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Résumé Chez l'homme, les dysfonctions des enzymes du cycle de Krebs liées à des mutations causent des encéphalopathies sévères du jeune enfant, ou différents types de tumeurs et de cancers touchant différents organes. Ainsi des mutations des gènes de la succinate-déshydrogénase ont été identifiées dans des formes familiales de phéochromocytomes et paragangliomes. Les mutations du gène de la fumarase s'observent dans des léiomyomes utérins en association avec des cancers rénaux, mais des cas d'encéphalopathies de l'enfant ont aussi été observés. Les mutations des gènes de l'isocitrate-déshydrogénase peuvent représenter jusqu'à 20% des leucémies myéloïdes aiguës et jusqu'à 80% dans certains gliomes. On ne sait actuellement expliciter la diversité des conséquences possibles de ces mutations : encéphalopathies versus cancers, nature des tissus affectés, cinétique variable d'apparition des symptômes. Il semble en revanche établi que les anomalies dans les équilibres métaboliques qui résultent de ces mutations soient à la base des processus tumoraux en modulant l'activité d'hydroxylases susceptibles d'affecter certains facteurs de transcriptions, mais aussi la méthylation de l'ADN et les histones. Dans ce contexte, il semble étonnant que soient largement utilisés certains fongicides dont l'effet est de bloquer une étape centrale du cycle de Krebs, la succinate-déshydrogénase. Le principe de précaution voudrait sans aucun doute de réévaluer urgemment la nécessité de l'usage de telles substances pour l'homme et l'environnement. In humans, dysfunctions of Krebs cycle enzymes caused by genetic mutations result either in severe encephalopathies of the young child, or in the appearance of different types of tumors and cancers affecting diverse organs. Thus mutations of the succinate dehydrogenase genes have been identified in familial forms of pheochromocytomas and paragangliomas. Most of the mutations in the fumarase gene are found in uterine leiomyomas in association with renal cancers, but cases of childhood encephalopathies have also been observed resulting from mutations affecting the same gene. Isocitrate dehydrogenase gene mutations account for up to 20% of acute myeloid leukemias, and up to 80% in some gliomas. So far we cannot explain the diversity of the possible consequences of these mutations : encephalopathies versus cancers, the different tissues that are affected and the variable kinetics of appearance of symptoms. On the other hand, it seems now established that the abnormalities in the metabolic equilibria that result from these mutations are at the basis of the tumor processes by modulating the activity of a number of hydroxylases that may affect the fate of certain transcription factors, but also the DNA and histone methylation. In this context, it seems surprising that some fungicides that are now widely used, block a central stage of the Krebs cycle, namely the succinate dehydrogenase. The principle of precaution would impose to urgently re-evaluate the need for the use of these substances in consideration of human health and the environment. [ABSTRACT FROM AUTHOR]

The bacterial GatCAB operon for tRNA-dependent amidotransferase (AdT) catalyzes the transamidation of mischarged glutamyl-tRNAGLN to glutaminyl-tRNAGLN. Here we describe the phenotype of temperature-sensitive (ts) mutants of GTF1, a gene proposed to code for subunit F of mitochondrial AdT in Saccharomyces cerevisiae. The ts gtfl mutants accumulate an electrophoretic variant of the mitochondrially encoded Cox2p subunit of cytochrome oxidase and an unstable form of the Atp8p subunit of the F1-F0 ATP synthase that is degraded, thereby preventing assembly of the F0 sector. Allotopic expression of recoded A TP8 and COX2 did not significantly improve growth of gtfl mutants on respiratory substrates. However, ts gftl mutants are partially rescued by overexpression of PET1J2 and HER2 that code for the yeast homologues of the catalytic subunits of bacterial AdT. Additionally, B66, a her2 point mutant has a phenotype similar to that of gtfl mutants. These results provide genetic support for the essentiality, in vivo, of the GatF subunit of the heterotrimeric AdT that catalyzes formation of glutaminyl-tRNAGLN (Frechin, M., Senger, B., Brayé, M., Kern, D., Martin, R. P., and Becker, H. D. (2009) Genes Dev. 23, 1119 -1130). [ABSTRACT FROM AUTHOR]

NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F1 coupled to F0-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease. [ABSTRACT FROM AUTHOR]

Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of ρ-/ρ0 petites issued from large deletions in mtDNA could be restricted to 20–30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion. [ABSTRACT FROM AUTHOR]

Within the mitochondrial F1F0-ATP synthase, the nucleus-encoded δ-F1 subunit plays a critical role in coupling the enzyme proton translocating and ATP synthesis activities. In Saccharomyces cerevisiae, deletion of the δ subunit gene (Δδ) was shown to result in a massive destabilization of the mitochondrial genome (mitochondrial DNA; mtDNA) in the form of 100% ρ-/ρ° petites (i.e. cells missing a large portion (>50%) of the mtDNA (ρ-) or totally devoid of mtDNA (ρ°)). Previous work has suggested that the absence of complete mtDNA (ρ+) in Δδ yeast is a consequence of an uncoupling of the ATP synthase in the form of a passive proton transport through the enzyme (i.e. not coupled to ATP synthesis). However, it was unclear why or how this ATP synthase defect destabilized the mtDNA. We investigated this question using a nonrespiratory gene (ARG8m) inserted into the mtDNA. We first show that retention of functional mtDNA is lethal to Δδ yeast. We further show that combined with a nuclear mutation (Δatp4) preventing the ATP synthase proton channel assembly, a lack of 6 subunit fails to destabilize the mtDNA, and ρ+ Δδ cells become viable. We conclude that Δδ yeast cannot survive when it has the ability to synthesize the ATP synthase proton channel. Accordingly, the ρ-/ρ° mutation can be viewed as a rescuing event, because this mutation prevents the synthesis of the two mtDNA-encoded subunits (Atp6p and Atp9p) forming the core of this channel. This is the first report of what we have called a "petite obligate" mutant of S. cerevisiae. [ABSTRACT FROM AUTHOR]

In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F0F1-ATP synthase at elevated temperatures. The Δfmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37 °C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the Δfmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports α-ketoglutarate and α-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the α-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system. [ABSTRACT FROM AUTHOR]

Very little is known about biogenesis of mitochondrial ribosomes. The GTPases encoded by the nuclear MTG1 and MTG2 genes of Saccharomyces cerevisiae have been reported to play a role in assembly of the ribosomal 54 S subunit. In the present study biochemical screens of a collection of respiratory deficient yeast mutants have enabled us to identify a third gene essential for expression of mitochondrial ribosomes. This gene codes for a member of the YqeH family of GTPases, which we have named MTG3 in keeping with the earlier convention. Mutations in MTG3 cause the accumulation of the 15 S rRNA precursor, previously shown to have an 80-nucleotide 5' extension. Sucrose gradient sedimentation of mitochondrial ribosomes from temperature- sensitive mtg3 mutants grown at the permissive and restrictive temperatures, combined with immunobloting with subunit-specific antibodies, indicate that Mtg3p is required for assembly of the 30 S but not 54 S ribosomal subunit. The respiratory deficient growth phenotype of an mtg3 null mutant is partially rescued by overexpression of the Mrpl4p constituent located at the peptide exit site of the 54 S subunit. The rescue is accompanied by an increase in processed 15 S rRNA. This suggests that Mtg3p and Mrpl4p jointly regulate assembly of the small subunit by modulating processing of the 15 S rRNA precursor. [ABSTRACT FROM AUTHOR]