Jean Velours

Absence of the Mitochondrial AAA Protease Yme1p Restores F0-ATPase Subunit Accumulation in anoxa1 Deletion Mutant of Saccharomyces cerevisiae

The nuclear gene OXA1 encodes a protein located within the mitochondrial inner membrane that is required for the biogenesis of both cytochrome c oxidase (Cox) and ATPase. In the absence of Oxa1p, the translocation of the mitochondrially encoded subunit Cox2p to the intermembrane space (also referred to as export) is prevented, and it has been proposed that Oxa1p could be a component of a general mitochondrial export machinery. We have examined the role of Oxa1p in light of its relationships with two mitochondrial proteases, the matrix protease Afg3p-Rca1p and the intermembrane space protease Yme1p, by analyzing the assembly and activity of the Cox and ATPase complexes inΔoxa1,Δoxa1Δafg3, andΔoxa1Δyme1 mutants. We show that membrane subunits of both complexes are specifically degraded in the absence of Oxa1p. Neither Afg3p nor Yme1p is responsible for the degradation of Cox subunits. However, the F0 subunits Atp4p, Atp6p, and Atp17p are stabilized in theΔoxa1Δyme1 double mutant, and oligomycin-sensitive ATPase activity is restored, showing that the increased stability of the ATPase subunits allows significant translocation and assembly to occur even in the absence of Oxa1p. These results suggest that Oxa1p is not essential for the export of ATPase subunits. In addition, although respiratory function is dispensable inSaccharomyces cerevisiae, we show that the simultaneous inactivation of AFG3 and YME1 is lethal and that the essential function does not reside in their protease activity.

Mitochondrial F1F0-ATP synthase and organellar internal architecture

The mitochondrial F(1)F(0)-ATP synthase adopts supramolecular structures. The interaction domains between monomers involve components belonging to the F(0) domains. In Saccharomyces cerevisiae, alteration of these components destabilizes the oligomeric structures, leading concomitantly to the appearance of monomeric species of ATP synthase and anomalous mitochondrial morphologies in the form of onion-like structures. The mitochondrial ultrastructure at the cristae level is thus modified. Electron microscopy on cross-sections of wild type mitochondria display many short cristae with narrowed intra-cristae space, whereas yeast mutants defected in supramolecular ATP synthases assembly present a low number of large lamellar cristae of constant thickness and traversing the whole organelle. The growth of these internal structures leads finally to mitochondria with sphere-like structures with a mean diameter of 1 microm that are easily identified by epifluorescence microscopy. As a result, ATP synthase is an actor of the mitochondrial ultrastructure in yeast. This paper reviews the ATP synthase components whose modifications lead to anomalous mitochondrial morphology and also provides a schema showing the formation of the so-called onion-like structures.

Preparation of an inner membrane-matrix fraction from yeast mitochondria

Abstract Treatment of yeast mitochondria with digitonin was used in order to prepare an inner membrane-matrix fraction preserving its permeability properties. The incubation time of mitochondria with digitonin was an essential parameter for the selective solubilization of the outer membrane. The incubation of mitochondria for l min at different concentrations of digitonin led to a three-step release of mitochondrial enzymes: (a) at low concentrations of digitonin, adenylate kinase was released; (b) higher concentrations were required to solubilize kynurenine hydroxylase, an outer membrane marker; (c) inner membrane markers (succinate dehydrogenase and oligomycin-sensitive adenosine triphosphatase) and matrix markers (fumarase and isocitrate dehydrogenase) were significantly released at concentrations of digitonin higher than 0.4 mg/mg of protein. The electron microscopic aspects of yeast mitoplasts (inner membrane-matrix fraction obtained by treatment with 0.4 mg of digitonin) showed an orthodox and a twisted configuration. These new organelles retained respiratory control when assayed with ethanol as the substrate. Their selective permeability properties were preserved as shown by isoosmotic swelling in potassium or ammonium salt solutions.

Protection of yeast mitochondrial structure by phosphate and other H+-donating anions

The energy-dependent uptake of cations by mammalian mitochondria can be balanced by an equivalent counterflux of other cations. In the case of the valinomycin-mediated uptake of K’ the coun- terion is H* [ 1,2] . The pH difference accross the membrane, increased by this process, can be utilized to accumulate Pi via the Pi-carrier (which catalyses a H+-anion symport) or other H’-donating anions as acetate (which cross the membrane under the undisso- ciated form) [3-51 . Some tricarboxylic acid cycle intermediates can also enter the matrix compartment by a series of exchange-diffusion carriers which require Pi [5,6] ; therefore, the accumulation of these anions is also electroneutral and ApH-dependent [3,4] . The permeability properties of the yeast mitochondrial membrane are not basically different from those of mammalian mitochondria [7-lo]. Brierley showed that an energy-linked pH increase in the matrix of isolated beef heart mitochondria modified the permeability of inner membrane to Cl- and other non-permeant anions [ II]. As shown in this paper a similar effect can lead to irreversible structural damage in yeast mitochondria. A protective effect of Pi, when it crosses the membrane via the Pi- carrier. is also demonstrated.

The yeast ATP synthase subunit 4: structure and function

The structure of ATP synthase subunit 4 was determined by using the oligonucleotide probe procedure. This subunit is the fourth polypeptide of the complex when classifying subunits in order of decreasing molecular mass. Its relative molecular mass is 25 kDa. The ATP4 gene was isolated and sequenced. The nucleotide sequence predicts that subunit 4 is probably derived from a precursor protein 244 amino acids long. Mature subunit 4 contains 209 amino acid residues and the predicted molecular mass is 23250 kDa. Subunit 4 shows homology with the b-subunit of Escherichia coli ATP synthase and the b-subunit of beef heart mitochondrial ATP synthase. By using homologous transformation, a mutant lacking wild subunit 4 was constructed. This mutant is devoid of oxidative phosphorylation and F1 is loosely bound to the membrane. Our data are in favor of a structural relationship between subunit 4 and the mitochondrially-translated subunit 6 during biogenesis of F0.

Proteolipids of yeast mitochondria: Discrimination between the phosphate-binding and the dicyclohexylcarbodiimide-binding proteolipids☆

Abstract Proteins soluble in organic solvents were synthesized on cyto- and mitoribosomes. These proteins (proteolipids) migrated in sodium dodecyl sulfate gel electrophoresis with an apparent molecular weight of 8000. Using two extraction procedures (Method I: M. Guerin and C. Napias, 1978 , Biochemistry17, 2510–2516; and Method II: K. J. Cattel, C. R. Lindop, I. G. Knight and R. B. Beechey, 1971 , Biochem. J.125, 169–177) it was shown that 2.3% of the total radioactivity incorporated into mitochondria was soluble in organic solvents, 0.3% was incorporated into a product of cytoribosomal system, and 1.9% was incorporated into products synthesized on mitoribosomes. These proteolipids were tentatively characterized by their mobility on thin-layer chromatography plates and their ability to bind specific ligands. The cytoplasmically translated product did not migrate on thin-layer chromatography. The mitochondrially translated products show several spots; one of them remained at the start of the chromatogram. It appeared that this nonmigrating mitochondrially translated product (which represented 0.2% of the total radioactivity) was able to bind inorganic phosphate. The major migrating spot was characterized as the dicyclohexylcarbodiimide-binding protein, and represented 1.1% of the total radioactivity incorporated into mitochondria. The dicyclohexyl-carbodiimide-binding protein which was recovered only by Method II was purified on a Sephadex LH-60 column using chloroform:methanol (2:1) as eluant. The Pi-binding protein was extracted by both methods and could be separated from minor contaminants and neutral lipids on Sephadex LH-60 with chloroform as eluant; however, in this case, the Pi-binding protein cochromatographed with the cytoribosomal translated protein.

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.

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.

Actions de carbures cancérogénes sur les lipides des membranes protoplastiques et des mitochondries de Saccharomyces cerev1siae

Incubation of Saccharomyces cerevisiae with carcinogenic hydrocarbons (benzo(a)pyrene, dibenzanthracenes) profoundly affects the nature and the amount of lipids, especially phospholipids: 1. 1. The amount of total lipids is strongly reduced. 2. 2. While neutral lipids are slightly affected, the decrease of phospholipids can reach 90% of their normal value and is almost balanced by an increase of monoglycerides. 3. 3. The individual phospholipids, determined as phospholipid phosphorus, are affected in a like manner. 4. 4. The trends, observed in whole cells, are found again in plasma membrane and in mitochondria. 5. 5. The strongly carcinogenic benzo(a)pyrene and dibenzanthracene (ah) have a similar action, while the anomalies are less with the weakly carcinogenic dibenzanthracene(ac). 6. 6. Using [3H]benzo(a)pyrene, it appears that the reagent penetrates into the cell where it is partially metabolised; some of the carcinogen and its by-products are found in the plasma membrane and in mitochondria. 7. 7. When the yeast, previously incubated with benzo(a)pyrene, is afterwards grown in carcinogen-free medium, the proportion of phospholipid increases, but does not reach normal. 8. 8. Benzo(a)pyrene does not alter the phospholipids of Escherichia coli.