Méthode d'estimation de la concentration des cytochromes dans les cellules entières de levure

The mitochondrial solute carriers Mrs3p and Mrs4p were originally isolated as multicopy suppressors of intron splicing defects. We show here that MRS4 is coregulated with the iron regulon genes, and up-regulated in a strain deficient for Yfh1p, the yeast homologue of human frataxin. Using in vivo 55 Fe cell radiolabeling we show that in glucose-grown cells mitochondrial iron accumulation is 5-15 times higher in ⌬YFH1 than in wildtype strain. However, although in a ⌬YFH1⌬MRS3⌬MRS4 strain, the intracellular 55 Fe content is extremely high, the mitochondrial iron concentration is decreased to almost wild-type levels. Moreover, ⌬YFH1⌬MRS3⌬MRS4 cells grown in high iron media do not lose their mitochondrial genome. Conversely, a ⌬YFH1 strain overexpressing MRS4 has an increased mitochondrial iron content and no mitochondrial genome. Therefore, MRS4 is required for mitochondrial iron accumulation in ⌬YFH1 cells. Expression of the iron regulon and intracellular 55 Fe content are higher in a ⌬MRS3⌬MRS4 strain than in the wild type. Nevertheless, the mitochondrial 55 Fe content, a balance between iron uptake and exit, is decreased by a factor of two. Moreover, 55Fe incorporation into heme by ferrochelatase is increased in an MRS4-overexpressing strain. The function of MRS4 in iron import into mitochondria is discussed.Mitochondria utilize most of the cellular iron. First, the mitochondrial ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX, the heme precursor of cytochromes. Second, the mitochondrial matrix and respiratory chain contain several iron-sulfur proteins. Moreover, it has recently been discovered that iron-sulfur clusters are synthesized inside mitochondria by a specific machinery involving more than 10 proteins that have orthologues in bacteria (1, 2). These iron-sulfur clusters are used for both mitochondrial and cytosolic proteins, and their export into the cytosol is probably mediated by the ABC transporter Atm1p (3). Based on their capacity to restore the low iron growth defect of an erg25 mutant (4), two homologous transporters belonging to the cation efflux transporter family (5), Mmt1p and Mmt2p, have been

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“…Low temperature cytochrome absorption spectra were carried out, and cytochrome concentration was estimated as reported (22).…”

Section: Methodsmentioning

confidence: 99%

Deletion of the Mitochondrial Carrier Genes MRS3 andMRS4 Suppresses Mitochondrial Iron Accumulation in a Yeast Frataxin-deficient Strain

Foury

Roganti

2002

Journal of Biological Chemistry

161

162

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“…The growth of petite mutants is limited to fermentable carbon sources such as glucose or sucrose, but they fail to grow on glycerol and ethanol (2,34). Cytochrome determinations show that petite mutants are lacking either cytochrome b or cytochrome aa 3 or both but always retain cytochrome c (6,10,35). The molecular basis of the petite phenotype is mutation or deletion of mitochondrial genes (9).…”

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confidence: 99%

Candida albicansMutants Deficient in Respiration Are Resistant to the Small Cationic Salivary Antimicrobial Peptide Histatin 5

Gyurko

Lendenmann

Troxler

et al. 2000

Antimicrob Agents Chemother

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Histatins are a group of small cationic peptides in human saliva which are well known for their antibacterial and antifungal activities. In a previous study we demonstrated that histatin 5 kills both blastoconidia and germ tubes of Candida albicans in a time-and concentration-dependent manner at 37°C, whereas no killing was detected at 4°C. This indicated that killing activity depends on cellular energy. To test histatin 5 killing activity at lower cellular ATP levels at 37°C, respiratory mutants, or so-called petite mutants, of C. albicans were prepared. These mutants are deficient in respiration due to mutations in mitochondrial DNA. Mutants were initially identified by their small colony size and were further characterized with respect to colony morphology, growth characteristics, respiratory activity, and cytochrome spectra. The killing activity of histatin 5 at the highest concentration was only 28 to 30% against respiratory mutants, whereas 98% of the wild-type cells were killed. Furthermore, histatin 5 killing activity was also tested on wild-type cells in the presence of the respiratory inhibitor sodium azide or, alternatively, the uncoupler carbonyl cyanide m-chlorophenylhydrazone. In both cases histatin 5 killing activity was significantly reduced. Additionally, supernatants and pellets of cells incubated with histatin 5 in the presence or absence of inhibitors of mitochondrial ATP synthesis were analyzed by sodium dodecyl sulfate gel electrophoresis. It was observed that wild-type cells accumulated large amounts of histatin 5, while wild-type cells treated with inhibitors or petite mutants did not accumulate significant amounts of the peptide. These data showed first that cellular accumulation of histatin 5 is necessary for killing activity and second that accumulation of histatin 5 depends on the availability of cellular energy. Therefore, mitochondrial ATP synthesis is required for effective killing activity of histatin 5.

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“…Cytochrome absorption spectra were recorded for whole cells in liquid nitrogen as described previously (23).…”

Section: Methodsmentioning

confidence: 99%

Mutations in the Yeast MRF1 Gene Encoding Mitochondrial Release Factor Inhibit Translation on Mitochondrial Ribosomes

Towpik

Chacińska

Cieśla

et al. 2004

Journal of Biological Chemistry

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Although the control of mitochondrial translation in the yeast Saccharomyces cerevisiae has been studied extensively, the mechanism of termination remains obscure. Ten mutations isolated in a genetic screen for read-through of premature stop codons in mitochondrial genes were localized in the chromosomal gene encoding the mitochondrial release factor mRF1. The mrf1-13 and mrf1-780 mutant genes, in contrast to other alleles, caused a non-respiratory phenotype that correlated with decreased expression of mitochondrial genes as well as a reporter ARG8 m gene inserted into mitochondrial DNA. The steady-state levels of several mitochondrially encoded proteins, but not their mRNAs, were dramatically decreased in mrf1-13 and mrf1-780 cells. Structural models of mRF1 were constructed, allowing localization of residues substituted in the mrf1 mutants and offering an insight into the possible mechanism by which these mutations change the mitochondrial translation termination fidelity. Inhibition of mitochondrial translation in mrf1-13 and mrf1-780 correlated with the three-dimensional localization of the mutated residues close to the PST motif presumably involved in the recognition of stop codons in mitochondrial mRNA.The mitochondrial genetic system of the yeast Saccharomyces cerevisiae has been a subject of intensive studies. Seven genes in the yeast mitochondrial DNA (mtDNA) 1 encode integral membrane proteins that are assembled together with subunits encoded in the nuclear DNA into multimeric respiratory complexes. Maintenance and expression of mtDNA require approximately 200 proteins encoded in the nuclear DNA and imported to mitochondria from the cytosol (1, 2). Among them are mitochondrial ribosomal proteins, general translation factors, and translational activators. Current analysis of the proteome of yeast mitochondria allowed for identification of 65 mitoribosomal proteins (2); however, only ϳ60% of these have recognizable homology with bacterial ribosomal proteins (3). Multiple mitoribosome-specific proteins in yeast and mammals demonstrate the considerable divergence of mitochondrial ribosomes from their prokaryotic ancestors (4, 5). The mechanisms that control mitochondrial translation are difficult to study because no in vitro system for the expression of translation products encoded in mtDNA has yet been established. In contrast to the biochemical difficulties, the yeast S. cerevisiae, being a facultative anaerobe, offers distinct advantages for mitochondrial genetic studies. Several hundred point mutations or small deletions called mit Ϫ have been genetically mapped and assigned to genes in mtDNA. We have long been involved in a search for suppressors of mit Ϫ mutants that act at the level of mitochondrial translation. Analogous suppressor approach applied to chromosomal mutants allowed identification of proteins responsible for translation fidelity in the yeast cytosolic system. Ribosomal proteins from the decoding center of the ribosome and translation termination factors represented two classes of prote...

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Méthode d'estimation de la concentration des cytochromes dans les cellules entières de levure

Cited by 108 publications

References 30 publications

Deletion of the Mitochondrial Carrier Genes MRS3 andMRS4 Suppresses Mitochondrial Iron Accumulation in a Yeast Frataxin-deficient Strain

Deletion of the Mitochondrial Carrier Genes MRS3 andMRS4 Suppresses Mitochondrial Iron Accumulation in a Yeast Frataxin-deficient Strain

Candida albicansMutants Deficient in Respiration Are Resistant to the Small Cationic Salivary Antimicrobial Peptide Histatin 5

Mutations in the Yeast MRF1 Gene Encoding Mitochondrial Release Factor Inhibit Translation on Mitochondrial Ribosomes

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