Mitochondrial AAA-type protease Yme1p is involved in Bax effects on cytochrome c oxidase.

Expression of the pro-apoptotic protein Bax in yeast Saccharomyces cerevisiae induces a release of cytochrome c accompanied by a decrease of the amount of cytochrome c oxidase. Here we show that the decrease of cytochrome c oxidase is due to the activation of mitochondrial protease Yme1p, of which cytochrome c oxidase subunit 2 (Cox2p) is a substrate. The absence of Yme1p slightly delays Bax-induced cell death, suggesting a role of this protease in yeast cell death and thus of its mammalian homologue in apoptosis.

Aging and oxidative stress: studies of some genes involved both in aging and in response to oxidative stress.

Aging is a complex physiological phenomenon and several theories have been developed about its origin. Among such theories, the 'mitochondrial theory of aging' has been supported by numerous studies and reviews. Cell oxidative damage, in particular the accumulation of mtDNA mutations, is determined by the rate of reactive oxygen species production and degradation induced by the antioxidant defense systems. In this review, data from our laboratory and from the recent literature have been examined to provide arguments that reinforce the crucial role of mitochondria in aging. Various genes that affect life span have been described in numerous organisms. Some of them encode signal transduction proteins and participate in the regulation of mitochondrial metabolism.

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.

Yeast mitochondrial dehydrogenases are associated in a supramolecular complex.

Separation of yeast mitochondrial complexes by colorless native polyacrylamide gel electrophoresis led to the identification of a supramolecular structure exhibiting NADH-dehydrogenase activity. Components of this complex were identified by N-terminal Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The complex was found to contain the five known intermembrane space-facing dehydrogenases, namely two external NADH-dehydrogenases Nde1p and Nde2p, glycerol-3-phosphate dehydrogenase Gut2p, D- and L-lactate-dehydrogenases Dld1p and Cyb2p, the matrix-facing NADH-dehydrogenase Ndi1p, two probable flavoproteins YOR356Wp and YPR004Cp, four tricarboxylic acids cycle enzymes (malate dehydrogenase Mdh1p, citrate synthase Cit1p, succinate dehydrogenase Sdh1p, and fumarate hydratase Fum1p), and the acetaldehyde dehydrogenase Ald4p. The association of these proteins is discussed in terms of NADH-channeling.

The 'SUN' family: yeast SUN4/SCW3 is involved in cell septation.

SUN4 is the fourth member of the SUN gene family from S. cerevisiae, whose products display high homology in their 258 amino acid C-terminal domain. SIM1, UTH1, NCA3 (the founding members) are involved in different cellular processes (DNA replication, ageing, mitochondrial biogenesis) and it is shown herein that SUN4 plays a role in the cell septation process. sun4 delta cells are larger than wild-type and begin a new cell cycle before they have separated from their mother cell. This phenotype is more pronounced in sun4Delta cells also deleted for UTH1. FACS analysis shows apparent polyploidy which disappears when the cell cycle is arrested by mating factor or nocodazole, indicating that cell septation is delayed without modification of the doubling time. Elutriated sun4 delta uth1 delta daughter cells are born larger, and therefore enter S phase sooner than their wild-type counterpart. S phase duration, as well as timing of Clb2 degradation, is normal, but cell septation is delayed. Sun4p/Scw3p was recently described as a cell wall protein (Cappellaro et al., 1998) and, consistent with this notion, electron micrographs of sun4 delta cells show defects in the final steps of cell wall septation. Our data suggest that Sun4p and Uth1p might contribute to the regulated process of cell wall morphogenesis and septation.

The "SUN" family: UTH1, an ageing gene, is also involved in the regulation of mitochondria biogenesis in Saccharomyces cerevisiae.

Since it was shown in previous work that NCA3 (one of the four genes of the SUN family) is involved in mitochondrial protein synthesis regulation, the effect of the other members of this gene family was tested. UTH1 (but not SUN4 or SIM1) was also shown to interfere with mitochondria biogenesis. In Deltauth1 cells, cytochromes aa(3), c, and b were lowered by 25 and 15%, respectively. In the double-null mutant Deltauth1Deltanca3, only cytochrome aa(3) was lowered by 50% relative to the wild type. However, the ratio of cellular respiration to cytochrome oxidase was greatly enhanced in the double-null mutant. Measurements on whole lysed cells showed that another mitochondrial enzyme, citrate synthase, was also lowered in Deltauth1 and Deltauth1Deltanca3 whereas hexokinase was not. Electron micrographs showed no difference in global mitochondria content in Deltauth1Deltanca3, but mitochondria appeared less dense to electrons compared to the wild type. Cardiolipin and mtDNA were equivalent in parental and mutant strains. Measurements on isolated mitochondria showed that the cyt aa(3)/cyt b ratio was also lowered in Deltauth1Deltanca3, but the control exerted by the oxidase on the respiratory flux was higher. The activity of other mitochondrial complexes versus oxidase was equivalent in mutants compared to the wild type. These results suggest that the protein equipment could be lowered in mitochondria from strains inactivated for UTH1.

The SUN family of Saccharomyces cerevisiae: the double knock-out of UTH1 and SIM1 promotes defects in nucleus migration and increased drug sensitivity.

UTH1 and SIM1 are two of four 'SUN' genes (SIM1, UTH1, NCA3 and SUN4/SCW3) whose products are involved in different cellular processes such as DNA replication, lifespan, mitochondrial biogenesis or cell septation. UTH1 or SIM1 inactivation did not affect cell growth, shape or nuclear migration, whereas the double null mutant presented phenotypes of numerous binucleate cells and benomyl sensitivity, suggesting that microtubule function could be altered; the uth1Deltasim1Delta strain also presented defects which could be related to the Ras/cAMP pathway: pet phenotype, heat shock sensitivity, inability to store glycogen, sensitivity to starvation and failure of spores to germinate. These observations suggested that Uth1p could be involved as a connection step between pathways controlling growth and those controlling division.

Comparison of the effects of bax-expression in yeast under fermentative and respiratory conditions: investigation of the role of adenine nucleotides carrier and cytochrome c.

A new system for bax-expression in yeast has been devised to investigate bax's effect under fermentative and respiro-fermentative conditions. This has allowed us to show unambiguously that the ability of bax to kill yeast is higher under respiratory conditions than under purely fermentative conditions. The extent of killing under respiro-fermentative conditions (non-repressive sugars) is intermediate. It has been proposed that the two proteins adenine nucleotides carrier (ANC) and cytochrome c play a crucial role in bax-induced cell death. We have investigated the effects of deletion of the genes encoding the two proteins on the toxicity induced by bax, using this new system. The absence of ANC did not modify bax-induced lethality in any way. Moreover, the absence of cytochrome c also did not prevent bax-induced death. Only the kinetics of lethality were altered. All these effects are prevented by co-expression of bcl-xL.

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.

Investigation of bax-induced release of cytochrome c from yeast mitochondria permeability of mitochondrial membranes, role of VDAC and ATP requirement.

Recent studies that attempt to explore the action of pro- and anti-apoptotic proteins of the bcl2 family demonstrate the crucial role of relocalization of cytochrome c from the mitochondrial intermembrane space to the cytosol. This early event of apoptosis can be mimicked in the yeast Saccharomyces cerevisiae following expression of bax. In mammalian mitochondria, the mechanism of relocalization is thought to involve the opening of the so-called permeability transition pore. We show in this paper: (a) that bax-induced release of cytochrome c in yeast does not involve any permeability transition of the inner mitochondrial membrane but involves a general alteration of the permeability of the outer mitochondrial membrane to macromolecules. This suggests that a permeability transition of the inner mitochondrial membrane is not an event required for the relocalization of cytochrome c in yeast. (b) The outer-membrane voltage-dependent anion channel (VDAC), a putative component of the permeability transition pore, is not involved in bax-induced release of cytochrome c or in the prevention of this release by bcl-xL. (c) Bax devoid of its C-terminal putative hydrophobic alpha-helix is as efficient as full-length bax to allow the relocalization of cytochrome c, demonstrating this segment of the protein is not required for membrane-targeting. (d) We finally observe that the action of bax on the outer mitochondrial membrane requires the presence of ATP both in vitro and in vivo, and it is shown that ATP directly increases the amount of bax inserted to mitochondria.

Role of the C-terminal domain of Bax and Bcl-XL in their localization and function in yeast cells.

It has been suggested that the C-terminal domain of Bcl-2 family members may contain a signal anchor sequence that targets these proteins to the mitochondrial outer membrane. We have investigated the consequence of deleting this domain upon cytochrome c release in yeast strains that coexpress truncated forms of Bax (i.e. BaxA) and Bcl-X(L) (i.e. Bcl-X(L)delta). We find that (i) Bax(delta) is as efficient as full-length Bax in promoting cytochrome c release, but Bcl-x(L)delta has remarkably reduced rescuing ability compared to full-length Bcl-x(L); (ii) full-length Bcl-X(L) protein acts by relocalizing Bax from the mitochondrial fraction to the soluble cytosolic fraction; (iii) Bax undergoes N-terminal cleavage when expressed in yeast, which is prevented by coexpression of Bcl-X(L), suggesting that Bcl-x(L) may mask the cleavage site of Bax through a direct physical interaction of the two proteins.

Isolation of supernumerary yeast ATP synthase subunits e and i. Characterization of subunit i and disruption of its structural gene ATP18.

Two subunits of the yeast ATP synthase have been isolated. Subunit e was found loosely associated to the complex. Triton X-100 at a 1% concentration removed this subunit from the ATP synthase. The N-terminal sequencing of subunit i has been performed. The data are in agreement with the sequence of the predicted product of a DNA fragment of Saccharomyces cerevisiae chromosome XIII. The ATP18 gene encodes subunit i, which is 59 amino acids long and corresponds to a calculated mass of 6687 Da. Its pI is 9.73. It is an amphiphilic protein having a hydrophobic N-terminal part and a hydrophilic C-terminal part. It is not apparently related to any subunit described in other ATP synthases. The null mutant showed low growth on nonfermentable medium. Mutant mitochondria display a low ADP/O ratio and a decrease with time in proton pumping after ATP addition. Subunit i is associated with the complex; it is not a structural component of the enzyme but rather is involved in the oxidative phosphorylations. Similar amounts of ATP synthase were measured for wild-type and null mutant mitochondria. Because 2-fold less specific ATPase activity was measured for the null mutant than for the wild-type mitochondria, we make the hypothesis that the observed decrease in the turnover of the mutant enzyme could be linked to a proton translocation defect through F0.

Topography of the yeast ATP synthase F0 sector.

The interaction between the hydrophilic C-terminal part of subunit 4 (subunit b) and OSCP, which are two components of the connecting stalk of the yeast ATP synthase, was shown after reconstitution of the two over-expressed proteins and by the two-hybrid method. The organization of a part of the F0 sector was studied by the use of mutants containing cysteine residues in a loop connecting the two N-terminal postulated membrane-spanning segments. Labelling of the mutated subunits 4 by a maleimide fluorescent probe revealed that the sulfhydryl groups were modified upon incubation of intact mitochondria. In addition, non-permeant maleimide reagents labeled subunit 4D54C, thus showing a location of this residue in the intermembrane space. Cross-linking experiments revealed the proximity of subunits 4 and f. In addition, a disulfide bridge between subunit 4D54C and subunit 6 was evidenced, thus demonstrating near-neighbor relationships of the two subunits and a location of the N-terminal part of the mitochondrially-encoded subunit 6 in the intermembrane space.

NCA2, a second nuclear gene required for the control of mitochondrial synthesis of subunits 6 and 8 of ATP synthase in Saccharomyces cerevisiae.

Respiratory-competent nuclear mutants have been isolated which presented a cryosensitive phenotype on a non-fermentable carbon source, due to a dysfunction of the mitochondrial F1-Fo ATP synthase. This defect results from an alteration of the mtDNA-encoded protein synthesis level of subunits 6 and 8 of the Fo sector, due to the simultaneous presence of a mutation in two unlinked nuclear genes. These mutations promote a modification of the expression of the cotranscript ATP8-ATP6 (formerly denoted AAP1-OL12): this mRNA undergoes a maturation at a unique site reaching to two cotranscripts of 5.2 and 4.6 kb in length: in the mutant, the relative amount of 5.2 kb cotranscript was greatly lowered. NCA2 was isolated from a wild-type yeast genomic library by genetic complementation. The relative level of the 5.2 kb transcript, as the synthesis of subunits 6 and 8, was partly restored in the transformed strain. A 1848 nucleotide open reading frame was depicted that encoded an amphiphilic protein of 70,816 Da. Disruption of chromosomal DNA within the reading frame promoted a dramatic decrease of the 5.2 kb mRNA but did not abolish the respiratory competence of a wild-type strain. Hybridization analyses indicated that NCA2 is located on chromosome XVI and produces a single 2750 base transcript.

NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the Fo-F1 ATP synthase of S. cerevisiae.

Respiratory-competent nuclear mutants have been isolated which presented a cryosensitive phenotype on a non-fermentative carbon source, due to a dysfunctioning of the mitochondrial F1-Fo ATP synthase which results from a relative defect in subunits 6 and 8 of the Fo sector. Both proteins are mtDNA-encoded, but the defect is due to the simultaneous presence of a mutation in two unlinked nuclear genes (NCA2 and NCA3, for Nuclear Control of ATPase) promoting a modification of the expression of the ATP8-ATP6 co-transcript (formerly denoted AAP1-OLI2). This co-transcript matures at a unique site to give two cotranscripts of 5.2 and 4.6 kb in length: in the mutant, the 5.2-kb co-transcript was greatly lowered. NCA3 was isolated from a wild-type yeast genomic library by genetic complementation. The level of the 5.2-kb transcript, like the synthesis of subunits 6 and 8, was partly restored in the transformed strain. A 1011-nucleotide ORF was identified that encodes an hydrophilic protein of 35417 Da. Disruption of chromosomal DNA within the reading frame promoted a dramatic decrease of the 5.2-kb mRNA but did not abolish the respiratory competence of a wild-type strain. NCA3 is located on chromosome IV and produces a single 1780-b transcript.

Partitioning of electron flux between the respiratory chains of the yeast Candida parapsilosis: parallel working of the two chains.

Partitioning of the electron flux between the classical and the alternative respiratory chains of the yeast Candida parapsilosis, was measured as a function of the oxidation rate and of the Q-pool redox poise. At low respiration rate, electrons from external NADH travelled preferentially through the alternative pathway as indicated by the antimycin A-insensitivity of electron flow. Inhibition of the alternative pathway by SHAM restored full antimycin A-sensitivity to the remaining electro flow. The dependence of the respiratory rate on the redox poise of the quinone pool was investigated when the electron flux was mediated either by the main respiratory chain (growth in the absence of antimycin A) or by the second respiratory chain (growth in the presence of antimycin A). In the former case, a linear relationship was found between these two parameters. In contrast, in the latter case, the relationship between Q-pool reduction level and electron flux was non-linear, but it could be resolved into two distinct curves. This second quinone is not reducible in the presence of antimycin A but only in the presence of high concentrations of myxothiazol or cyanide. Since two quinone species exist in C. parapsilosis, UQ9 and Qx (C33H54O4), we hypothesized that these two curves could correspond to the functioning of the second quinone engaged during the alternative pathway activity. Partitioning of electrons between both respiratory chains could occur upstream of complex III with the second chain functioning in parallel to the main one, and with the additional possibility of merging into the main one at the complex IV level.

Isolation, characterization and function of the two cytochromes c of the yeast Candida parapsilosis.

Candida parapsilosis is a strictly aerobic yeast which possesses two respiratory chains with a peculiar organisation, different from that of plant mitochondria. Besides the classical electron transport pathway, mitochondria of C. parapsilosis develops an alternative pathway, which does not branch off at the ubiquinone level, but merges at the complex IV level. Two pools of cytochromes c were distinguished by their spectrometric and potentiometric properties: (i) sequential cytochrome c reduction was promoted by two substrates, PMS (Em = 70 mV) and TMPD (Em = 280 mV). TMPD promoted the reduction of a cytochrome c with maxima at 551.9 and 417.3 nm for the alpha and the Soret bands, respectively, whereas cytochrome c reducible by PMS exhibited maxima at 549.7 and 419.9 nm; (ii) two midpoint redox potentials were resolved at 180 mV and 280 mV, respectively. The two cytochromes c were copurified by ion-exchange chromatography on Amberlite; after this step, the two cytochromes c can always be differentiated by TMPD and PMS, these reductants promoting different absorption bands. The two cytochromes c were separated by reverse-phase HPLC; this last purification step resolved two proteins with the same relative molecular mass of 13600 but a different amino-acid composition. Comparison of N-terminal sequences revealed differences between the two proteins. It was hypothesized that one cytochrome c is implicated in the functioning of the main chain and the other in that of the secondary pathway.

Regulation by nuclear genes of the mitochondrial synthesis of subunits 6 and 8 of the ATP synthase of Saccharomyces cerevisiae.

The nuclear mutant AB1-4A/8/100, a respiratory-competent strain altered in the regulation of ATP synthesis, has been shown to be modified in the relative stoichiometry of the mtDNA-encoded proteolipids of the F0 sector of ATP synthase: the ratios mutant/wild type of the proteolipids were equal to 0.4/0.7/1 for Su8/Su6/Su9, respectively. This defect results from the simultaneous presence of two nuclear genes which promote a cryosensitive phenotype on a nonfermentable carbon source. Measurements of mitochondrial protein synthesis carried out "in vivo" and "in organello" evidenced a specific defect in the synthesis of subunits 6 and 8. Measurements of the steady state levels of mitochondrial mRNA showed that the defect in subunits 6 and 8 was correlated with a modification of the expression of a cotranscript ATP8-ATP6. This cotranscript is matured at a unique site to give two cotranscripts of 4600 and 5200 bases in length. In mutant mitochondria, the ratio between both cotranscripts, 5200/4600, was lowered. In parallel, expression of the whole mitochondrial transcription unit supporting the genes COXI, ATP8, ATP6, and RF3 was enhanced. However, despite this over expression, the amount of the long cotranscript ATP8-ATP6 remained lower than in wild type mitochondria.

Biochemical studies carried out on different groups of Candida parapsilosis and Candida rhagii strains by comparing some cellular and mitochondrial activities.

A comparative biochemical study was performed on some strains of Candida rhagii and on strains belonging to different subgroups of Candida parapsilosis. Measurements of alcohol dehydrogenase activity, resistance to drugs and occurrence of an alternative pathway enabled us to confirm the classification between several subgroups within the C. parapsilosis species.

The antimycin-A-insensitive respiratory pathway of Candida parapsilosis: evidence for a second quinone involved specifically in its functioning.

The involvement of a quinone in the antimycin A-insensitive electron transfer from NADH-dehydrogenase to cytochrome c via the alternative respiratory chain of Candida parapsilosis, by-passing complex II, has been studied. After a partial extraction of quinones, the residual respiration was fully antimycin-A-sensitive, but reincorporation of the organic extract partially restored an antimycin A-insensitive respiration. Analysis of quinone content by HPLC, after purification by thin-layer chromatography, evidenced another quinone species in a very low amount. Myxothiazol and stigmatellin were shown to inhibit the alternative pathway but at a higher concentration than required to inhibit the classical pathway. Cytochrome spectra analysis showed that, in the presence of high myxothiazol concentrations, cytochromes c and aa3 were not reduced, while they were in the presence of antimycin A. It is suggested that the secondary pathway of C. parapsilosis involved a specific quinone pool which can be displaced from its binding site by high concentrations of myxothiazol or analogous compounds.