Martine Guerin

Perméabilité de la membrane interne des mitochondries de levure: étude des relations entre structure et activité

Summary In order to investigate the possible relations between the anionic permeability and the functions (or the structure) of the inner mitochondrial membrane, three types of organelles isolated from S. cerevisiae were tested: mitochondria (aerobic culture), promitochondria (anaerobic culture) and CAP-mitochondria (aerobic culture with chloramphenicol added). By using the technique of swelling in isoosmotic potassium salts, after a determination of the isotonic conditions, it was possible to discriminate between an electrogenic (valinomycin induced) or an electroneutral (both valinomycin and uncoupler induced) translocation. o 1) Mitochondria : The permeability properties of mitochondria are energy dependent: a) Respiring mitochondria are permeable to Cl − ; Mg 2+ , however, inhibits this translocation. Phosphate transport seems to be exclusively electrogenic and mersalyl insensitive, but swelling inhibition by that thiol reagent is restored by Mg 2+ . b) Non respiring mitochondria are impermeable to Cl − , but ATP addition restores the permeability. Thiocyanate permeates as the anionic form and acetate as the undissociated form. The phosphate transport, sensitive to mersalyl, seems to be partially electrogenic. 2) Promitochondria : Deficient of respiratory enzymes but containing an oligomycin sensitive ATPase, they are impermeable to Cl − only when Mg 2+ is added. In these conditions, an electrogenic phosphate transport, sensitive to mersalyl, is observed. 3) CAP-mitochondria : Although CAP-mitochondria are cytochrome deficient and contain an oligomycin insensitive ATPase, they are also impermeable to Cl − in presence of Mg 2+ . As in fully differenciated mitochondria, an electroneutral phosphate entry is observed; Mg 2+ is required for mersalyl sensitivity.

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 co-transcripts 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.

Evidence for three different electrophoretic pathways in yeast mitochondria : ion specificity and inhibitor sensitivity

AbstractWe identified three electrophoretic pathways by spectrophotometrically following the swelling of isolated yeast mitochondria:--An anion uniport whose activity could only be detected after depletion of divalent cations from the matrix by treatment with 1,10-phenanthroline. This uniport was inhibited by Mg2+ and dicyclohexylcarbodiimide.--A K+(Na+) uniport which was detected only when mitochondria were suspended at low pH and low temperature. This uniport was sensitive to ruthenium red and oleic acid.--A K+ selective uniport which was activated by alkaline pH and ATP depletion. This pathway was sensitive to glibenclamide and to various amphiphilic cations.nSimilarities and differences between these three electrophoretic pathways and the electrophoretic systems described in mammalian and plant mitochondria are discussed.

Studies on anionic transport in yeast mitochondria and promitochondria. Swelling in ammonium phosphate, glutamate, succinate and fumarate solutions

The transport of some anionic components into mitochondria appeared to be mediated by a series of exchange-diffusion carriers, localized in the internal membrane. Moreover, rat liver mitochondria have been shown to contain at least eight specific carriers [ 141 . One of the most useful tools, in identifying some of these carriers was the osmotic swelling of mitochondria suspended in the isotonic ammonium salt of the anion to be tested [S] . For instance, rat liver mitochondria spontaneously swelled in ammonium phosphate or glutamate, suggesting an anion-hydroxyl exchange. But, with succinate or malate, phosphate was necessary before swelling occurred. However, only the existence of specific inhibitors is a probe for a mediated-carrier transport. With yeast mitochondria and promitochondria , Kolarov et al. [6] noted likewise a swelling in ammonium phosphate, but they reported that succinate uptake did not require phosphate as it did for malate: these data suggested the presence of a succinate carrier distinct of the malate carrier. In order to determine if, in yeast mitochondria (aerobically grown cells) and promitochondria (anaerofically grown cells), the phosphate uptake was carrier mediated, we studied the mersalyl effects on the swelling. We also examined the glutamate, succinate and fumarate transport in these organelles.

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.

Effects of physiological manipulation on the kinetics of mitochondrial phosphate transport in Saccharomyces cerevisiae.

Abstract The kinetics of [ 32 P]phosphate uptake has been studied in different types of Saccharomyces cerevisiae mitochondria. Mitochondria were isolated from yeast grown aerobically on 2% lactate (Lac-mitochondria), 2% galactose (Gal-mitochondria), 5.4% glucose (Glu-mitochondria) or from yeast grown anaerobically on 2% galactose (Promitochondria). The effect of chloramphenicol was also studied by adding it to the growth medium of yeast grown aerobically on 2% galactose (chloramphenicol-mitochondria). [ 32 P]Phosphate uptake followed an oscillatory pattern in Lac, Gal-mitochondria and Promitochondria. Saturation kinetics were detected in fully differenciated mitochondria and in Promitochondria, but not in chloramphenicol-mitochondria. Glu-mitochondria did not translocate phosphate as shown both by lack of [ 32 P]phosphate uptake and lack of swelling in isoosmotic potassium solution. Repressed yeast cells were incubated in a resting cell medium and mitochondria were isolated at different times of incubation. The rate of respiration and the oligomycin-sensitive ATPase increased during the course of the incubation. After 2h, a mitochondrial mersalyl-sensitive swelling in an isoosmotic potassium phosphate solution was detected. As expected, no increase of the rate of respiration was observed when chloramphenicol was added in the derepression medium. But the oligomycin-sensitive ATPase decreased. Chloramphenicol did not affect the phosphate transport activity as measured by the swelling of mitochondria, but the [ 32 P]phosphate uptake did not follow saturation kinetics. A complete derepression of the inorganic phosphate-carrier activity was achieved by a 4 h incubation of the repressed cells in the presence of chloramphenicol, followed by a 6 h incubation in presence of cycloheximide. These data strongly suggest that the mitochondrial protein-synthesis system is required for the normal function of the inorganic phosphate-carrier.

The second respiratory chain of Candida parapsilosis: a comprehensive study.

The yeast C. parapsilosis CBS7157 is strictly dependent on oxidative metabolism for growth since it lacks a fermentative pathway. It is nevertheless able to grow on high glucose concentrations and also on a glycerol medium supplemented with antimycin A or drugs acting at the level of mitochondrial protein synthesis. Besides its normal respiratory chain C. parapsilosis develops a second electron transfer chain antimycin A-insensitive which allows the oxidation of cytoplasmic NAD(P)H resulting from glycolytic and hexose monophosphate pathways functioning through a route different from the NADH-coenzyme Q oxidoreductase described in S. cerevisiae or from the alternative pathways described in numerous plants and microorganisms. The second respiratory chain of C. parapsilosis involves 2 dehydrogenases specific for NADH and NADPH respectively, which are amytal and mersalyl sensitive and located on the outer face of the inner membrane. Since this antimycin A-insensitive pathway is fully inhibited by myxothiazol, it was hypothesized that electrons are transferred to a quinone pool that is different from the classical coenzyme Q-cytochrome b cycle. Two inhibitory sites were evidenced with myxothiazol, one related to the classical pathway, the other to the second pathway and thus, the second quinone pool could bind to a Q-binding protein at a specific site. Elimination of this second pool leads to a fully antimycin A-sensitive NADH oxidation, whereas its reincorporation in mitochondria allows recovery of an antimycin A-insensitive, myxothiazol sensitive NADH oxidation. The third step in this second respiratory chain involves a specific pool of cytochrome c which can deliver electrons either to a third phosphorylation site or to an alternative oxidase, cytochrome 590. This cytochrome is inhibited by high cyanide concentrations and salicylhydroxamates.

ATP opens an electrophoretic potassium transport pathway in respiring yeast mitochondria

In the presence of KCl and only at low phosphate concentrations, ATP stimulated state 4 of the respiration of isolated yeast mitochondria. This effect could be related to a partial collapse of the transmembrane potential which was created by the respiratory chain or the F0F7‐ATPase. Sodium and lithium could not replace potassium ion. Atractyloside prevented the opening of this K+ pathway, suggesting that only matricial ATP operated. All these effects were inhibited by increasing phosphate concentration, or by adding propranolol, quinine, Zn2+ or Mg2+.

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

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.