGC-1α in conversion of fast glycolytic fibers to slow and oxidative fiber through enhanced mitochondrial respiration
Non-Alcoholic Fatty Liver Disease.
Engin, Atilla
2017-01-01
Non-alcoholic fatty liver disease (NAFLD) is in parallel with the obesity epidemic and it is the most common cause of liver diseases. The development of hepatic steatosis in majority of patients is linked to dietary fat ingestion. NAFLD is characterized by excess accumulation of triglyceride in the hepatocyte due to both increased inflow of free fatty acids and de novo hepatic lipogenesis. Insulin resistance with the deficiency of insulin receptor substrate-2 (IRS-2)-associated phosphatidylinositol 3-kinase (PI3K) activity causes an increase in intracellular fatty acid-derived metabolites such as diacylglycerol, fatty acyl CoA or ceramides. Lipotoxicity-related mechanism of NAFLD could be explained still best by the "double-hit" hypothesis. Insulin resistance is the major mechanism in the development and progression of NAFLD/Non-alcoholic steatohepatitis (NASH). Metabolic oxidative stress, autophagy, and inflammation induce NASH progression. In the "first hit" the hepatic concentrations of diacylglycerol increase with rising saturated liver fat content in human NAFLD. Activities of mitochondrial respiratory chain complexes are decreased in liver tissue of patients with NASH. Furthermore, hepatocyte lipoapoptosis is a critical feature of NASH. In "second hit" reduced glutathione levels due to oxidative stress lead to overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling that induces cell death in the steatotic liver. Accumulation of toxic levels of reactive oxygen species (ROS) is caused by the ineffectual cycling of the endoplasmic reticulum (ER) oxidoreductin (Ero1)-protein disulfide isomerase oxidation cycle through the downstream of the inner membrane mitochondrial oxidative metabolism and Kelch like-ECH-associated protein 1 (Keap1)- Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway.
Control mechanisms in mitochondrial oxidative phosphorylation☆
Hroudová, Jana; Fišar, Zdeněk
2013-01-01
Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5’- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5’-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by “second control mechanisms,” such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5’-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production. PMID:25206677
Control mechanisms in mitochondrial oxidative phosphorylation.
Hroudová, Jana; Fišar, Zdeněk
2013-02-05
Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism-firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.
Gao, Qi; Cao, Xuan; Huang, Yu-Ying; Yang, Jing-Lin; Chen, Jun; Wei, Liu-Jing; Hua, Qiang
2018-05-18
Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y. lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated β-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y. lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.
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
Glycosylceramide modifies the flavor and metabolic characteristics of sake yeast.
Ferdouse, Jannatul; Yamamoto, Yuki; Taguchi, Seiga; Yoshizaki, Yumiko; Takamine, Kazunori; Kitagaki, Hiroshi
2018-01-01
In the manufacture of sake, Japanese traditional rice wine, sake yeast is fermented with koji, which is steamed rice fermented with the non-pathogenic fungus Aspergillus oryzae . During fermentation, sake yeast requires lipids, such as unsaturated fatty acids and sterols, in addition to substances provided by koji enzymes for fermentation. However, the role of sphingolipids on the brewing characteristics of sake yeast has not been studied. In this study, we revealed that glycosylceramide, one of the sphingolipids abundant in koji, affects yeast fermentation. The addition of soy, A. oryzae , and Grifola frondosa glycosylceramide conferred a similar effect on the flavor profiles of sake yeast. In particular, the addition of A. oryzae and G. frondosa glycosylceramide were very similar in terms of the decreases in ethyl caprylate and ethyl 9-decenoate. The addition of soy glycosylceramide induced metabolic changes to sake yeast such as a decrease in glucose, increases in ethanol and glycerol and changes in several amino acids and organic acids concentrations. Tricarboxylic acid (TCA) cycle, pyruvate metabolism, starch and sucrose metabolism, and glycerolipid metabolism were overrepresented in the cultures incubated with sake yeast and soy glycosylceramide. This is the first study of the effect of glycosylceramide on the flavor and metabolic profile of sake yeast.
Glycosylceramide modifies the flavor and metabolic characteristics of sake yeast
Taguchi, Seiga; Yoshizaki, Yumiko; Takamine, Kazunori
2018-01-01
In the manufacture of sake, Japanese traditional rice wine, sake yeast is fermented with koji, which is steamed rice fermented with the non-pathogenic fungus Aspergillus oryzae. During fermentation, sake yeast requires lipids, such as unsaturated fatty acids and sterols, in addition to substances provided by koji enzymes for fermentation. However, the role of sphingolipids on the brewing characteristics of sake yeast has not been studied. In this study, we revealed that glycosylceramide, one of the sphingolipids abundant in koji, affects yeast fermentation. The addition of soy, A. oryzae, and Grifola frondosa glycosylceramide conferred a similar effect on the flavor profiles of sake yeast. In particular, the addition of A. oryzae and G. frondosa glycosylceramide were very similar in terms of the decreases in ethyl caprylate and ethyl 9-decenoate. The addition of soy glycosylceramide induced metabolic changes to sake yeast such as a decrease in glucose, increases in ethanol and glycerol and changes in several amino acids and organic acids concentrations. Tricarboxylic acid (TCA) cycle, pyruvate metabolism, starch and sucrose metabolism, and glycerolipid metabolism were overrepresented in the cultures incubated with sake yeast and soy glycosylceramide. This is the first study of the effect of glycosylceramide on the flavor and metabolic profile of sake yeast. PMID:29761062
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.
Aconitase couples metabolic regulation to mitochondrial DNA maintenance.
Chen, Xin Jie; Wang, Xiaowen; Kaufman, Brett A; Butow, Ronald A
2005-02-04
Mitochondrial DNA (mtDNA) is essential for cells to maintain respiratory competency and is inherited as a protein-DNA complex called the nucleoid. We have identified 22 mtDNA-associated proteins in yeast, among which is mitochondrial aconitase (Aco1p). We show that this Krebs-cycle enzyme is essential for mtDNA maintenance independent of its catalytic activity. Regulation of ACO1 expression by the HAP and retrograde metabolic signaling pathways directly affects mtDNA maintenance. When constitutively expressed, Aco1p can replace the mtDNA packaging function of the high-mobility-group protein Abf2p. Thus, Aco1p may integrate metabolic signals and mtDNA maintenance.
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.
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.
Mattiazzi Ušaj, M.; Brložnik, M.; Kaferle, P.; Žitnik, M.; Wolinski, H.; Leitner, F.; Kohlwein, S.D.; Zupan, B.; Petrovič, U.
2015-01-01
Pex11 is a peroxin that regulates the number of peroxisomes in eukaryotic cells. Recently, it was found that a mutation in one of the three mammalian paralogs, PEX11β, results in a neurological disorder. The molecular function of Pex11, however, is not known. Saccharomyces cerevisiae Pex11 has been shown to recruit to peroxisomes the mitochondrial fission machinery, thus enabling proliferation of peroxisomes. This process is essential for efficient fatty acid β-oxidation. In this study, we used high-content microscopy on a genome-wide scale to determine the subcellular localization pattern of yeast Pex11 in all non-essential gene deletion mutants, as well as in temperature-sensitive essential gene mutants. Pex11 localization and morphology of peroxisomes was profoundly affected by mutations in 104 different genes that were functionally classified. A group of genes encompassing MDM10, MDM12 and MDM34 that encode the mitochondrial and cytosolic components of the ERMES complex was analyzed in greater detail. Deletion of these genes caused a specifically altered Pex11 localization pattern, whereas deletion of MMM1, the gene encoding the fourth, endoplasmic-reticulum-associated component of the complex, did not result in an altered Pex11 localization or peroxisome morphology phenotype. Moreover, we found that Pex11 and Mdm34 physically interact and that Pex11 plays a role in establishing the contact sites between peroxisomes and mitochondria through the ERMES complex. Based on these results, we propose that the mitochondrial/cytosolic components of the ERMES complex establish a direct interaction between mitochondria and peroxisomes through Pex11. PMID:25769804
Curbo, Sophie; Lagier-Tourenne, Clotilde; Carrozzo, Rosalba; Palenzuela, Lluis; Lucioli, Simona; Hirano, Michio; Santorelli, Filippo; Arenas, Joaquin; Karlsson, Anna; Johansson, Magnus
2006-03-01
Pyrophosphatases (PPases) catalyze the hydrolysis of inorganic pyrophosphate generated in several cellular enzymatic reactions. A novel human pyrophosphatase cDNA encoding a 334-amino-acid protein approximately 60% identical to the previously identified human cytosolic PPase was cloned and characterized. The novel enzyme, named PPase-2, was enzymatically active and catalyzed hydrolysis of pyrophosphate at a rate similar to that of the previously identified PPase-1. A functional mitochondrial import signal sequence was identified in the N-terminus of PPase-2, which targeted the enzyme to the mitochondrial matrix. The human pyrophosphatase 2 gene (PPase-2) was mapped to chromosome 4q25 and the 1.4-kb mRNA was ubiquitously expressed in human tissues, with highest levels in muscle, liver, and kidney. The yeast homologue of the mitochondrial PPase-2 is required for mitochondrial DNA maintenance and yeast cells lacking the enzyme exhibit mitochondrial DNA depletion. We sequenced the PPA2 gene in 13 patients with mitochondrial DNA depletion syndromes (MDS) of unknown cause to determine if mutations in the PPA2 gene of these patients were associated with this disease. No pathogenic mutations were identified in the PPA2 gene of these patients and we found no evidence that PPA2 gene mutations are a common cause of MDS in humans.
Yi Lo, Jennifer Chi; O'Connor, Anne E; Andrews, Zane B; Lo, Camden; Tiganis, Tony; Watt, Matthew J; O'Bryan, Moira K
2016-12-01
Fatty liver, or hepatic steatosis, is an alarmingly common pathology in western societies, in large part because if left unheeded, it can lead to life-threatening forms of nonalcoholic fatty liver disease, including nonalcoholic steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. As such, it is essential that we attain a greater understanding of the pathways that control energy partitioning in the liver and ultimately how they are impacted by environmental factors. Here, we define the essential requirement for a member of the Ras-related protein in the brain (RAB)-like (RABL) clade of small GTPases, RABL2, in fatty acid metabolism including in microtubule-associated mitochondrial movement within the liver. RABL2 dysfunction, even in mice fed a low-fat chow diet, leads to retarded hepatic mitochondria movement associated with and a cascading phenotype of interrelated metabolic defects reminiscent of a type 2 diabetic state: hepatic steatosis, insulin resistance, glucose intolerance, and adult onset obesity. RABL2 dysfunction does not, however, alter mitochondrial content, or the inherent respiratory capacity of individual mitochondria per se. Rather, it is associated with a decreased capacity for fatty oxidation in the context of the intact cell, suggesting a complex, and important, role for mitochondrial movement in metabolic health. Our data highlight the importance of RABL2 and mitochondrial dynamics in hepatic fatty acid oxidation and in the achievement of metabolic balance.
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
Nesterov, S V; Skorobogatova, Iu A; Iaguzhinskiĭ, L S
2014-01-01
This paper represents the study of endogenous and exogenous fatty acids affecting the mitochondrial phosphorylation system effectiveness depending on temperature. The experiment was set up under conditions in which the oxidative phosphorylation system operates as a supercomplex. Rat liver mitochondria were isolated without purposive fatty acids removal from membranes, then studied in hypotonic medium (120 mOsm). We managed to detect a very narrow interval 19 ± 1°C where the fatty acid uncoupling effect is weak up to disappearing. At the same small temperature range, a structural rearrangement that takes place in the enzyme system is accompanied with denser packing of membrane protein complexes. Thus, at the temperatures close to 19°C the supercomplex works in the specific regime protected (or partially protected) from the uncoupling effect of fatty acids. Here we also discuss a physiological significance of the increased ATP-synthesis effectiveness at lower temperatures and the most probable character of structural rearrangement taking place at 19°C in the enzymes in the mitochondrial membrane.
Browne, Christopher M.; Samir, Parimal; Fites, J. Scott; Villarreal, Seth A.
2013-01-01
Using affinity purifications coupled with mass spectrometry and yeast two-hybrid assays, we show the Saccharomyces cerevisiae translation initiation factor complex eukaryotic translation initiation factor 2B (eIF2B) and the very-long-chain fatty acid (VLCFA) synthesis keto-reductase enzyme YBR159W physically interact. The data show that the interaction is specifically between YBR159W and eIF2B and not between other members of the translation initiation or VLCFA pathways. A ybr159wΔ null strain has a slow-growth phenotype and a reduced translation rate but a normal GCN4 response to amino acid starvation. Although YBR159W localizes to the endoplasmic reticulum membrane, subcellular fractionation experiments show that a fraction of eIF2B cofractionates with lipid membranes in a YBR159W-independent manner. We show that a ybr159wΔ yeast strain and other strains with null mutations in the VLCFA pathway cause eIF2B to appear as numerous foci throughout the cytoplasm. PMID:23263984
Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts.
Guo, Yi-Cheng; Zhang, Lin; Dai, Shao-Xing; Li, Wen-Xing; Zheng, Jun-Juan; Li, Gong-Hua; Huang, Jing-Fei
2016-01-01
Dekkera yeasts have often been considered as alternative sources of ethanol production that could compete with S. cerevisiae. The two lineages of yeasts independently evolved traits that include high glucose and ethanol tolerance, aerobic fermentation, and a rapid ethanol fermentation rate. The Saccharomyces yeasts attained these traits mainly through whole genome duplication approximately 100 million years ago (Mya). However, the Dekkera yeasts, which were separated from S. cerevisiae approximately 200 Mya, did not undergo whole genome duplication (WGD) but still occupy a niche similar to S. cerevisiae. Upon analysis of two Dekkera yeasts and five closely related non-WGD yeasts, we found that a massive loss of cis-regulatory elements occurred in an ancestor of the Dekkera yeasts, which led to improved mitochondrial functions similar to the S. cerevisiae yeasts. The evolutionary analysis indicated that genes involved in the transcription and translation process exhibited faster evolution in the Dekkera yeasts. We detected 90 positively selected genes, suggesting that the Dekkera yeasts evolved an efficient translation system to facilitate adaptive evolution. Moreover, we identified that 12 vacuolar H+-ATPase (V-ATPase) function genes that were under positive selection, which assists in developing tolerance to high alcohol and high sugar stress. We also revealed that the enzyme PGK1 is responsible for the increased rate of glycolysis in the Dekkera yeasts. These results provide important insights to understand the independent adaptive evolution of the Dekkera yeasts and provide tools for genetic modification promoting industrial usage.
Independent Evolution of Winner Traits without Whole Genome Duplication in Dekkera Yeasts
Dai, Shao-Xing; Li, Wen-Xing; Zheng, Jun-Juan; Li, Gong-Hua; Huang, Jing-Fei
2016-01-01
Dekkera yeasts have often been considered as alternative sources of ethanol production that could compete with S. cerevisiae. The two lineages of yeasts independently evolved traits that include high glucose and ethanol tolerance, aerobic fermentation, and a rapid ethanol fermentation rate. The Saccharomyces yeasts attained these traits mainly through whole genome duplication approximately 100 million years ago (Mya). However, the Dekkera yeasts, which were separated from S. cerevisiae approximately 200 Mya, did not undergo whole genome duplication (WGD) but still occupy a niche similar to S. cerevisiae. Upon analysis of two Dekkera yeasts and five closely related non-WGD yeasts, we found that a massive loss of cis-regulatory elements occurred in an ancestor of the Dekkera yeasts, which led to improved mitochondrial functions similar to the S. cerevisiae yeasts. The evolutionary analysis indicated that genes involved in the transcription and translation process exhibited faster evolution in the Dekkera yeasts. We detected 90 positively selected genes, suggesting that the Dekkera yeasts evolved an efficient translation system to facilitate adaptive evolution. Moreover, we identified that 12 vacuolar H+-ATPase (V-ATPase) function genes that were under positive selection, which assists in developing tolerance to high alcohol and high sugar stress. We also revealed that the enzyme PGK1 is responsible for the increased rate of glycolysis in the Dekkera yeasts. These results provide important insights to understand the independent adaptive evolution of the Dekkera yeasts and provide tools for genetic modification promoting industrial usage. PMID:27152421
Sanz, Alberto; Soikkeli, Mikko; Portero-Otín, Manuel; Wilson, Angela; Kemppainen, Esko; McIlroy, George; Ellilä, Simo; Kemppainen, Kia K.; Tuomela, Tea; Lakanmaa, Matti; Kiviranta, Essi; Stefanatos, Rhoda; Dufour, Eric; Hutz, Bettina; Naudí, Alba; Jové, Mariona; Zeb, Akbar; Vartiainen, Suvi; Matsuno-Yagi, Akemi; Yagi, Takao; Rustin, Pierre; Pamplona, Reinald; Jacobs, Howard T.
2010-01-01
Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling. PMID:20435911
Darvishi, Farshad; Ariana, Mehdi; Marella, Eko Roy; Borodina, Irina
2018-07-01
Oleaginous yeast Yarrowia lipolytica is an important industrial host for the production of enzymes, oils, fragrances, surfactants, cosmetics, and pharmaceuticals. More recently, improved synthetic biology tools have allowed more extensive engineering of this yeast species, which lead to the production of non-native metabolites. In this review, we summarize the recent advances of genome editing tools for Y. lipolytica, including the application of CRISPR/Cas9 system and discuss case studies, where Y. lipolytica was engineered to produce various non-native chemicals: short-chain fatty alcohols and alkanes as biofuels, polyunsaturated fatty acids for nutritional and pharmaceutical applications, polyhydroxyalkanoates and dicarboxylic acids as precursors for biodegradable plastics, carotenoid-type pigments for food and feed, and campesterol as a precursor for steroid drugs.
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.
Deryabina, Y I; Isakova, E P; Shurubor, E I; Zvyagilskaya, R A
2004-09-01
Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.
Moreno-García, Jaime; García-Martínez, Teresa; Moreno, Juan; Millán, M Carmen; Mauricio, Juan Carlos
2014-02-17
Saccharomyces cerevisiae "flor" yeast shows a strong tolerance to high ethanol concentrations and develops a velum (biofilm) on the wine surface after the alcoholic fermentation of grape must. This velum remains along several years during the so called "biological aging" process in the elaboration of some special wines carried out in specific regions around the world and it contributes to the typical organoleptic characteristics of these wines. In order to grow in this condition, flor yeast has to elaborate a response where the mitochondrial function is essential. The objective of this study is to elucidate the role of the mitochondria in the response of a flor yeast, S. cerevisiae G1, growing in a controlled velum formation condition. For this purpose, proteome and metabolome were characterized by comparing data with those from an initial fermentative condition used as reference. The obtained proteomic profiles show more mitochondrial proteins related with the ethanol resistance (13), cell respiration (18), mitochondrial genome maintenance (13), and apoptosis (2) detected under the velum formation condition. Also, the finger-printing obtained by means of the exo-metabolites directly related with the quality of fermented beverages and quantified in the velum condition shows important differences from those obtained in the reference condition. Copyright © 2013 Elsevier B.V. All rights reserved.
ERIC Educational Resources Information Center
Waisbren, Susan E.; Landau, Yuval; Wilson, Jenna; Vockley, Jerry
2013-01-01
Mitochondrial fatty acid oxidation disorders include conditions in which the transport of activated acyl-Coenzyme A (CoA) into the mitochondria or utilization of these substrates is disrupted or blocked. This results in a deficit in the conversion of fat into energy. Most patients with fatty acid oxidation defects are now identified through…
Advanced tools for the analysis of protein phosphorylation in yeast mitochondria.
Walter, Corvin; Gonczarowska-Jorge, Humberto; Sickmann, Albert; Zahedi, René P; Meisinger, Chris; Schmidt, Oliver
2018-05-24
The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn 2+ -Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods. Copyright © 2018. Published by Elsevier Inc.
Yeast Biomass Production in Brewery's Spent Grains Hemicellulosic Hydrolyzate
NASA Astrophysics Data System (ADS)
Duarte, Luís C.; Carvalheiro, Florbela; Lopes, Sónia; Neves, Ines; Gírio, Francisco M.
Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery's spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h-1, 0.61 g g-1, and 0.56 g 1-1 h-1, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.
Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria
Kathiresan, Meena; Martins, Dorival; English, Ann M.
2014-01-01
In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453
Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.
Kathiresan, Meena; Martins, Dorival; English, Ann M
2014-12-09
In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.
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
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
Influence of aeration during propagation of pitching yeast on fermentation and beer flavor.
Cheong, Chul; Wackerbauer, Karl; Kang, Soon Ah
2007-02-01
The effect of yeast propagated at different aeration conditions on yeast physiology, fermentation ability, and beer quality was investigated using three strains of Saccharomyces cerevisiae. It was shown that yeast cells grown under continuous aeration conditions during propagation were almost two times higher as compared with discontinuous aeration conditions. The maximum of cell growth of all samples reached between 36 h and 48 h. The concentration of trehalose was increased under continuous aerated yeasts, whereas glycogen was decreased. It was also observed that the concentration of glycogen and trehalose in yeast cells had no direct effect on subsequent fermentation ability. The effect of yeast propagated under different aeration conditions on subsequent fermentation ability was different from yeast strains, in which the influence will be most pronounced at the first fermentation. Later, the yeasts might regain its original characteristics in the following fermentations. Generally, continuously propagated yeast had a positive effect on beer quality in subsequent fermentation. Hence, the concentration of aroma compounds obtained with yeast propagated under 6 1/h for 48 h aeration was lower than those grown under other aeration conditions in the bottom yeasts; in particular, the amounts of phenylethyl alcohol, ester, and fatty acids were decreased.
PARP10 (ARTD10) modulates mitochondrial function
Nagy, Lilla; Vida, András; Kis, Gréta; Brunyánszki, Attila; Antal, Miklós; Lüscher, Bernhard; Bai, Péter
2018-01-01
Poly(ADP-ribose) polymerase (PARP)10 is a PARP family member that performs mono-ADP-ribosylation of target proteins. Recent studies have linked PARP10 to metabolic processes and metabolic regulators that prompted us to assess whether PARP10 influences mitochondrial oxidative metabolism. The depletion of PARP10 by specific shRNAs increased mitochondrial oxidative capacity in cellular models of breast, cervical, colorectal and exocrine pancreas cancer. Upon silencing of PARP10, mitochondrial superoxide production decreased in line with increased expression of antioxidant genes pointing out lower oxidative stress upon PARP10 silencing. Improved mitochondrial oxidative capacity coincided with increased AMPK activation. The silencing of PARP10 in MCF7 and CaCo2 cells decreased the proliferation rate that correlated with increased expression of anti-Warburg enzymes (Foxo1, PGC-1α, IDH2 and fumarase). By analyzing an online database we showed that lower PARP10 expression increases survival in gastric cancer. Furthermore, PARP10 expression decreased upon fasting, a condition that is characterized by increases in mitochondrial biogenesis. Finally, lower PARP10 expression is associated with increased fatty acid oxidation. PMID:29293500
Carrau, Francisco M; Medina, Karina; Farina, Laura; Boido, Eduardo; Henschke, Paul A; Dellacassa, Eduardo
2008-11-01
The contribution of yeast fermentation metabolites to the aromatic profile of wine is well documented; however, the biotechnological application of this knowledge, apart from strain selection, is still rather limited and often contradictory. Understanding and modeling the relationship between nutrient availability and the production of desirable aroma compounds by different strains must be one of the main objectives in the selection of industrial yeasts for the beverage and food industry. In order to overcome the variability in the composition of grape juices, we have used a chemically defined model medium for studying yeast physiological behavior and metabolite production in response to nitrogen supplementation so as to identify an appropriate yeast assimilable nitrogen level for strain differentiation. At low initial nitrogen concentrations, strain KU1 produced higher quantities of esters and fatty acids whereas M522 produced higher concentrations of isoacids, gamma-butyrolactone, higher alcohols and 3-methylthio-1-propanol. We propose that although strains KU1 and M522 have a similar nitrogen consumption profile, they represent useful models for the chemical characterization of wine strains in relation to wine quality. The differential production of aroma compounds by the two strains is discussed in relation to their capacity for nitrogen usage and their impact on winemaking. The results obtained here will help to develop targeted metabolic footprinting methods for the discrimination of industrial yeasts.
Comparison of nitrogen depletion and repletion on lipid production in yeast and fungal species
Yang, Shihui; Wang, Wei; Wei, Hui; ...
2016-08-29
Although it is well known that low nitrogen stimulates lipid accumulation, especially for algae and some oleaginous yeast, few studies have been conducted in fungal species, especially on the impact of different nitrogen deficiency strategies. In this study, we use two promising consolidated bioprocessing (CBP) candidates to examine the impact of two nitrogen deficiency strategies on lipid production, which are the extensively investigated oleaginous yeast Yarrowia lipolytica, and the commercial cellulase producer Trichoderma reesei. We first utilized bioinformatics approaches to reconstruct the fatty acid metabolic pathway and demonstrated the presence of a triacylglycerol (TAG) biosynthesis pathway in Trichoderma reesei. Wemore » then examined the lipid production of Trichoderma reesei and Y. lipomyces in different media using two nitrogen deficiency strategies of nitrogen natural repletion and nitrogen depletion through centrifugation. Our results demonstrated that nitrogen depletion was better than nitrogen repletion with about 30% lipid increase for Trichoderma reesei and Y. lipomyces, and could be an option to improve lipid production in both oleaginous yeast and filamentous fungal species. The resulting distinctive lipid composition profiles indicated that the impacts of nitrogen depletion on yeast were different from those for fungal species. Under three types of C/N ratio conditions, C16 and C18 fatty acids were the predominant forms of lipids for both Trichoderma reesei and Y. lipolytica. In addition, while the overall fatty acid methyl ester (FAME) profiles of Trichoderma reesei were similar, the overall FAME profiles of Y. lipolytica observed a shift. The fatty acid metabolic pathway reconstructed in this work supports previous reports of lipid production in T. reesei, and provides a pathway for future omics studies and metabolic engineering efforts. Further investigation to identify the genetic targets responsible for the effect of nitrogen depletion
Comparison of nitrogen depletion and repletion on lipid production in yeast and fungal species
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yang, Shihui; Wang, Wei; Wei, Hui
Although it is well known that low nitrogen stimulates lipid accumulation, especially for algae and some oleaginous yeast, few studies have been conducted in fungal species, especially on the impact of different nitrogen deficiency strategies. In this study, we use two promising consolidated bioprocessing (CBP) candidates to examine the impact of two nitrogen deficiency strategies on lipid production, which are the extensively investigated oleaginous yeast Yarrowia lipolytica, and the commercial cellulase producer Trichoderma reesei. We first utilized bioinformatics approaches to reconstruct the fatty acid metabolic pathway and demonstrated the presence of a triacylglycerol (TAG) biosynthesis pathway in Trichoderma reesei. Wemore » then examined the lipid production of Trichoderma reesei and Y. lipomyces in different media using two nitrogen deficiency strategies of nitrogen natural repletion and nitrogen depletion through centrifugation. Our results demonstrated that nitrogen depletion was better than nitrogen repletion with about 30% lipid increase for Trichoderma reesei and Y. lipomyces, and could be an option to improve lipid production in both oleaginous yeast and filamentous fungal species. The resulting distinctive lipid composition profiles indicated that the impacts of nitrogen depletion on yeast were different from those for fungal species. Under three types of C/N ratio conditions, C16 and C18 fatty acids were the predominant forms of lipids for both Trichoderma reesei and Y. lipolytica. In addition, while the overall fatty acid methyl ester (FAME) profiles of Trichoderma reesei were similar, the overall FAME profiles of Y. lipolytica observed a shift. The fatty acid metabolic pathway reconstructed in this work supports previous reports of lipid production in T. reesei, and provides a pathway for future omics studies and metabolic engineering efforts. Further investigation to identify the genetic targets responsible for the effect of nitrogen depletion
Physiology, ecology and industrial applications of aroma formation in yeast
Dzialo, Maria C; Park, Rahel; Steensels, Jan; Lievens, Bart; Verstrepen, Kevin J
2017-01-01
Abstract Yeast cells are often employed in industrial fermentation processes for their ability to efficiently convert relatively high concentrations of sugars into ethanol and carbon dioxide. Additionally, fermenting yeast cells produce a wide range of other compounds, including various higher alcohols, carbonyl compounds, phenolic compounds, fatty acid derivatives and sulfur compounds. Interestingly, many of these secondary metabolites are volatile and have pungent aromas that are often vital for product quality. In this review, we summarize the different biochemical pathways underlying aroma production in yeast as well as the relevance of these compounds for industrial applications and the factors that influence their production during fermentation. Additionally, we discuss the different physiological and ecological roles of aroma-active metabolites, including recent findings that point at their role as signaling molecules and attractants for insect vectors. PMID:28830094
Ruggiero, Christine; Elks, Carrie M.; Kruger, Claudia; Cleland, Ellen; Addison, Kaity; Noland, Robert C.
2014-01-01
Albuminuria is associated with metabolic syndrome and diabetes. It correlates with the progression of chronic kidney disease, particularly with tubular atrophy. The fatty acid load on albumin significantly increases in obesity, presenting a proinflammatory environment to the proximal tubules. However, little is known about changes in the redox milieu during fatty acid overload and how redox-sensitive mechanisms mediate cell death. Here, we show that albumin with fatty acid impurities or conjugated with palmitate but not albumin itself compromised mitochondrial and cell viability, membrane potential and respiration. Fatty acid overload led to a redox imbalance which deactivated the antioxidant protein peroxiredoxin 2 and caused a peroxide-mediated apoptosis through the redox-sensitive pJNK/caspase-3 pathway. Transfection of tubular cells with peroxiredoxin 2 was protective and mitigated apoptosis. Mitochondrial fatty acid entry and ceramide synthesis modulators suggested that mitochondrial β oxidation but not ceramide synthesis may modulate lipotoxic effects on tubular cell survival. These results suggest that albumin overloaded with fatty acids but not albumin itself changes the redox environment in the tubules, inducing a peroxide-mediated redox-sensitive apoptosis. Thus, mitigating circulating fatty acid levels may be an important factor in both preserving redox balance and preventing tubular cell damage in proteinuric diseases. PMID:24500687
Aoun, Manar; Michel, Francoise; Fouret, Gilles; Schlernitzauer, Audrey; Ollendorff, Vincent; Wrutniak-Cabello, Chantal; Cristol, Jean-Paul; Carbonneau, Marie-Annette; Coudray, Charles; Feillet-Coudray, Christine
2011-08-01
Accumulation of muscle TAG content and modification of muscle phospholipid fatty acid pattern may have an impact on lipid metabolism, increasing the risk of developing diabetes. Some polyphenols have been reported to modulate lipid metabolism, in particular those issued from red grapes. The present study was designed to determine whether a grape polyphenol extract (PPE) modulates skeletal muscle TAG content and phospholipid fatty acid composition in high-fat-high-sucrose (HFHS) diet-fed rats. Muscle plasmalemmal and mitochondrial fatty acid transporters, GLUT4 and lipid metabolism pathways were also explored. The PPE decreased muscle TAG content in HFHS/PPE diet-fed rats compared with HFHS diet-fed rats and induced higher proportions of n-3 PUFA in phospholipids. The PPE significantly up-regulated GLUT4 mRNA expression. Gene and protein expression of muscle fatty acid transporter cluster of differentiation 36 (CD36) was increased in HFHS diet-fed rats but returned to control values in HFHS/PPE diet-fed rats. Carnitine palmitoyltransferase 1 protein expression was decreased with the PPE. Mitochondrial β-hydroxyacyl CoA dehydrogenase was increased in HFHS diet-fed rats and returned to control values with PPE supplementation. Lipogenesis, mitochondrial biogenesis and mitochondrial activity were not affected by the PPE. In conclusion, the PPE modulated membrane phospholipid fatty acid composition and decreased muscle TAG content in HFHS diet-fed rats. The PPE lowered CD36 gene and protein expression, probably decreasing fatty acid transport and lipid accumulation within skeletal muscle, and increased muscle GLUT4 expression. These effects of the PPE are in favour of a better insulin sensibility.
Xu, Jing; Nie, Hong-gang; Zhang, Xiao-dong; Tian, Ye; Yu, Bo
2011-08-01
The majority of experimental and clinical studies indicates that the hypertrophied and failing myocardium are characterized by changes in energy and substrate metabolism that attributed to failing heart changes at the genomic level, in fact, heart failure is caused by various diseases, their energy metabolism and substrate are in different genetic variations, then the potential significance of the molecular mechanisms for the aetiology of heart failure is necessary to be evaluated. Persistent viral infection (especially coxsackievirus group B3) of the myocardium in viral myocarditis and viral dilated cardiomyopathy has never been neglected by experts. This study aimed to explore the role and regulatory mechanism of the altered gene expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism in viral dilated cardiomyopathy. cDNA Microarray technology was used to evaluate the expression of >35,852 genes in a mice model of viral dilated cardiomyopathy. In total 1385 highly different genes expression, we analyzed 33 altered genes expression for energy metabolism involved in mitochondrial oxidative phosphorylation, fatty acid metabolism and further selected real-time-PCR for quantity one of regulatory mechanisms for energy including fatty acid metabolism-the UCP2 and assayed cytochrome C oxidase activity by Spectrophotometer to explore mitochondrial oxidative phosphorylation function. We found obviously different expression of 33 energy metabolism genes associated with mitochondria oxidative phosphorylation, fatty acid metabolism in cardiomyopathy mouse heart, the regulatory gene for energy metabolism: UCP2 was down-regulated and cytochrome C oxidase activity was decreased. Genes involved in both fatty acid metabolism and mitochondrial oxidative phosphorylation were down-regulated, mitochondrial uncoupling proteins (UCP2) expression did not increase but decrease which might be a kind of adaptive protection response to
ERIC Educational Resources Information Center
Rea, Shane L.; Graham, Brett H.; Nakamaru-Ogiso, Eiko; Kar, Adwitiya; Falk, Marni J.
2010-01-01
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.…
Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa
2017-01-01
Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier protein family and mediate regulated proton leak across the inner mitochondrial membrane. Free fatty acids, aldehydes such as hydroxynonenal, and retinoids activate UCPs. However, there are some controversies about the effective action of retinoids and aldehydes alone; thus, only free fatty acids are commonly accepted positive effectors of UCPs. Purine nucleotides such as GTP inhibit UCP-mediated mitochondrial proton leak. In turn, membranous coenzyme Q may play a role as a redox state-dependent metabolic sensor that modulates the complete activation/inhibition of UCPs. Such regulation has been observed for UCPs in microorganisms, plant and animal UCP1 homologues, and UCP1 in mammalian brown adipose tissue. The origin of UCPs is still under debate, but UCP homologues have been identified in all systematic groups of eukaryotes. Despite the differing levels of amino acid/DNA sequence similarities, functional studies in unicellular and multicellular organisms, from amoebae to mammals, suggest that the mechanistic regulation of UCP activity is evolutionarily well conserved. This review focuses on the regulatory feedback loops of UCPs involving free fatty acids, aldehydes, retinoids, purine nucleotides, and coenzyme Q (particularly its reduction level), which may derive from the early stages of evolution as UCP first emerged. Copyright © 2016 Elsevier B.V. All rights reserved.
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.
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
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.
The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders
DOE Office of Scientific and Technical Information (OSTI.GOV)
Ndukwe Erlingsson, Uzochi Chimdinma; Iacobazzi, Francesco; Department of Basic Medical Sciences, University of Bari, Piazza Giulio Cesare 11, Policlinico, I-70124 Bari
Highlights: •Valinomycin can cause mitochondrial stress and stimulate fatty acid oxidation. •Cells with VLCAD deficiency fail to increase fatty acid oxidation in response to valinomycin. •Response to valinomycin can help in the diagnosis of VLCAD deficiency. -- Abstract: Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacitymore » of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP{sup +}) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP{sup +} accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [{sup 14}C]-palmitate oxidation (measured as [{sup 14}C]O{sub 2} release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.« less
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.
Kieliszek, Marek; Jermacz, Karolina; Błażejak, Stanisław
2017-01-01
The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua, Debaryomyces hansenii, Kluyveromyces marxianus, Kazachstania unispora, and Zygotorulaspora florentina. We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L−1. Kazachstania unispora was able to accumulate the high amount of palmitoleic acid. PMID:29098157
Gientka, Iwona; Kieliszek, Marek; Jermacz, Karolina; Błażejak, Stanisław
2017-01-01
The search for efficient oleaginous microorganisms, which can be an alternative to fossil fuels and biofuels obtained from oilseed crops, has been going on for many years. The suitability of microorganisms in this regard is determined by their ability to biosynthesize lipids with preferred fatty acid profile along with the concurrent utilization of energy-rich industrial waste. In this study, we isolated, characterized, and identified kefir yeast strains using molecular biology techniques. The yeast isolates identified were Candida inconspicua , Debaryomyces hansenii , Kluyveromyces marxianus , Kazachstania unispora , and Zygotorulaspora florentina . We showed that deproteinated potato wastewater, a starch processing industry waste, supplemented with various carbon sources, including lactose and glycerol, is a suitable medium for the growth of yeast, which allows an accumulation of over 20% of lipid substances in its cells. Fatty acid composition primarily depended on the yeast strain and the carbon source used, and, based on our results, most of the strains met the criteria required for the production of biodiesel. In particular, this concerns a significant share of saturated fatty acids, such as C16:0 and C18:0, and unsaturated fatty acids, such as C18:1 and C18:2. The highest efficiency in lipid biosynthesis exceeded 6.3 g L -1 . Kazachstania unispora was able to accumulate the high amount of palmitoleic acid.
Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas
2012-01-01
Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.
Krill Oil Ameliorates Mitochondrial Dysfunctions in Rats Treated with High-Fat Diet.
Ferramosca, Alessandra; Conte, Annalea; Zara, Vincenzo
2015-01-01
In recent years, several studies focused their attention on the role of dietary fats in the pathogenesis of hepatic steatosis. It has been demonstrated that a high-fat diet is able to induce hyperglycemia, hyperinsulinemia, obesity, and nonalcoholic fatty liver disease. On the other hand, krill oil, a novel dietary supplement of n-3 PUFAs, has the ability to improve lipid and glucose metabolism, exerting possible protective effects against hepatic steatosis. In this study we have investigated the effects of krill oil on mitochondrial energetic metabolism in animals fed a high-fat diet. To this end, male Sprague-Dawley rats were divided into three groups and fed for 4 weeks with a standard diet (control group), a diet with 35% fat (HF group), or a high-fat diet supplemented with 2.5% krill oil (HF+KO group). The obtained results suggest that krill oil promotes the burning of fat excess introduced by the high-fat diet. This effect is obtained by stimulating mitochondrial metabolic pathways such as fatty acid oxidation, Krebs cycle, and respiratory chain complexes activity. Modulation of the expression of carrier proteins involved in mitochondrial uncoupling was also observed. Overall, krill oil counteracts the negative effects of a high-fat diet on mitochondrial energetic metabolism.
Burr, Risa; Stewart, Emerson V; Espenshade, Peter J
2017-03-31
The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Hsueh, Tun-Yun; Baum, Jamie I; Huang, Yan
2018-01-01
Polyunsaturated fatty acids are important nutrients for human health, especially omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been found to play positive roles in the prevention of various diseases. However, previous studies have reported that excessive omega-3 fatty acids supplement during pregnancy caused side effects such as slower neural transmission times and postnatal growth restriction. In this study, we investigated the effect of EPA and DHA on mitochondrial function and gene expression in C2C12 myoblasts during skeletal muscle differentiation. C2C12 myoblasts were cultured to confluency and then treated with differentiation medium that contained fatty acids (50-µM EPA and DHA). After 72 h of myogenic differentiation, mRNA was collected, and gene expression was analyzed by real-time PCR. Microscopy was used to examine cell morphology following treatment with fatty acids. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XF24 Analyzer. Cells treated with fatty acids had fewer myotubes formed ( P ≤ 0.05) compared with control cells. The expression of the genes related to myogenesis was significantly lower ( P ≤ 0.05) in cells treated with fatty acids, compared with control cells. Genes associated with adipogenesis had higher ( P ≤ 0.05) expression after treatment with fatty acids. Also, the mitochondrial biogenesis decreased with lower ( P ≤ 0.05) gene expression and lower ( P ≤ 0.05) mtDNA/nDNA ratio in cells treated with fatty acids compared with control cells. However, the expression of genes related to peroxisome biosynthesis was higher ( P ≤ 0.05) in cells treated with fatty acids. Moreover, fatty-acid treatment reduced ( P ≤ 0.05) oxygen consumption rate under oligomycin-inhibited (reflecting proton leak) and uncoupled conditions. Our data imply that fatty acids might reduce myogenesis and increase adipogenesis in myotube formation. Fatty acids
The Effects of Physical Exercise on Fatty Liver Disease
van der Windt, Dirk J.; Sud, Vikas; Zhang, Hongji; Tsung, Allan; Huang, Hai
2018-01-01
The increasing prevalence of obesity has made nonalcoholic fatty liver disease (NAFLD) the most common chronic liver disease. As a consequence, NAFLD and especially its inflammatory form nonalcoholic steatohepatitis (NASH) are the fastest increasing etiology of end-stage liver disease and hepatocellular carcinoma. Physical inactivity is related to the severity of fatty liver disease irrespective of body weight, supporting the hypothesis that increasing physical activity through exercise can improve fatty liver disease. This review summarizes the evidence for the effects of physical exercise on NAFLD and NASH. Several clinical trials have shown that both aerobic and resistance exercise reduce the hepatic fat content. From clinical and basic scientific studies, it is evident that exercise affects fatty liver disease through various pathways. Improved peripheral insulin resistance reduces the excess delivery of free fatty acids and glucose for free fatty acid synthesis to the liver. In the liver, exercise increases fatty acid oxidation, decreases fatty acid synthesis, and prevents mitochondrial and hepatocellular damage through a reduction of the release of damage-associated molecular patterns. In conclusion, physical exercise is a proven therapeutic strategy to improve fatty liver disease. PMID:29212576
Lis, Paweł; Jurkiewicz, Paweł; Cal-Bąkowska, Magdalena; Ko, Young H; Pedersen, Peter L; Goffeau, Andre; Ułaszewski, Stanisław
2016-03-01
In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP.
Coudray, Charles; Fouret, Gilles; Lambert, Karen; Ferreri, Carla; Rieusset, Jennifer; Blachnio-Zabielska, Agnieszka; Lecomte, Jérôme; Ebabe Elle, Raymond; Badia, Eric; Murphy, Michael P; Feillet-Coudray, Christine
2016-04-14
The prevalence of the metabolic syndrome components including abdominal obesity, dyslipidaemia and insulin resistance is increasing in both developed and developing countries. It is generally accepted that the development of these features is preceded by, or accompanied with, impaired mitochondrial function. The present study was designed to analyse the effects of a mitochondrial-targeted lipophilic ubiquinone (MitoQ) on muscle lipid profile modulation and mitochondrial function in obesogenic diet-fed rats. For this purpose, twenty-four young male Sprague-Dawley rats were divided into three groups and fed one of the following diets: (1) control, (2) high fat (HF) and (3) HF+MitoQ. After 8 weeks, mitochondrial function markers and lipid metabolism/profile modifications in skeletal muscle were measured. The HF diet was effective at inducing the major features of the metabolic syndrome--namely, obesity, hepatic enlargement and glucose intolerance. MitoQ intake prevented the increase in rat body weight, attenuated the increase in adipose tissue and liver weights and partially reversed glucose intolerance. At the muscle level, the HF diet induced moderate TAG accumulation associated with important modifications in the muscle phospholipid classes and in the fatty acid composition of total muscle lipid. These lipid modifications were accompanied with decrease in mitochondrial respiration. MitoQ intake corrected the lipid alterations and restored mitochondrial respiration. These results indicate that MitoQ protected obesogenic diet-fed rats from some features of the metabolic syndrome through its effects on muscle lipid metabolism and mitochondrial activity. These findings suggest that MitoQ is a promising candidate for future human trials in the metabolic syndrome prevention.
Determination of free fatty acids in beer wort.
Bravi, Elisabetta; Benedetti, Paolo; Marconi, Ombretta; Perretti, Giuseppe
2014-05-15
The importance of free fatty acids (FFAs) in wort has been known for a long time because of their influence on beer quality and yeast metabolism. Lipids have a beneficial effect on yeast growth during fermentation as well as negative effects on beer quality. Lipids content of beer affects the ability to form a stable head of foam and plays an important role in beer staling. Moreover, the ratio of unsaturated and saturated fatty acids seems to be related to gushing problems. A novel, simple, and reliable procedure for quantitative analysis of FFAs in wort was developed and validated. The determination of FFAs in wort was achieved via liquid-liquid cartridge extraction, purification of FFA fraction by solid phase extraction, boron trifluoride in methanol methylation, and injection into GC-FID system. The proposed method has high accuracy (<0.3%, expressed as the bias), high precision (<1.2%, RSD), and recoveries ranging from 74% to 98%. The method was tested on two different wort samples (9° and 12° Plato). Copyright © 2013 Elsevier Ltd. All rights reserved.
Reconstitution of the protein insertion machinery of the mitochondrial inner membrane.
Haucke, V; Schatz, G
1997-01-01
We have reconstituted the protein insertion machinery of the yeast mitochondrial inner membrane into proteoliposomes. The reconstituted proteoliposomes have a distinct morphology and protein composition and correctly insert the ADP/ATP carrier (AAC) and Tim23p, two multi-spanning integral proteins of the mitochondrial inner membrane. The reconstituted system requires a membrane potential, but not Tim44p or mhsp70, both of which are required for the ATP-driven translocation of proteins into the matrix. The protein insertion machinery can thus operate independently of the energy-transducing Tim44p-mhsp70 complex. PMID:9303300
Fatty acids in energy metabolism of the central nervous system.
Panov, Alexander; Orynbayeva, Zulfiya; Vavilin, Valentin; Lyakhovich, Vyacheslav
2014-01-01
In this review, we analyze the current hypotheses regarding energy metabolism in the neurons and astroglia. Recently, it was shown that up to 20% of the total brain's energy is provided by mitochondrial oxidation of fatty acids. However, the existing hypotheses consider glucose, or its derivative lactate, as the only main energy substrate for the brain. Astroglia metabolically supports the neurons by providing lactate as a substrate for neuronal mitochondria. In addition, a significant amount of neuromediators, glutamate and GABA, is transported into neurons and also serves as substrates for mitochondria. Thus, neuronal mitochondria may simultaneously oxidize several substrates. Astrocytes have to replenish the pool of neuromediators by synthesis de novo, which requires large amounts of energy. In this review, we made an attempt to reconcile β-oxidation of fatty acids by astrocytic mitochondria with the existing hypothesis on regulation of aerobic glycolysis. We suggest that, under condition of neuronal excitation, both metabolic pathways may exist simultaneously. We provide experimental evidence that isolated neuronal mitochondria may oxidize palmitoyl carnitine in the presence of other mitochondrial substrates. We also suggest that variations in the brain mitochondrial metabolic phenotype may be associated with different mtDNA haplogroups.
Fatty Acids in Energy Metabolism of the Central Nervous System
Orynbayeva, Zulfiya; Vavilin, Valentin; Lyakhovich, Vyacheslav
2014-01-01
In this review, we analyze the current hypotheses regarding energy metabolism in the neurons and astroglia. Recently, it was shown that up to 20% of the total brain's energy is provided by mitochondrial oxidation of fatty acids. However, the existing hypotheses consider glucose, or its derivative lactate, as the only main energy substrate for the brain. Astroglia metabolically supports the neurons by providing lactate as a substrate for neuronal mitochondria. In addition, a significant amount of neuromediators, glutamate and GABA, is transported into neurons and also serves as substrates for mitochondria. Thus, neuronal mitochondria may simultaneously oxidize several substrates. Astrocytes have to replenish the pool of neuromediators by synthesis de novo, which requires large amounts of energy. In this review, we made an attempt to reconcile β-oxidation of fatty acids by astrocytic mitochondria with the existing hypothesis on regulation of aerobic glycolysis. We suggest that, under condition of neuronal excitation, both metabolic pathways may exist simultaneously. We provide experimental evidence that isolated neuronal mitochondria may oxidize palmitoyl carnitine in the presence of other mitochondrial substrates. We also suggest that variations in the brain mitochondrial metabolic phenotype may be associated with different mtDNA haplogroups. PMID:24883315
Genome sequence of the lager brewing yeast, an interspecies hybrid.
Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko
2009-04-01
This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production.
Genome Sequence of the Lager Brewing Yeast, an Interspecies Hybrid
Nakao, Yoshihiro; Kanamori, Takeshi; Itoh, Takehiko; Kodama, Yukiko; Rainieri, Sandra; Nakamura, Norihisa; Shimonaga, Tomoko; Hattori, Masahira; Ashikari, Toshihiko
2009-01-01
This work presents the genome sequencing of the lager brewing yeast (Saccharomyces pastorianus) Weihenstephan 34/70, a strain widely used in lager beer brewing. The 25 Mb genome comprises two nuclear sub-genomes originating from Saccharomyces cerevisiae and Saccharomyces bayanus and one circular mitochondrial genome originating from S. bayanus. Thirty-six different types of chromosomes were found including eight chromosomes with translocations between the two sub-genomes, whose breakpoints are within the orthologous open reading frames. Several gene loci responsible for typical lager brewing yeast characteristics such as maltotriose uptake and sulfite production have been increased in number by chromosomal rearrangements. Despite an overall high degree of conservation of the synteny with S. cerevisiae and S. bayanus, the syntenies were not well conserved in the sub-telomeric regions that contain lager brewing yeast characteristic and specific genes. Deletion of larger chromosomal regions, a massive unilateral decrease of the ribosomal DNA cluster and bilateral truncations of over 60 genes reflect a post-hybridization evolution process. Truncations and deletions of less efficient maltose and maltotriose uptake genes may indicate the result of adaptation to brewing. The genome sequence of this interspecies hybrid yeast provides a new tool for better understanding of lager brewing yeast behavior in industrial beer production. PMID:19261625
Wu, Baojun; Hao, Weilong
2014-04-16
Group I introns are highly dynamic and mobile, featuring extensive presence-absence variation and widespread horizontal transfer. Group I introns can invade intron-lacking alleles via intron homing powered by their own encoded homing endonuclease gene (HEG) after horizontal transfer or via reverse splicing through an RNA intermediate. After successful invasion, the intron and HEG are subject to degeneration and sequential loss. It remains unclear whether these mechanisms can fully address the high dynamics and mobility of group I introns. Here, we found that HEGs undergo a fast gain-and-loss turnover comparable with introns in the yeast mitochondrial 21S-rRNA gene, which is unexpected, as the intron and HEG are generally believed to move together as a unit. We further observed extensively mosaic sequences in both the introns and HEGs, and evidence of gene conversion between HEG-containing and HEG-lacking introns. Our findings suggest horizontal transfer and gene conversion can accelerate HEG/intron degeneration and loss, or rescue and propagate HEG/introns, and ultimately result in high HEG/intron turnover rate. Given that up to 25% of the yeast mitochondrial genome is composed of introns and most mitochondrial introns are group I introns, horizontal transfer and gene conversion could have served as an important mechanism in introducing mitochondrial intron diversity, promoting intron mobility and consequently shaping mitochondrial genome architecture.
CLUH couples mitochondrial distribution to the energetic and metabolic status.
Wakim, Jamal; Goudenege, David; Perrot, Rodolphe; Gueguen, Naig; Desquiret-Dumas, Valerie; Chao de la Barca, Juan Manuel; Dalla Rosa, Ilaria; Manero, Florence; Le Mao, Morgane; Chupin, Stephanie; Chevrollier, Arnaud; Procaccio, Vincent; Bonneau, Dominique; Logan, David C; Reynier, Pascal; Lenaers, Guy; Khiati, Salim
2017-06-01
Mitochondrial dynamics and distribution are critical for supplying ATP in response to energy demand. CLUH is a protein involved in mitochondrial distribution whose dysfunction leads to mitochondrial clustering, the metabolic consequences of which remain unknown. To gain insight into the role of CLUH on mitochondrial energy production and cellular metabolism, we have generated CLUH-knockout cells using CRISPR/Cas9. Mitochondrial clustering was associated with a smaller cell size and with decreased abundance of respiratory complexes, resulting in oxidative phosphorylation (OXPHOS) defects. This energetic impairment was found to be due to the alteration of mitochondrial translation and to a metabolic shift towards glucose dependency. Metabolomic profiling by mass spectroscopy revealed an increase in the concentration of some amino acids, indicating a dysfunctional Krebs cycle, and increased palmitoylcarnitine concentration, indicating an alteration of fatty acid oxidation, and a dramatic decrease in the concentrations of phosphatidylcholine and sphingomyeline, consistent with the decreased cell size. Taken together, our study establishes a clear function for CLUH in coupling mitochondrial distribution to the control of cell energetic and metabolic status. © 2017. Published by The Company of Biologists Ltd.
Boulet, Aren; Vest, Katherine E.; Maynard, Margaret K.; Gammon, Micah G.; Russell, Antoinette C.; Mathews, Alexander T.; Cole, Shelbie E.; Zhu, Xinyu; Phillips, Casey B.; Kwong, Jennifer Q.; Dodani, Sheel C.; Leary, Scot C.; Cobine, Paul A.
2018-01-01
Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2. Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo. PMID:29237729
Recovery of Fuel-Precursor Lipids from Oleaginous Yeast
Kruger, Jacob S.; Cleveland, Nicholas S.; Yeap, Rou Yi; ...
2018-01-24
Bio-derived lipids offer a potentially promising intermediate to displace petroleum-derived diesel. One of the key challenges for the production of lipids via microbial cell mass is that these products are stored intracellularly and must be extracted and recovered efficiently and economically. Thus, improved methods of cell lysis and lipid extraction are needed. In this study, we examine lipid extraction from wet oleaginous yeast in combination with seven different cell lysis approaches encompassing both physical and chemical techniques (high-pressure homogenization, microwave and conventional thermal treatments, bead beating, acid, base, and enzymatic treatments) to facilitate lipid extraction from a model oleaginous yeastmore » strain, Lipomyces starkeyi. Of the seven techniques investigated, acid treatment led to the highest lipid recovery yields. Further exploration of acid treatment and integration with an economic model revealed that treatment at 170 degrees C for 60 min at 1 wt% H 2SO 4 and 8 wt% yeast solids represents a viable option for both lipid recovery yield and process economics, enabling experimental lipid recovery yields of 88.5-93.0% to be achieved at a corresponding estimated minimum fuel selling price (MFSP) of $5.13-$5.61/gallon of gasoline equivalent (GGE). The same acid treatment conditions applied to two other strains of oleaginous yeast (Cutaneotrichosporon curvatus and Rhodotorula toruloides) resulted in similar lipid recovery yields. In pretreatment experiments scaled up to 300 mL, slightly lower temperatures or shorter pretreatment times, along with higher yeast solids loading, resulted in higher lipid yields than the conditions identified from the small-scale runs. Two replicate runs carried out at 170 degrees C for 30 min using 1 wt% H2SO4 and 19 wt% yeast solids achieved an average lipid recovery of 96.1% at a corresponding estimated MFSP of $4.89/GGE. In all cases, the lipids are primarily triglycerides and free fatty acids
Recovery of Fuel-Precursor Lipids from Oleaginous Yeast
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kruger, Jacob S.; Cleveland, Nicholas S.; Yeap, Rou Yi
Bio-derived lipids offer a potentially promising intermediate to displace petroleum-derived diesel. One of the key challenges for the production of lipids via microbial cell mass is that these products are stored intracellularly and must be extracted and recovered efficiently and economically. Thus, improved methods of cell lysis and lipid extraction are needed. In this study, we examine lipid extraction from wet oleaginous yeast in combination with seven different cell lysis approaches encompassing both physical and chemical techniques (high-pressure homogenization, microwave and conventional thermal treatments, bead beating, acid, base, and enzymatic treatments) to facilitate lipid extraction from a model oleaginous yeastmore » strain, Lipomyces starkeyi. Of the seven techniques investigated, acid treatment led to the highest lipid recovery yields. Further exploration of acid treatment and integration with an economic model revealed that treatment at 170 degrees C for 60 min at 1 wt% H 2SO 4 and 8 wt% yeast solids represents a viable option for both lipid recovery yield and process economics, enabling experimental lipid recovery yields of 88.5-93.0% to be achieved at a corresponding estimated minimum fuel selling price (MFSP) of $5.13-$5.61/gallon of gasoline equivalent (GGE). The same acid treatment conditions applied to two other strains of oleaginous yeast (Cutaneotrichosporon curvatus and Rhodotorula toruloides) resulted in similar lipid recovery yields. In pretreatment experiments scaled up to 300 mL, slightly lower temperatures or shorter pretreatment times, along with higher yeast solids loading, resulted in higher lipid yields than the conditions identified from the small-scale runs. Two replicate runs carried out at 170 degrees C for 30 min using 1 wt% H2SO4 and 19 wt% yeast solids achieved an average lipid recovery of 96.1% at a corresponding estimated MFSP of $4.89/GGE. In all cases, the lipids are primarily triglycerides and free fatty acids
Wang, Mingxuan; Chen, Haiqin; Gu, Zhennan; Zhang, Hao; Chen, Wei; Chen, Yong Q
2013-12-01
The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.
Targeted exome sequencing of suspected mitochondrial disorders
Lieber, Daniel S.; Calvo, Sarah E.; Shanahan, Kristy; Slate, Nancy G.; Liu, Shangtao; Hershman, Steven G.; Gold, Nina B.; Chapman, Brad A.; Thorburn, David R.; Berry, Gerard T.; Schmahmann, Jeremy D.; Borowsky, Mark L.; Mueller, David M.; Sims, Katherine B.
2013-01-01
Objective: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity. Methods: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines. Results: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency. Conclusion: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting. PMID:23596069
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.
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.
Regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes
Bender, Tom; Pena, Gabrielle; Martinou, Jean-Claude
2015-01-01
At the pyruvate branch point, the fermentative and oxidative metabolic routes diverge. Pyruvate can be transformed either into lactate in mammalian cells or into ethanol in yeast, or transported into mitochondria to fuel ATP production by oxidative phosphorylation. The recently discovered mitochondrial pyruvate carrier (MPC), encoded by MPC1, MPC2, and MPC3 in yeast, is required for uptake of pyruvate into the organelle. Here, we show that while expression of Mpc1 is not dependent on the carbon source, expression of Mpc2 and Mpc3 is specific to fermentative or respiratory conditions, respectively. This gives rise to two alternative carrier complexes that we have termed MPCFERM and MPCOX. By constitutively expressing the two alternative complexes in yeast deleted for all three endogenous genes, we show that MPCOX has a higher transport activity than MPCFERM, which is dependent on the C-terminus of Mpc3. We propose that the alternative MPC subunit expression in yeast provides a way of adapting cellular metabolism to the nutrient availability. PMID:25672363
Mitochondrial Protein Synthesis, Import, and Assembly
Fox, Thomas D.
2012-01-01
The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899
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.
Nitric Oxide and Mitochondrial Function in Neurological Diseases.
Ghasemi, Mehdi; Mayasi, Yunis; Hannoun, Anas; Eslami, Seyed Majid; Carandang, Raphael
2018-04-15
Mitochondria are key cellular organelles that play crucial roles in the energy production and regulation of cellular metabolism. Accumulating evidence suggests that mitochondrial activity can be modulated by nitric oxide (NO). As a key neurotransmitter in biologic systems, NO mediates the majority of its function through activation of the cyclic guanylyl cyclase (cGC) signaling pathway and S-nitrosylation of a variety of proteins involved in cellular functioning including those involved in mitochondrial biology. Moreover, excess NO or the formation of reactive NO species (RNS), e.g., peroxynitrite (ONOO - ), impairs mitochondrial functioning and this, in conjunction with nuclear events, eventually affects neuronal cell metabolism and survival, contributing to the pathogenesis of several neurodegenerative diseases. In this review we highlight the possible mechanisms underlying the noxious effects of excess NO and RNS on mitochondrial function including (i) negative effects on electron transport chain (ETC); (ii) ONOO - -mediated alteration in mitochondrial permeability transition; (iii) enhanced mitochondrial fragmentation and autophagy through S-nitrosylation of key proteins involved in this process such as dynamin-related protein 1 (DRP-1) and Parkin/PINK1 (protein phosphatase and tensin homolog-induced kinase 1) complex; (iv) alterations in the mitochondrial metabolic pathways including Krebs cycle, glycolysis, fatty acid metabolism, and urea cycle; and finally (v) mitochondrial ONOO - -induced nuclear toxicity and subsequent release of apoptosis-inducing factor (AIF) from mitochondria, causing neuronal cell death. These proposed mechanisms highlight the multidimensional nature of NO and its signaling in the mitochondrial function. Understanding the mechanisms by which NO mediates mitochondrial (dys)function can provide new insights into the treatment of neurodegenerative diseases. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.
PDE 5 inhibitor improves insulin sensitivity by enhancing mitochondrial function in adipocytes.
Yu, Hea Min; Chung, Hyo Kyun; Kim, Koon Soon; Lee, Jae Min; Hong, Jun Hwa; Park, Kang Seo
2017-11-04
Adipocytes are involved in many metabolic disorders. It was recently reported that phosphodiesterase type 5 (PDE5) is expressed in human adipose tissue. In addition, PDE5 inhibitors have been shown to improve insulin sensitivity in humans. However, the mechanism underlying the role of PDE5 inhibitors as an insulin sensitizer remains largely unknown. The present study was undertaken to investigate the role of the PDE5 inhibitor udenafil in insulin signaling in adipocytes and whether this is mediated through the regulation of mitochondrial function. To study the mechanism underlying the insulin sensitizing action of PDE5 inhibitors, we evaluated quantitative changes in protein or mRNA levels of mitochondrial oxidative phosphorylation (OxPhos) complex, oxygen consumption rate (OCR), and fatty acid oxidation with varying udenafil concentrations in 3T3-L1 cells. Our cell study suggested that udenafil enhanced the insulin signaling pathway in 3T3-L1 cells. Following udenafil treatment, basal mitochondrial OCR, maximal OxPhos capacity, and OxPhos gene expression significantly increased. Finally, we examined whether udenafil can affect the fatty acid oxidation process. Treatment of 3T3-L1 cells with udenafil (10 and 20 μM) significantly increased fatty acid oxidation rate in a dose-dependent manner. In addition, the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) significantly increased. We demonstrated that the PDE5 inhibitor udenafil enhances insulin sensitivity by improving mitochondrial function in 3T3-L1 cells. This might be the mechanism underlying the PDE5 inhibitor-enhanced insulin signaling in adipocytes. This also suggests that udenafil may provide benefit in the treatment of type 2 diabetes and other related cardiovascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.
Nguyen, Tammy T; Lewandowska, Agnieszka; Choi, Jae-Yeon; Markgraf, Daniel F; Junker, Mirco; Bilgin, Mesut; Ejsing, Christer S; Voelker, Dennis R; Rapoport, Tom A; Shaw, Janet M
2012-01-01
In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology. PMID:22409400
Seifert, Erin L; Estey, Carmen; Xuan, Jian Y; Harper, Mary-Ellen
2010-02-19
Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels.
Seifert, Erin L.; Estey, Carmen; Xuan, Jian Y.; Harper, Mary-Ellen
2010-01-01
Oxidative stress in skeletal muscle is a hallmark of various pathophysiologic states that also feature increased reliance on long-chain fatty acid (LCFA) substrate, such as insulin resistance and exercise. However, little is known about the mechanistic basis of the LCFA-induced reactive oxygen species (ROS) burden in intact mitochondria, and elucidation of this mechanistic basis was the goal of this study. Specific aims were to determine the extent to which LCFA catabolism is associated with ROS production and to gain mechanistic insights into the associated ROS production. Because intermediates and by-products of LCFA catabolism may interfere with antioxidant mechanisms, we predicted that ROS formation during LCFA catabolism reflects a complex process involving multiple sites of ROS production as well as modified mitochondrial function. Thus, we utilized several complementary approaches to probe the underlying mechanism(s). Using skeletal muscle mitochondria, our findings indicate that even a low supply of LCFA is associated with ROS formation in excess of that generated by NADH-linked substrates. Moreover, ROS production was evident across the physiologic range of membrane potential and was relatively insensitive to membrane potential changes. Determinations of topology and membrane potential as well as use of inhibitors revealed complex III and the electron transfer flavoprotein (ETF) and ETF-oxidoreductase, as likely sites of ROS production. Finally, ROS production was sensitive to matrix levels of LCFA catabolic intermediates, indicating that mitochondrial export of LCFA catabolic intermediates can play a role in determining ROS levels. PMID:20032466
Sun, Xiaocun; Zemel, Michael B
2009-01-01
Background The effects of dairy on energy metabolism appear to be mediated, in part, by leucine and calcium which regulate both adipocyte and skeletal muscle energy metabolism. We recently demonstrated that leucine and calcitriol regulate fatty acid oxidation in skeletal muscle cells in vitro, with leucine promoting and calcitriol suppressing fatty acid oxidation. Moreover, leucine coordinately regulated adipocyte lipid metabolism to promote flux of lipid to skeletal muscle and regulate metabolic flexibility. We have now investigated the role of mitochondrial biogenesis in mediating these effects. Methods We tested the effect of leucine, calcitriol and calcium in regulation of mitochondrial mass using a fluorescence method and tested mitochondrial biogenesis regulatory genes as well mitochondrial component genes using real-time PCR. We also evaluated the effect of leucine on oxygen consumption with a modified perfusion system. Results Leucine (0.5 mM) increased mitochondrial mass by 30% and 53% in C2C12 myocytes and 3T3-L1 adipocytes, respectively, while calcitriol (10 nM) decreased mitochondrial abundance by 37% and 27% (p < 0.02). Leucine also stimulated mitochondrial biogenesis genes SIRT-1, PGC-1α and NRF-1 as well as mitochondrial component genes UCP3, COX, and NADH expression by 3–5 fold in C2C12 cells (p < 0.003). Adipocyte-conditioned medium reduced mitochondrial abundance (p < 0.001) and decreased UCP3 but increased PGC-1α expression in myocytes, suggesting a feedback stimulation of mitochondrial biogenesis. Similar data were observed in C2C12 myocytes co-cultured with adipocytes, with co-culture markedly suppressing mitochondrial abundance (p < 0.02). Leucine stimulated oxygen consumption in both C2C12 cells and adipocytes compared with either control or valine-treated cells. Transfection of C2C12 myocytes with SIRT-1 siRNA resulted in parallel suppression of SIRT-1 expression and leucine-induced stimulation of PGC-1α and NRF-1, indicating that SIRT
Stöckl, Petra; Zankl, Christina; Hütter, Eveline; Unterluggauer, Hermann; Laun, Peter; Heeren, Gino; Bogengruber, Edith; Herndler-Brandstetter, Dietmar; Breitenbach, Michael; Jansen-Dürr, Pidder
2007-09-15
The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.
Padman, Benjamin S; Bach, Markus; Lucarelli, Giuseppe; Prescott, Mark; Ramm, Georg
2013-11-01
Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.
Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes
Dimmer, Kai S
2018-01-01
Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged. PMID:29923829
The control of translational accuracy is a determinant of healthy ageing in yeast.
von der Haar, Tobias; Leadsham, Jane E; Sauvadet, Aimie; Tarrant, Daniel; Adam, Ilectra S; Saromi, Kofo; Laun, Peter; Rinnerthaler, Mark; Breitenbach-Koller, Hannelore; Breitenbach, Michael; Tuite, Mick F; Gourlay, Campbell W
2017-01-01
Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing. © 2017 The Authors.
The control of translational accuracy is a determinant of healthy ageing in yeast
Leadsham, Jane E.; Sauvadet, Aimie; Tarrant, Daniel; Adam, Ilectra S.; Saromi, Kofo; Laun, Peter; Rinnerthaler, Mark; Breitenbach-Koller, Hannelore; Breitenbach, Michael; Tuite, Mick F.; Gourlay, Campbell W.
2017-01-01
Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing. PMID:28100667
Wilfling, F; Weber, A; Potthoff, S; Vögtle, F-N; Meisinger, C; Paschen, S A; Häcker, G
2012-08-01
During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.
Kwon, Young-Yon; Lee, Sung-Keun; Lee, Cheol-Koo
2017-04-01
Caloric restriction (CR) has been shown to extend lifespan and prevent cellular senescence in various species ranging from yeast to humans. Many effects of CR may contribute to extend lifespan. Specifically, CR prevents oxidative damage from reactive oxygen species (ROS) by enhancing mitochondrial function. In this study, we characterized 33 single electron transport chain (ETC) gene-deletion strains to identify CR-induced chronological lifespan (CLS) extension mechanisms. Interestingly, defects in 17 of these 33 ETC gene-deleted strains showed loss of both respiratory function and CR-induced CLS extension. On the contrary, the other 16 respiration-capable mutants showed increased CLS upon CR along with increased mitochondrial membrane potential (MMP) and intracellular adenosine triphosphate (ATP) levels, with decreased mitochondrial superoxide generation. We measured the same parameters in the 17 non-respiratory mutants upon CR. CR simultaneously increased MMP and mitochondrial superoxide generation without altering intracellular ATP levels. In conclusion, respiration is essential for CLS extension by CR and is important for balancing MMP, ROS, and ATP levels.
Effects of soya fatty acids on cassava ethanol fermentation.
Xiao, Dongguang; Wu, Shuai; Zhu, Xudong; Chen, Yefu; Guo, Xuewu
2010-01-01
Ethanol tolerance is a key trait of microbes in bioethanol production. Previous studies have shown that soya flour contributed to the increase of ethanol tolerance of yeast cells. In this paper, the mechanism of this ethanol tolerance improvement was investigated in cassava ethanol fermentation supplemented with soya flour or defatted soya flour, respectively. Experiment results showed that ethanol tolerance of cells from soya flour supplemented medium increased by 4-6% (v/v) than the control with defatted soya flour. Microscopic observation found that soya flour can retain the cell shape while dramatic elongations of cells were observed with the defatted soya flour supplemented medium. Unsaturated fatty acids (UFAs) compositions of cell membrane were analyzed and the UFAs amounts increased significantly in all tested strains grown in soya flour supplemented medium. Growth study also showed that soya flour stimulated the cell growth rate by approximately tenfolds at 72-h fermentation. All these results suggested that soya fatty acids play an important role to protect yeast cells from ethanol stress during fermentation process.
Hsueh, Tun-Yun; Baum, Jamie I.; Huang, Yan
2018-01-01
Polyunsaturated fatty acids are important nutrients for human health, especially omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which have been found to play positive roles in the prevention of various diseases. However, previous studies have reported that excessive omega-3 fatty acids supplement during pregnancy caused side effects such as slower neural transmission times and postnatal growth restriction. In this study, we investigated the effect of EPA and DHA on mitochondrial function and gene expression in C2C12 myoblasts during skeletal muscle differentiation. C2C12 myoblasts were cultured to confluency and then treated with differentiation medium that contained fatty acids (50-µM EPA and DHA). After 72 h of myogenic differentiation, mRNA was collected, and gene expression was analyzed by real-time PCR. Microscopy was used to examine cell morphology following treatment with fatty acids. The effect of EPA and DHA on cellular oxygen consumption was measured using a Seahorse XF24 Analyzer. Cells treated with fatty acids had fewer myotubes formed (P ≤ 0.05) compared with control cells. The expression of the genes related to myogenesis was significantly lower (P ≤ 0.05) in cells treated with fatty acids, compared with control cells. Genes associated with adipogenesis had higher (P ≤ 0.05) expression after treatment with fatty acids. Also, the mitochondrial biogenesis decreased with lower (P ≤ 0.05) gene expression and lower (P ≤ 0.05) mtDNA/nDNA ratio in cells treated with fatty acids compared with control cells. However, the expression of genes related to peroxisome biosynthesis was higher (P ≤ 0.05) in cells treated with fatty acids. Moreover, fatty-acid treatment reduced (P ≤ 0.05) oxygen consumption rate under oligomycin-inhibited (reflecting proton leak) and uncoupled conditions. Our data imply that fatty acids might reduce myogenesis and increase adipogenesis in myotube formation. Fatty acids may also
Lis, Paweł; Jurkiewicz, Paweł; Cal-Bąkowska, Magdalena; Ko, Young H.; Pedersen, Peter L.; Goffeau, Andre; Ułaszewski, Stanisław
2016-01-01
In this study the detailed characteristic of the anti-cancer agent 3-bromopyruvate (3-BP) activity in the yeast Saccharomyces cerevisiae model is described, with the emphasis on its influence on energetic metabolism of the cell. It shows that 3-BP toxicity in yeast is strain-dependent and influenced by the glucose-repression system. Its toxic effect is mainly due to the rapid depletion of intracellular ATP. Moreover, lack of the Whi2p phosphatase results in strongly increased sensitivity of yeast cells to 3-BP, possibly due to the non-functional system of mitophagy of damaged mitochondria through the Ras-cAMP-PKA pathway. Single deletions of genes encoding glycolytic enzymes, the TCA cycle enzymes and mitochondrial carriers result in multiple effects after 3-BP treatment. However, it can be concluded that activity of the pentose phosphate pathway is necessary to prevent the toxicity of 3-BP, probably due to the fact that large amounts of NADPH are produced by this pathway, ensuring the reducing force needed for glutathione reduction, crucial to cope with the oxidative stress. Moreover, single deletions of genes encoding the TCA cycle enzymes and mitochondrial carriers generally cause sensitivity to 3-BP, while totally inactive mitochondrial respiration in the rho0 mutant resulted in increased resistance to 3-BP. PMID:26862728
Baruffini, Enrico; Dallabona, Cristina; Invernizzi, Federica; Yarham, John W; Melchionda, Laura; Blakely, Emma L; Lamantea, Eleonora; Donnini, Claudia; Santra, Saikat; Vijayaraghavan, Suresh; Roper, Helen P; Burlina, Alberto; Kopajtich, Robert; Walther, Anett; Strom, Tim M; Haack, Tobias B; Prokisch, Holger; Taylor, Robert W; Ferrero, Ileana; Zeviani, Massimo; Ghezzi, Daniele
2013-11-01
We report three families presenting with hypertrophic cardiomyopathy, lactic acidosis, and multiple defects of mitochondrial respiratory chain (MRC) activities. By direct sequencing of the candidate gene MTO1, encoding the mitochondrial-tRNA modifier 1, or whole exome sequencing analysis, we identified novel missense mutations. All MTO1 mutations were predicted to be deleterious on MTO1 function. Their pathogenic role was experimentally validated in a recombinant yeast model, by assessing oxidative growth, respiratory activity, mitochondrial protein synthesis, and complex IV activity. In one case, we also demonstrated that expression of wt MTO1 could rescue the respiratory defect in mutant fibroblasts. The severity of the yeast respiratory phenotypes partly correlated with the different clinical presentations observed in MTO1 mutant patients, although the clinical outcome was highly variable in patients with the same mutation and seemed also to depend on timely start of pharmacological treatment, centered on the control of lactic acidosis by dichloroacetate. Our results indicate that MTO1 mutations are commonly associated with a presentation of hypertrophic cardiomyopathy, lactic acidosis, and MRC deficiency, and that ad hoc recombinant yeast models represent a useful system to test the pathogenic potential of uncommon variants, and provide insight into their effects on the expression of a biochemical phenotype. © 2013 The Authors. *Human Mutation published by Wiley Periodicals, Inc.
Burstein, Michelle T.; Koupaki, Olivia; Gomez-Perez, Alejandra; Levy, Sean; Pluska, Lukas; Mattie, Sevan; Rafeh, Rami; Iouk, Tatiana; Sheibani, Sara; Greenwood, Michael; Vali, Hojatollah; Titorenko, Vladimir I.
2013-01-01
Macromitophagy controls mitochondrial quality and quantity. It involves the sequestration of dysfunctional or excessive mitochondria within double-membrane autophagosomes, which then fuse with the vacuole/lysosome to deliver these mitochondria for degradation. To investigate a physiological role of macromitophagy in yeast, we examined how the atg32Δ-dependent mutational block of this process influences the chronological lifespan of cells grown in a nutrient-rich medium containing low (0.2%) concentration of glucose. Under these longevity-extending conditions of caloric restriction (CR) yeast cells are not starving. We also assessed a role of macromitophagy in lifespan extension by lithocholic acid (LCA), a bile acid that prolongs yeast longevity under CR conditions. Our findings imply that macromitophagy is a longevity assurance process underlying the synergistic beneficial effects of CR and LCA on yeast lifespan. Our analysis of how the atg32Δ mutation influences mitochondrial morphology, composition and function revealed that macromitophagy is required to maintain a network of healthy mitochondria. Our comparative analysis of the membrane lipidomes of organelles purified from wild-type and atg32Δ cells revealed that macromitophagy is required for maintaining cellular lipid homeostasis. We concluded that macromitophagy defines yeast longevity by modulating vital cellular processes inside and outside of mitochondria. PMID:23553280
Murgasova, Renata; Tor Carreras, Ester; Bourgailh, Julien
2018-05-03
The present study was designed to validate the functional assay that enables rapid screening of therapeutic candidates for their effect on mitochondrial fatty acid oxidation. The two whole-cell systems (tissue homogenates and hepatocytes) have been evaluated to monitor the total beta-oxidation flux of physiologically important 3 H-palmitic acid by measurement of tritiated water enrichment in incubations using UPLC coupled on-line to radioactivity monitoring and mass spectrometry. Our results with several known inhibitors of fatty acid oxidation showed that this simple assay could correctly predict a potential in alteration of mitochondrial function by drug candidates. Since the beta-oxidation of palmitic acid takes place almost exclusively in mitochondria of human hepatocytes, this model can be also utilized to distinguish between the mitochondrial and peroxisomal routes of this essential metabolic pathway in some cases. The present work offers a new in vitro screen of changes in mitochondrial beta-oxidation by xenobiotics as well as a model to study the mechanism of this pathway.
Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver
Satapati, Santhosh; Kucejova, Blanka; Duarte, Joao A.G.; Fletcher, Justin A.; Reynolds, Lacy; Sunny, Nishanth E.; He, Tianteng; Nair, L. Arya; Livingston, Kenneth; Fu, Xiaorong; Merritt, Matthew E.; Sherry, A. Dean; Malloy, Craig R.; Shelton, John M.; Lambert, Jennifer; Parks, Elizabeth J.; Corbin, Ian; Magnuson, Mark A.; Browning, Jeffrey D.; Burgess, Shawn C.
2015-01-01
Mitochondria are critical for respiration in all tissues; however, in liver, these organelles also accommodate high-capacity anaplerotic/cataplerotic pathways that are essential to gluconeogenesis and other biosynthetic activities. During nonalcoholic fatty liver disease (NAFLD), mitochondria also produce ROS that damage hepatocytes, trigger inflammation, and contribute to insulin resistance. Here, we provide several lines of evidence indicating that induction of biosynthesis through hepatic anaplerotic/cataplerotic pathways is energetically backed by elevated oxidative metabolism and hence contributes to oxidative stress and inflammation during NAFLD. First, in murine livers, elevation of fatty acid delivery not only induced oxidative metabolism, but also amplified anaplerosis/cataplerosis and caused a proportional rise in oxidative stress and inflammation. Second, loss of anaplerosis/cataplerosis via genetic knockdown of phosphoenolpyruvate carboxykinase 1 (Pck1) prevented fatty acid–induced rise in oxidative flux, oxidative stress, and inflammation. Flux appeared to be regulated by redox state, energy charge, and metabolite concentration, which may also amplify antioxidant pathways. Third, preventing elevated oxidative metabolism with metformin also normalized hepatic anaplerosis/cataplerosis and reduced markers of inflammation. Finally, independent histological grades in human NAFLD biopsies were proportional to oxidative flux. Thus, hepatic oxidative stress and inflammation are associated with elevated oxidative metabolism during an obesogenic diet, and this link may be provoked by increased work through anabolic pathways. PMID:26571396
Crichton, Paul G.; Affourtit, Charles; Moore, Anthony L.
2006-01-01
In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ∼16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria. PMID:16999687
Boulet, Aren; Vest, Katherine E; Maynard, Margaret K; Gammon, Micah G; Russell, Antoinette C; Mathews, Alexander T; Cole, Shelbie E; Zhu, Xinyu; Phillips, Casey B; Kwong, Jennifer Q; Dodani, Sheel C; Leary, Scot C; Cobine, Paul A
2018-02-09
Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Monroig, Oscar; Navarro, Juan C; Dick, James R; Alemany, Frederic; Tocher, Douglas R
2012-08-01
Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.
Fatty Acids Change the Conformation of Uncoupling Protein 1 (UCP1)*
Divakaruni, Ajit S.; Humphrey, Dickon M.; Brand, Martin D.
2012-01-01
UCP1 catalyzes proton leak across the mitochondrial inner membrane to disengage substrate oxidation from ATP production. It is well established that UCP1 is activated by fatty acids and inhibited by purine nucleotides, but precisely how this regulation occurs remains unsettled. Although fatty acids can competitively overcome nucleotide inhibition in functional assays, fatty acids have little effect on purine nucleotide binding. Here, we present the first demonstration that fatty acids induce a conformational change in UCP1. Palmitate dramatically changed the binding kinetics of 2′/3′-O-(N-methylanthraniloyl)-GDP, a fluorescently labeled nucleotide analog, for UCP1. Furthermore, palmitate accelerated the rate of enzymatic proteolysis of UCP1. The altered kinetics of both processes indicate that fatty acids change the conformation of UCP1, reconciling the apparent discrepancy between existing functional and ligand binding data. Our results provide a framework for how fatty acids and nucleotides compete to regulate the activity of UCP1. PMID:22952235
Catoni, Elisabetta; Desimone, Marcelo; Hilpert, Melanie; Wipf, Daniel; Kunze, Reinhard; Schneider, Anja; Flügge, Ulf-Ingo; Schumacher, Karin; Frommer, Wolf B
2003-01-01
Background Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. Results High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources. Conclusion AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds. PMID:12517306
Miltefosine has post-antifungal effect and induces apoptosis in Cryptococcus yeasts.
Spadari, Cristina de Castro; Vila, Taissa; Rozental, Sonia; Ishida, Kelly
2018-05-29
Cryptococcus spp. are common opportunistic fungal pathogens, particularly in HIV patients. The approved drug miltefosine (MFS) has potential as an alternative antifungal against cryptococcosis; however, the mechanism of action of MFS in Cryptococcus is poorly understood. Here, we examined the effects of MFS on C. neoformans and C. gattii yeasts (planktonic and biofilm lifestyles), to clarify its mechanism of action. MFS presented inhibitory and fungicidal effects against planktonic Cryptococcus cells, with similar activity against dispersion biofilm cells, while sessile biofilm cells were less sensitive to MFS. Interestingly, MFS had post-antifungal effect on Cryptococcus , with a proliferation delay of up to 8.15 h after short exposure to fungicidal doses. MFS at fungicidal concentrations increased plasma membrane permeability, likely due to direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. Moreover, MFS reduced the mitochondrial membrane potential, increased ROS production, and induced DNA fragmentation and condensation, all of which are hallmarks of apoptosis. Transmission electron microscopy analysis showed that MFS-treated yeasts had a reduced mucopolysaccharide capsule (confirmed by morphometry in light microscopy), plasma membrane irregularities, mitochondrial swelling and a less conspicuous cell wall. Our results suggest that MFS increases plasma membrane permeability in Cryptococcus via interaction with ergosterol, and also affects the mitochondrial membrane, eventually leading to apoptosis, in line with its fungicidal activity. These findings confirm the potential of MFS as an antifungal against C. neoformans and C. gattii, and warrants further studies to establish clinical protocols for MFS use against cryptococcosis. Copyright © 2018 American Society for Microbiology.
Regulatory link between steryl ester formation and hydrolysis in the yeast Saccharomyces cerevisiae.
Ploier, Birgit; Korber, Martina; Schmidt, Claudia; Koch, Barbara; Leitner, Erich; Daum, Günther
2015-07-01
Steryl esters and triacylglycerols are the major storage lipids of the yeast Saccharomyces cerevisiae. Steryl esters are formed in the endoplasmic reticulum by the two acyl-CoA:sterol acyltransferases Are1p and Are2p, whereas steryl ester hydrolysis is catalyzed by the three steryl ester hydrolases Yeh1p, Yeh2p and Tgl1p. To shed light on the regulatory link between steryl ester formation and hydrolysis in the maintenance of cellular sterol and free fatty acid levels we employed yeast mutants which lacked the enzymes catalyzing the degradation of steryl esters. These studies revealed feedback regulation of steryl ester formation by steryl ester hydrolysis although in a Δtgl1Δyeh1Δyeh2 triple mutant the gene expression levels of ARE1 and ARE2 as well as protein levels and stability of Are1p and Are2p were not altered. Nevertheless, the capacity of the triple mutant to synthesize steryl esters was significantly reduced as shown by in vitro and in vivo labeling of lipids with [(14)C]oleic acid and [(14)C]acetate. Enzymatic analysis revealed that inhibition of steryl ester formation occurred at the enzyme level. As the amounts and the formation of sterols and fatty acids were also decreased in the triple mutant we concluded that defects in steryl ester hydrolysis also caused feedback inhibition on the formation of sterols and fatty acids which serve as precursors for steryl ester formation. In summary, this study demonstrates a regulatory link within the steryl ester metabolic network which contributes to non-polar lipid homeostasis in yeast cells. Copyright © 2014 Elsevier B.V. All rights reserved.
A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria.
Diffley, J F; Stillman, B
1991-01-01
ABF2 (ARS-binding factor 2), a small, basic DNA-binding protein that binds specifically to the autonomously replicating sequence ARS1, is located primarily in the mitochondria of the yeast Saccharomyces cerevisiae. The abundance of ABF2 and the phenotype of abf2- null mutants argue that this protein plays a key role in the structure, maintenance, and expression of the yeast mitochondrial genome. The predicted amino acid sequence of ABF2 is closely related to the high-mobility group proteins HMG1 and HMG2 from vertebrate cell nuclei and to several other DNA-binding proteins. Additionally, ABF2 and the other HMG-related proteins are related to a globular domain from the heat shock protein hsp70 family. ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression. Images PMID:1881919
Kellogg, James A.; Bankert, David A.; Chaturvedi, Vishnu
1998-01-01
The ability of the rapid, computerized Microbial Identification System (MIS; Microbial ID, Inc.) to identify a variety of clinical isolates of yeast species was compared to the abilities of a combination of tests including the Yeast Biochemical Card (bioMerieux Vitek), determination of microscopic morphology on cornmeal agar with Tween 80, and when necessary, conventional biochemical tests and/or the API 20C Aux system (bioMerieux Vitek) to identify the same yeast isolates. The MIS chromatographically analyzes cellular fatty acids and compares the results with the fatty acid profiles in its database. Yeast isolates were subcultured onto Sabouraud dextrose agar and were incubated at 28°C for 24 h. The resulting colonies were saponified, methylated, extracted, and chromatographically analyzed (by version 3.8 of the MIS YSTCLN database) according to the manufacturer’s instructions. Of 477 isolates of 23 species tested, 448 (94%) were given species names by the MIS and 29 (6%) were unidentified (specified as “no match” by the MIS). Of the 448 isolates given names by the MIS, only 335 (75%) of the identifications were correct to the species level. While the MIS correctly identified only 102 (82%) of 124 isolates of Candida glabrata, the predictive value of an MIS identification of unknown isolates as C. glabrata was 100% (102 of 102) because no isolates of other species were misidentified as C. glabrata. In contrast, while the MIS correctly identified 100% (15 of 15) of the isolates of Saccharomyces cerevisiae, the predictive value of an MIS identification of unknown isolates as S. cerevisiae was only 47% (15 of 32), because 17 isolates of C. glabrata were misidentified as S. cerevisiae. The low predictive values for accuracy associated with MIS identifications for most of the remaining yeast species indicate that the procedure and/or database for the system need to be improved. PMID:9574676
Rapamycin enhances survival in a Drosophila model of mitochondrial disease.
Wang, Adrienne; Mouser, Jacob; Pitt, Jason; Promislow, Daniel; Kaeberlein, Matt
2016-12-06
Pediatric mitochondrial disorders are a devastating category of diseases caused by deficiencies in mitochondrial function. Leigh Syndrome (LS) is the most common of these diseases with symptoms typically appearing within the first year of birth and progressing rapidly until death, usually by 6-7 years of age. Our lab has recently shown that genetic inhibition of the mechanistic target of rapamycin (TOR) rescues the short lifespan of yeast mutants with defective mitochondrial function, and that pharmacological inhibition of TOR by administration of rapamycin significantly rescues the shortened lifespan, neurological symptoms, and neurodegeneration in a mouse model of LS. However, the mechanism by which TOR inhibition exerts these effects, and the extent to which these effects can extend to other models of mitochondrial deficiency, are unknown. Here, we probe the effects of TOR inhibition in a Drosophila model of complex I deficiency. Treatment with rapamycin robustly suppresses the lifespan defect in this model of LS, without affecting behavioral phenotypes. Interestingly, this increased lifespan in response to TOR inhibition occurs in an autophagy-independent manner. Further, we identify a fat storage defect in the ND2 mutant flies that is rescued by rapamycin, supporting a model that rapamycin exerts its effects on mitochondrial disease in these animals by altering metabolism.
Rostovtseva, Tatiana K.; Gurnev, Philip A.; Protchenko, Olga; Hoogerheide, David P.; Yap, Thai Leong; Philpott, Caroline C.; Lee, Jennifer C.; Bezrukov, Sergey M.
2015-01-01
Participation of the small, intrinsically disordered protein α-synuclein (α-syn) in Parkinson disease (PD) pathogenesis has been well documented. Although recent research demonstrates the involvement of α-syn in mitochondrial dysfunction in neurodegeneration and suggests direct interaction of α-syn with mitochondria, the molecular mechanism(s) of α-syn toxicity and its effect on neuronal mitochondria remain vague. Here we report that at nanomolar concentrations, α-syn reversibly blocks the voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane that controls most of the metabolite fluxes in and out of the mitochondria. Detailed analysis of the blockage kinetics of VDAC reconstituted into planar lipid membranes suggests that α-syn is able to translocate through the channel and thus target complexes of the mitochondrial respiratory chain in the inner mitochondrial membrane. Supporting our in vitro experiments, a yeast model of PD shows that α-syn toxicity in yeast depends on VDAC. The functional interactions between VDAC and α-syn, revealed by the present study, point toward the long sought after physiological and pathophysiological roles for monomeric α-syn in PD and in other α-synucleinopathies. PMID:26055708
Kolodziejczak, Marta; Skibior-Blaszczyk, Renata; Janska, Hanna
2018-05-01
For optimal mitochondrial activity, the mitochondrial proteome must be properly maintained or altered in response to developmental and environmental stimuli. Based on studies of yeast and humans, one of the key players in this control are m-AAA proteases, mitochondrial inner membrane-bound ATP-dependent metalloenzymes. This study focuses on the importance of m-AAA proteases in plant mitochondria, providing their first experimentally proven physiological substrate. We found that the Arabidopsis m- AAA complexes composed of AtFTSH3 and/or AtFTSH10 are involved in the proteolytic maturation of ribosomal subunit L32. Consequently, in the double Arabidopsis ftsh3/10 mutant, mitoribosome biogenesis, mitochondrial translation and functionality of OXPHOS (oxidative phosphorylation) complexes are impaired. However, in contrast to their mammalian or yeast counterparts, plant m-AAA complexes are not critical for the survival of Arabidopsis under optimal conditions; ftsh3/10 plants are only slightly smaller in size at the early developmental stage compared with plants containing m-AAA complexes. Our data suggest that a lack of significant visible morphological alterations under optimal growth conditions involves mechanisms which rely on existing functional redundancy and induced functional compensation in Arabidopsis mitochondria.
Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast
Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y.; Strahl, Sabine; Clausen, Henrik
2015-01-01
Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575
Chao, Honglu; Liu, Yinlong; Fu, Xian; Xu, Xiupeng; Bao, Zhongyuan; Lin, Chao; Li, Zheng; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing
2018-02-01
iPLA 2 γ, calcium-independent phospholipase A 2 γ, discerningly hydrolyses glycerophospholipids to liberate free fatty acids. iPLA 2 γ-deficiency has been associated with abnormal mitochondrial function. More importantly, the iPLA 2 family is causative proteins in mitochondrial neurodegenerative disorders such as parkinsonian disorders. However, the mechanisms by which iPLA 2 γ affects Parkinson's disease (PD) remain unknown. Mitochondrion stress has a key part in rotenone-induced dopaminergic neuronal degeneration. The present evaluation revealed that lowered iPLA 2 γ function provokes the parkinsonian phenotype and leads to the reduction of dopamine and its metabolites, lowered survival, locomotor deficiencies, and organismal hypersensitivity to rotenone-induced oxidative stress. In addition, lowered iPLA 2 γ function escalated the amount of mitochondrial irregularities, including mitochondrial reactive oxygen species (ROS) regeneration, reduced ATP synthesis, reduced glutathione levels, and abnormal mitochondrial morphology. Further, lowered iPLA 2 γ function was tightly linked with strengthened lipid peroxidation and mitochondrial membrane flaws following rotenone treatment, which can cause cytochrome c release and eventually apoptosis. These results confirmed the important role of iPLA 2 γ, whereby decreasing iPLA 2 γ activity aggravates mitochondrial degeneration to induce neurodegenerative disorders in a rotenone rat model of Parkinson's disease. These findings may be useful in the design of rational approaches for the prevention and treatment of PD-associated symptoms. Copyright © 2017 Elsevier Inc. All rights reserved.
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
Apoptotic signals induce specific degradation of ribosomal RNA in yeast
Mroczek, Seweryn; Kufel, Joanna
2008-01-01
Organisms exposed to reactive oxygen species, generated endogenously during respiration or by environmental conditions, undergo oxidative stress. Stress response can either repair the damage or activate one of the programmed cell death (PCD) mechanisms, for example apoptosis, and finally end in cell death. One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. This process is most likely endonucleolytic, is correlated with stress response, and depends on the mitochondrial respiratory status: rRNA is less susceptible to degradation in respiring cells with functional defence against oxidative stress. In addition, RNA fragmentation is independent of two yeast apoptotic factors, metacaspase Yca1 and apoptosis-inducing factor Aif1, but it relies on the apoptotic chromatin condensation induced by histone H2B modifications. These data describe a novel phenotype for certain stress- and ageing-related PCD pathways in yeast. PMID:18385160
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sekharam, K.M.; Patel, J.M.; Block, E.R.
1990-12-01
Vitamin E, a dietary antioxidant, is presumed to be incorporated into the lipid bilayer of biological membranes to an extent proportional to the amount of polyunsaturated fatty acids or phospholipids in the membrane. In the present study we evaluated the distribution of incorporated polyunsaturated fatty acids (PUFA) and phosphatidylethanolamine (PE) in various membranes of pulmonary artery endothelial cells. We also studied whether incorporation of PUFA or PE is responsible for increased incorporation of (3H)-vitamin E into the membranes of these cells. Following a 24-hr incubation with linoleic acid (18:2), 18:2 was increased by 6.9-, 9.2-, and 13.2-fold in plasma, mitochondrial,more » and microsomal membranes, respectively. Incorporation of 18:2 caused significant increases in the unsaturation indexes of mitochondrial and microsomal polyunsaturated fatty acyl chains (P less than .01 versus control in both membranes). Incubation with arachidonic acid (20:4) for 24 hr resulted in 1.5-, 2.3-, and 2.4-fold increases in 20:4 in plasma, mitochondrial, and microsomal membranes, respectively. The unsaturation indexes of polyunsaturated fatty acyl chains of mitochondrial and microsomal membranes also increased (P less than .01 versus control in both membranes). Although incubations with 18:2 or 20:4 resulted in several-fold increases in membrane 18:2 or 20:4 fatty acids, incorporation of (3H)-vitamin E into these membranes was similar to that in controls. Following a 24-hr incubation with PE, membrane PE content was significantly increased, and (3H)-vitamin E incorporation was also increased to a comparable degree, i.e., plasma membrane greater than mitochondria greater than microsomes. Endogenous vitamin E content of the cells was not altered because of increased incorporation of PE and (3H)-vitamin E.« less
Zubareva, E V; Seferova, R I; Denisova, N A
1991-01-01
Under conditions of adaptation to heating lipid composition in mitochondrial membranes of rat inner tissues was altered as follows: an increase in relative concentration of plasmalogenous forms of phospholipids (kidney, heart) and in content of saturated fatty acids (liver tissue), a decrease in the index of fatty acids unsaturation and in the ratio of fatty acids omega-3/omega-6. The alterations observed enabled the membranes to keep sufficient amount of liquidity essential for functional activity of mitochondria in heating.
Najjar, Amal; Robert, Sylvie; Guérin, Clémence; Violet-Asther, Michèle; Carrière, Frédéric
2011-03-01
Lipase secretion, extracellular lipolysis, and fatty acid uptake were quantified in the yeast Yarrowia lipolytica grown in the presence of olive oil and/or glucose. Specific lipase assays, Western blot analysis, and ELISA indicated that most of the lipase activity measured in Y. lipolytica cultures resulted from the YLLIP2 lipase. Lipase production was triggered by olive oil and, during the first hours of culture, most of the lipase activity and YLLIP2 immunodetection remained associated with the yeast cells. YLLIP2 was then released in the culture medium before it was totally degraded by proteases. Olive oil triglycerides were largely degraded when the lipase was still attached to the cell wall. The fate of lipolysis products in the culture medium and inside the yeast cell, as well as lipid storage, was investigated simultaneously by quantitative TLC-FID and GC analysis. The intracellular levels of free fatty acids (FFA) and triglycerides increased transiently and were dependent on the carbon sources. A maximum fat storage of 37.8% w/w of yeast dry mass was observed with olive oil alone. A transient accumulation of saturated FFA was observed whereas intracellular triglycerides became enriched in unsaturated fatty acids. So far, yeasts have been mainly used for studying the intracellular synthesis, storage, and mobilization of neutral lipids. The present study shows that yeasts are also interesting models for studying extracellular lipolysis and fat uptake by the cell. The quantitative data obtained here allow for the first time to establish interesting analogies with gastrointestinal and vascular lipolysis in humans.
USDA-ARS?s Scientific Manuscript database
Enhanced control of species of Cryptococcus, non-fermentative yeast pathogens, was achieved by chemosensitization through co-application of certain compounds with a conventional antimicrobial drug. The species of Cryptococcus tested showed higher sensitivity to mitochondrial respiratory chain inhibi...
Obesity and Cancer Progression: Is There a Role of Fatty Acid Metabolism?
Balaban, Seher; Lee, Lisa S.; Schreuder, Mark; Hoy, Andrew J.
2015-01-01
Currently, there is renewed interest in elucidating the metabolic characteristics of cancer and how these characteristics may be exploited as therapeutic targets. Much attention has centered on glucose, glutamine and de novo lipogenesis, yet the metabolism of fatty acids that arise from extracellular, as well as intracellular, stores as triacylglycerol has received much less attention. This review focuses on the key pathways of fatty acid metabolism, including uptake, esterification, lipolysis, and mitochondrial oxidation, and how the regulators of these pathways are altered in cancer. Additionally, we discuss the potential link that fatty acid metabolism may serve between obesity and changes in cancer progression. PMID:25866768
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
The sites and topology of mitochondrial superoxide production
Brand, Martin D.
2010-01-01
Mitochondrial superoxide production is an important source of reactive oxygen species in cells, and may cause or contribute to ageing and the diseases of ageing. Seven major sites of superoxide production in mammalian mitochondria are known and widely accepted. In descending order of maximum capacity they are the ubiquinone binding sites in complex I (site IQ) and complex III (site IIIQo), glycerol 3-phosphate dehydrogenase, the flavin in complex I (site IF), the electron transferring flavoprotein:Q oxidoreductase (ETFQOR) of fatty acid beta oxidation, and pyruvate and 2-oxoglutarate dehydrogenases. None of these sites is fully characterized and for some we only have sketchy information. The topology of the sites is important because it determines whether or not a site will produce superoxide in the mitochondrial matrix and be able to damage mitochondrial DNA. All sites produce superoxide in the matrix; site IIIQo and glycerol 3-phosphate dehydrogenase also produce superoxide to the intermembrane space. The relative contribution of each site to mitochondrial reactive oxygen species generation in the absence of electron transport inhibitors is unknown in isolated mitochondria, in cells or in vivo, and may vary considerably with species, tissue, substrate, energy demand and oxygen tension. PMID:20064600
Impheng, Hathaichanok; Richert, Lysiane; Pekthong, Dumrongsak; Scholfield, C Norman; Pongcharoen, Sutatip; Pungpetchara, Ittipon; Srisawang, Piyarat
2015-01-01
The de novo fatty acid synthesis catalyzed by key lipogenic enzymes, including fatty acid synthase (FASN) has emerged as one of the novel targets of anti-cancer approaches. The present study explored the possible inhibitory efficacy of [6]-gingerol on de novo fatty acid synthesis associated with mitochondrial-dependent apoptotic induction in HepG2 cells. We observed a dissipation of mitochondrial membrane potential accompanied by a reduction of fatty acid levels. [6]-gingerol administration manifested inhibition of FASN expression, indicating FASN is a major target of [6]-gingerol inducing apoptosis in HepG2 cells. Indeed, we found that increased ROS generation could likely be a mediator of the anti-cancer effect of [6]-gingerol. A reduction of fatty acid levels and induction of apoptosis were restored by inhibition of acetyl-CoA carboxylase (ACC) activity, suggesting an accumulation of malonyl-CoA level could be the major cause of apoptotic induction of [6]-gingerol in HepG2 cells. The present study also showed that depletion of fatty acid following [6]-gingerol treatment caused an inhibitory effect on carnitine palmitoyltransferase-1 activity (CPT-1), whereas C75 augmented CPT-1 activity, indicating that [6]-gingerol exhibits the therapeutic benefit on suppression of fatty acid β-oxidation.
Santos-Ocaña, Carlos; Do, Thai Q; Padilla, Sergio; Navas, Placido; Clarke, Catherine F
2002-03-29
Coenzyme Q (Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells but also is present in other cellular membranes where it acts as an antioxidant. Because Q synthesis machinery in Saccharomyces cerevisiae is located in the mitochondria, the intracellular distribution of Q indicates the existence of intracellular Q transport. In this study, the uptake of exogenous Q(6) by yeast and its transport from the plasma membrane to mitochondria was assessed in both wild-type and in Q-less coq7 mutants derived from four distinct laboratory yeast strains. Q(6) supplementation of medium containing ethanol, a non-fermentable carbon source, rescued growth in only two of the four coq7 mutant strains. Following culture in medium containing dextrose, the added Q(6) was detected in the plasma membrane of each of four coq7 mutants tested. This detection of Q(6) in the plasma membrane was corroborated by measuring ascorbate stabilization activity, as catalyzed by NADH-ascorbate free radical reductase, a transmembrane redox activity that provides a functional assay of plasma membrane Q(6). These assays indicate that each of the four coq7 mutant strains assimilate exogenous Q(6) into the plasma membrane. The two coq7 mutant strains rescued by Q(6) supplementation for growth on ethanol contained mitochondrial Q(6) levels similar to wild type. However, the content of Q(6) in mitochondria from the non-rescued strains was only 35 and 8%, respectively, of that present in the corresponding wild-type parental strains. In yeast strains rescued by exogenous Q(6), succinate-cytochrome c reductase activity was partially restored, whereas non-rescued strains contained very low levels of activity. There was a strong correlation between mitochondrial Q(6) content, succinate-cytochrome c reductase activity, and steady state levels of the cytochrome c(1) polypeptide. These studies show that transport of extracellular Q(6) to the mitochondria operates in yeast but is
Non-toxic fluorescent phosphonium probes to detect mitochondrial potential.
Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X
2017-03-22
We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry-xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe's limitations.
Non-toxic fluorescent phosphonium probes to detect mitochondrial potential
NASA Astrophysics Data System (ADS)
Šarić, Ana; Crnolatac, Ivo; Bouillaud, Frédéric; Sobočanec, Sandra; Mikecin, Ana-Matea; Mačak Šafranko, Željka; Delgeorgiev, Todor; Piantanida, Ivo; Balog, Tihomir; Petit, Patrice X.
2017-03-01
We evaluated our phosphonium-based fluorescent probes for selective staining of mitochondria. Currently used probes for monitoring mitochondrial membrane potential show varying degrees of interference with cell metabolism, photo-induced damage and probe binding. Here presented probes are characterised by highly efficient cellular uptake and specific accumulation in mitochondria. Fluorescent detection of the probes was accomplished using flow cytometry and confocal microscopy imaging of yeast and mammalian cells. Toxicity analysis (impedimetry—xCELLigence for the cellular proliferation and Seahorse technology for respiratory properties) confirms that these dyes exhibit no-toxicity on mitochondrial or cellular functioning even for long time incubation. The excellent chemical and photophysical stability of the dyes makes them promising leads toward improved fluorescent probes. Therefore, the probes described here offer to circumvent the problems associated with existing-probe’s limitations.
Jenkins, Bethany J; Daly, Thomas M; Morrisey, Joanne M; Mather, Michael W; Vaidya, Akhil B; Bergman, Lawrence W
2016-01-01
Coenzyme Q (CoQ, ubiquinone) is a central electron carrier in mitochondrial respiration. CoQ is synthesized through multiple steps involving a number of different enzymes. The prevailing view that the CoQ used in respiration exists as a free pool that diffuses throughout the mitochondrial inner membrane bilayer has recently been challenged. In the yeast Saccharomyces cerevisiae, deletion of the gene encoding Coq10p results in respiration deficiency without inhibiting the synthesis of CoQ, suggesting that the Coq10 protein is critical for the delivery of CoQ to the site(s) of respiration. The precise mechanism by which this is achieved remains unknown at present. We have identified a Plasmodium orthologue of Coq10 (PfCoq10), which is predominantly expressed in trophozoite-stage parasites, and localizes to the parasite mitochondrion. Expression of PfCoq10 in the S. cerevisiae coq10 deletion strain restored the capability of the yeast to grow on respiratory substrates, suggesting a remarkable functional conservation of this protein over a vast evolutionary distance, and despite a relatively low level of amino acid sequence identity. As the antimalarial drug atovaquone acts as a competitive inhibitor of CoQ, we assessed whether over-expression of PfCoq10 altered the atovaquone sensitivity in parasites and in yeast mitochondria, but found no alteration of its activity.
Multilayered control of peroxisomal activity upon salt stress in Saccharomyces cerevisiae.
Manzanares-Estreder, Sara; Espí-Bardisa, Joan; Alarcón, Benito; Pascual-Ahuir, Amparo; Proft, Markus
2017-06-01
Peroxisomes are dynamic organelles and the sole location for fatty acid β-oxidation in yeast cells. Here, we report that peroxisomal function is crucial for the adaptation to salt stress, especially upon sugar limitation. Upon stress, multiple layers of control regulate the activity and the number of peroxisomes. Activated Hog1 MAP kinase triggers the induction of genes encoding enzymes for fatty acid activation, peroxisomal import and β-oxidation through the Adr1 transcriptional activator, which transiently associates with genes encoding fatty acid metabolic enzymes in a stress- and Hog1-dependent manner. Moreover, Na + and Li + stress increases the number of peroxisomes per cell in a Hog1-independent manner, which depends instead of the retrograde pathway and the dynamin related GTPases Dnm1 and Vps1. The strong activation of the Faa1 fatty acyl-CoA synthetase, which specifically localizes to lipid particles and peroxisomes, indicates that adaptation to salt stress requires the enhanced mobilization of fatty acids from internal lipid stores. Furthermore, the activation of mitochondrial respiration during stress depends on peroxisomes, mitochondrial acetyl-carnitine uptake is essential for salt resistance and the number of peroxisomes attached to the mitochondrial network increases during salt adaptation, which altogether indicates that stress-induced peroxisomal β-oxidation triggers enhanced respiration upon salt shock. © 2017 John Wiley & Sons Ltd.
The organization of repeating units in mitochondrial DNA from yeast petite mutants.
Bos, J L; Heyting, C; Van der Horst, G; Borst, P
1980-04-01
We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ρ(-) petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ρ(-) petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.
Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis.
Marí, Montserrat; Caballero, Francisco; Colell, Anna; Morales, Albert; Caballeria, Juan; Fernandez, Anna; Enrich, Carlos; Fernandez-Checa, José C; García-Ruiz, Carmen
2006-09-01
The etiology of progression from steatosis to steatohepatitis (SH) remains unknown. Using nutritional and genetic models of hepatic steatosis, we show that free cholesterol (FC) loading, but not free fatty acids or triglycerides, sensitizes to TNF- and Fas-induced SH. FC distribution in endoplasmic reticulum (ER) and plasma membrane did not cause ER stress or alter TNF signaling. Rather, mitochondrial FC loading accounted for the hepatocellular sensitivity to TNF due to mitochondrial glutathione (mGSH) depletion. Selective mGSH depletion in primary hepatocytes recapitulated the susceptibility to TNF and Fas seen in FC-loaded hepatocytes; its repletion rescued FC-loaded livers from TNF-mediated SH. Moreover, hepatocytes from mice lacking NPC1, a late endosomal cholesterol trafficking protein, or from obese ob/ob mice, exhibited mitochondrial FC accumulation, mGSH depletion, and susceptibility to TNF. Thus, we propose a critical role for mitochondrial FC loading in precipitating SH, by sensitizing hepatocytes to TNF and Fas through mGSH depletion.
Neutrophil depletion improves diet-induced non-alcoholic fatty liver disease in mice.
Ou, Rongying; Liu, Jia; Lv, Mingfen; Wang, Jingying; Wang, Jinmeng; Zhu, Li; Zhao, Liang; Xu, Yunsheng
2017-07-01
Non-alcoholic fatty liver disease is highly associated with morbidity and mortality in population. Although studies have already demonstrated that the immune response plays a pivotal role in the development of non-alcoholic fatty liver disease, the comprehensive regulation is unclear. Therefore, present study was carried out to investigate the non-alcoholic fatty liver disease development under neutrophil depletion. To achieve the aim of the study, C57BL/6 J mice were fed with high fat diet for 6 weeks before treated with neutrophil deplete antibody 1A8 or isotype control (200 μg/ mouse every week) for another 4 weeks. Treated with 1A8 antibody, obese mice exhibited better whole body metabolic parameters, including reduction of body weight gain and fasting blood glucose levels. Neutrophil depletion also effectively reduced hepatic structural disorders, dysfunction and lipid accumulation. Lipid β-oxidative markers, phosphorylated-AMP-activated protein kinase α and phosphorylated-acetyl-CoA carboxylase levels were increased in 1A8 antibody-treated obese mouse group. The mitochondrial number and function were also reversed after 1A8 antibody treatment, including increased mitochondrial number, reduced lipid oxidative damage and enhanced mitochondrial activity. Furthermore, the expression of inflammatory cytokines, tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 were obviously reduced after neutrophil depletion, accompanied with decreased F4/80 mRNA level and macrophage percentage in liver. The decreased NF-κB signaling activity was also involved in the beneficial effect of neutrophil depletion. Taken together, neutrophil depletion could attenuate metabolic syndromes and hepatic dysfunction.
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.
Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection
Dolezal, Pavel; Aili, Margareta; Tong, Janette; Jiang, Jhih-Hang; Marobbio, Carlo M.; Lee, Sau fung; Schuelein, Ralf; Belluzzo, Simon; Binova, Eva; Mousnier, Aurelie; Frankel, Gad; Giannuzzi, Giulia; Palmieri, Ferdinando; Gabriel, Kipros; Naderer, Thomas; Hartland, Elizabeth L.; Lithgow, Trevor
2012-01-01
The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms. PMID:22241989
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
Jin, Zixue; Wei, Wei; Yang, Marie; Du, Yang; Wan, Yihong
2014-01-01
SUMMARY Mitochondrial complex I (CI) deficiency is associated with multiple neurological and metabolic disorders. However, its effect on innate immunity and bone remodeling is unclear. Using deletion of the essential CI subunit Ndufs4 as a model for mitochondrial dysfunction, we report that mitochondria suppress macrophage activation and inflammation while promoting osteoclast differentiation and bone resorption via both cell-autonomous and systemic regulation. Global Ndufs4 deletion causes systemic inflammation and osteopetrosis. Hematopoietic Ndufs4 deletion causes an intrinsic lineage shift from osteoclast to macrophage. Liver Ndufs4 deletion causes a metabolic shift from fatty acid oxidation to glycolysis, accumulating fatty acids and lactate (FA/LAC) in circulation. FA/LAC further activates Ndufs4−/− macrophages via ROS induction, and diminishes osteoclast lineage commitment in Ndufs4−/− progenitors; both inflammation and osteopetrosis in Ndufs4−/− mice are attenuated by TLR4/2 deletion. Together, these findings reveal mitochondrial CI as a critical rheostat of innate immunity and skeletal homeostasis. PMID:25130399
Loss of macrophage fatty acid oxidation does not potentiate systemic metabolic dysfunction
Gonzalez-Hurtado, Elsie; Lee, Jieun; Choi, Joseph; Selen Alpergin, Ebru S.; Collins, Samuel L.; Horton, Maureen R.
2017-01-01
Fatty acid oxidation in macrophages has been suggested to play a causative role in high-fat diet-induced metabolic dysfunction, particularly in the etiology of adipose-driven insulin resistance. To understand the contribution of macrophage fatty acid oxidation directly to metabolic dysfunction in high-fat diet-induced obesity, we generated mice with a myeloid-specific knockout of carnitine palmitoyltransferase II (CPT2 Mϕ-KO), an obligate step in mitochondrial long-chain fatty acid oxidation. While fatty acid oxidation was clearly induced upon IL-4 stimulation, fatty acid oxidation-deficient CPT2 Mϕ-KO bone marrow-derived macrophages displayed canonical markers of M2 polarization following IL-4 stimulation in vitro. In addition, loss of macrophage fatty acid oxidation in vivo did not alter the progression of high-fat diet-induced obesity, inflammation, macrophage polarization, oxidative stress, or glucose intolerance. These data suggest that although IL-4-stimulated alternatively activated macrophages upregulate fatty acid oxidation, fatty acid oxidation is dispensable for macrophage polarization and high-fat diet-induced metabolic dysfunction. Macrophage fatty acid oxidation likely plays a correlative, rather than causative, role in systemic metabolic dysfunction. PMID:28223293
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.
Omega-3 fatty acid supplementation and cardiovascular disease
Jump, Donald B.; Depner, Christopher M.; Tripathy, Sasmita
2012-01-01
Epidemiological studies on Greenland Inuits in the 1970s and subsequent human studies have established an inverse relationship between the ingestion of omega-3 fatty acids [C20–22 ω 3 polyunsaturated fatty acids (PUFA)], blood levels of C20–22 ω 3 PUFA, and mortality associated with cardiovascular disease (CVD). C20–22 ω 3 PUFA have pleiotropic effects on cell function and regulate multiple pathways controlling blood lipids, inflammatory factors, and cellular events in cardiomyocytes and vascular endothelial cells. The hypolipemic, anti-inflammatory, anti-arrhythmic properties of these fatty acids confer cardioprotection. Accordingly, national heart associations and government agencies have recommended increased consumption of fatty fish or ω 3 PUFA supplements to prevent CVD. In addition to fatty fish, sources of ω 3 PUFA are available from plants, algae, and yeast. A key question examined in this review is whether nonfish sources of ω 3 PUFA are as effective as fatty fish-derived C20–22 ω 3 PUFA at managing risk factors linked to CVD. We focused on ω 3 PUFA metabolism and the capacity of ω 3 PUFA supplements to regulate key cellular events linked to CVD. The outcome of our analysis reveals that nonfish sources of ω 3 PUFA vary in their capacity to regulate blood levels of C20–22 ω 3 PUFA and CVD risk factors. PMID:22904344
Colak, Gozde; Pougovkina, Olga; Dai, Lunzhi; Tan, Minjia; te Brinke, Heleen; Huang, He; Cheng, Zhongyi; Park, Jeongsoon; Wan, Xuelian; Liu, Xiaojing; Yue, Wyatt W.; Wanders, Ronald J. A.; Locasale, Jason W.; Lombard, David B.; de Boer, Vincent C. J.; Zhao, Yingming
2015-01-01
The protein substrates of sirtuin 5-regulated lysine malonylation (Kmal) remain unknown, hindering its functional analysis. In this study, we carried out proteomic screening, which identified 4042 Kmal sites on 1426 proteins in mouse liver and 4943 Kmal sites on 1822 proteins in human fibroblasts. Increased malonyl-CoA levels in malonyl-CoA decarboxylase (MCD)-deficient cells induces Kmal levels in substrate proteins. We identified 461 Kmal sites showing more than a 2-fold increase in response to MCD deficiency as well as 1452 Kmal sites detected only in MCD−/− fibroblast but not MCD+/+ cells, suggesting a pathogenic role of Kmal in MCD deficiency. Cells with increased lysine malonylation displayed impaired mitochondrial function and fatty acid oxidation, suggesting that lysine malonylation plays a role in pathophysiology of malonic aciduria. Our study establishes an association between Kmal and a genetic disease and offers a rich resource for elucidating the contribution of the Kmal pathway and malonyl-CoA to cellular physiology and human diseases. PMID:26320211
Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids.
Leber, Christopher; Da Silva, Nancy A
2014-02-01
Carbon feedstocks from fossilized sources are being rapidly depleted due to rising demand for industrial and commercial applications. Many petroleum-derived chemicals can be directly or functionally substituted with chemicals derived from renewable feedstocks. Several short chain organic acids may fulfill this role using their functional groups as a target for chemical catalysis. Saccharomyces cerevisiae was engineered to produce short chain carboxylic acids (C6 to C10 ) from glucose using the heterologous Homo sapiens type I fatty acid synthase (hFAS). This synthase was activated by phosphopantetheine transfereases AcpS and Sfp from Escherichia coli and Bacillus subtilis, respectively, both in vitro and in vivo. hFAS was produced in the holo-form and produced carboxylic acids in vitro, confirmed by NADPH and ADIFAB assays. Overexpression of hFAS in a yeast FAS2 knockout strain, deficient in de novo fatty acid synthesis, demonstrated the full functional replacement of the native fungal FAS by hFAS. Two active heterologous short chain thioesterases (TEs) from Cuphea palustris (CpFatB1) and Rattus norvegicus (TEII) were evaluated for short chain fatty acid (SCFA) synthesis in vitro and in vivo. Three hFAS mutants were constructed: a mutant deficient in the native TE domain, a mutant with a linked CpFatB1 TE and a mutant with a linked TEII TE. Using the native yeast fatty acid synthase for growth, the overexpression of the hFAS mutants and the short-chain TEs (linked or plasmid-based) increased in vivo caprylic acid and total SCFA production up to 64-fold (63 mg/L) and 52-fold (68 mg/L), respectively, over the native yeast levels. Combined over-expression of the phosphopantetheine transferase with the hFAS mutant resulted in C8 titers of up to 82 mg/L and total SCFA titers of up to 111 mg/L. © 2013 Wiley Periodicals, Inc.
Mitochondrial function at extreme high altitude.
Murray, Andrew J; Horscroft, James A
2016-03-01
At high altitude, barometric pressure falls and with it inspired P(O2), potentially compromising O2 delivery to the tissues. With sufficient acclimatisation, the erythropoietic response increases red cell mass such that arterial O2 content (C(aO2)) is restored; however arterial P(O2)(P(aO2)) remains low, and the diffusion of O2 from capillary to mitochondrion is impaired. Mitochondrial respiration and aerobic capacity are thus limited, whilst reactive oxygen species (ROS) production increases. Restoration of P(aO2) with supplementary O2 does not fully restore aerobic capacity in acclimatised individuals, possibly indicating a peripheral impairment. With prolonged exposure to extreme high altitude (>5500 m), muscle mitochondrial volume density falls, with a particular loss of the subsarcolemmal population. It is not clear whether this represents acclimatisation or deterioration, but it does appear to be regulated, with levels of the mitochondrial biogenesis factor PGC-1α falling, and shows similarities to adapted Tibetan highlanders. Qualitative changes in mitochondrial function also occur, and do so at more moderate high altitudes with shorter periods of exposure. Electron transport chain complexes are downregulated, possibly mitigating the increase in ROS production. Fatty acid oxidation capacity is decreased and there may be improvements in biochemical coupling at the mitochondrial inner membrane that enhance O2 efficiency. Creatine kinase expression falls, possibly impairing high-energy phosphate transfer from the mitochondria to myofibrils. In climbers returning from the summit of Everest, cardiac energetic reserve (phosphocreatine/ATP) falls, but skeletal muscle energetics are well preserved, possibly supporting the notion that mitochondrial remodelling is a core feature of acclimatisation to extreme high altitude. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.
The function of yeast CAP family proteins in lipid export, mating, and pathogen defense.
Darwiche, Rabih; El Atab, Ola; Cottier, Stéphanie; Schneiter, Roger
2018-04-01
In their natural habitat, yeast cells are constantly challenged by changing environmental conditions and a fierce competition for limiting resources. To thrive under such conditions, cells need to adapt and divide quickly, and be able to neutralize the toxic compounds secreted by their neighbors. Proteins like the pathogen-related yeast, Pry proteins, which belong to the large CAP/SCP/TAPS superfamily, may have an important role in this function. CAP proteins are conserved from yeast to man and are characterized by a unique αβα sandwich fold. They are mostly secreted glycoproteins and have been implicated in many different physiological processes including pathogen defense, virulence, venom toxicity, and sperm maturation. Yeast members of this family bind and export sterols as well as fatty acids, and they render cells resistant to eugenol, an antimicrobial compound present in clove oil. CAP family members might thus exert their various physiological functions through binding, sequestration, and neutralization of such small hydrophobic compounds. © 2017 Federation of European Biochemical Societies.
Crichton, Paul G.; Lee, Yang; Ruprecht, Jonathan J.; Cerson, Elizabeth; Thangaratnarajah, Chancievan; King, Martin S.; Kunji, Edmund R. S.
2015-01-01
Mitochondrial carriers, including uncoupling proteins, are unstable in detergents, which hampers structural and mechanistic studies. To investigate carrier stability, we have purified ligand-free carriers and assessed their stability with a fluorescence-based thermostability assay that monitors protein unfolding with a thiol-reactive dye. We find that mitochondrial carriers from both mesophilic and thermophilic organisms exhibit poor stability in mild detergents, indicating that instability is inherent to the protein family. Trends in the thermostability of yeast ADP/ATP carrier AAC2 and ovine uncoupling protein UCP1 allow optimal conditions for stability in detergents to be established but also provide mechanistic insights into the interactions of lipids, substrates, and inhibitors with these proteins. Both proteins exhibit similar stability profiles across various detergents, where stability increases with the size of the associated detergent micelle. Detailed analysis shows that lipids stabilize carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilizes by direct interactions as well. Cardiolipin reverses destabilizing effects of ADP and bongkrekic acid on AAC2 and enhances large stabilizing effects of carboxyatractyloside, revealing that this lipid interacts in the m-state and possibly other states of the transport cycle, despite being in a dynamic interface. Fatty acid activators destabilize UCP1 in a similar way, which can also be prevented by cardiolipin, indicating that they interact like transport substrates. Our controls show that carriers can be soluble but unfolded in some commonly used detergents, such as the zwitterionic Fos-choline-12, which emphasizes the need for simple validation assays like the one used here. PMID:25653283