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A Goffeau

Publications and source records attributed to A Goffeau.

At least 181 records · Page 10Linked to original sources
PubMed1981

Electrogenic proton translocation coupled to ATP hydrolysis by the plasma membrane Mg2+-dependent ATPase of yeast in reconstituted proteoliposomes.

The purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe was incorporated in liposomes using a cholate-dialysis method. The ATPase activity of the incorporated enzyme was stimulated by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone and to a much lower extent of the K+-ionophore valinomycin in the presence of potassium. The K+/H+ exchanger nigericin (plus K+) did not stimulate ATPase activity, whereas the combined addition of both nigericin plus valinomycin was strongly stimulatory. The incorporated ATPase activity was controlled by the generated electrochemical H+ gradient since only conditions which collapse both the membrane potential and the pH gradient stimulated fully the ATPase activity of the incorporated enzyme. Direct measurement of proton movement with a pH glass electrode showed a fast and transient proton entry into the proteoliposomes upon addition of MgATP in the presence of the charge-compensating cation K+ (plus valinomycin). Moreover, during the steady state ATP hydrolysis, a H+ entry was again observed when the membrane potential was collapsed upon addition of valinomycin in the presence of K+. These data demonstrate that the plasma membrane ATPase of yeast cells is involved in electrogenic H+ translocation coupled to ATP hydrolysis since the purified enzyme incorporated in the liposomes is virtually free of mitochondrial F1F0-ATPase contaminant.

Adenosine Triphosphatases↗
PubMed1981

Electrogenic proton ejection coupled to electron transport through the energy-conserving site 2 and K+/H+ exchange in yeast mitochondria.

The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H+ ejection/2e- ratio approaches an average value of 3 when K+ (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H+ ejection/2e- ratio of 2 is observed. (3) The low stoichiometry of 3H+ ejected (instead of 4) per 2e- and the high rate of H+ back-decay (0.1615 ln delta (ngatom)H+/s and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K+/H+ antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.

Antimycin A↗
PubMed1981

Induction by glucose of an antimycin-insensitive, azide-sensitive respiration in the yeast Kluyveromyces lactis.

Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (QO2 of 80 microliter O2 . h-1 . mg-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 microliter O2 . h-2 . mg-1 dry weight. Antimycin A inhibits the growth of K. lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced. It can be concluded that a minimal antimycin-insensitive QO2 is necessary for cellular growth when the normal respiratory pathway is not functional. The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 microM azide or 1 mM cyanide. These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements. A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells. The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.

Antimycin A↗
PubMed1981

Large-scale purification and phosphorylation of a detergent-treated adenosine triphosphatase complex from plasma membrane of Saccharomyces cerevisiae.

A new procedure for large-scale preparation of plasma-membrane-bound ATPase from Saccharomyces cerevisiae is described. The crude membrane fraction is purified by selective extraction with three successive detergents: deoxycholate (0.25 mg/mg protein), Triton X-100 (0.25%) and lysophosphatidylcholine (1 mg/mg protein). These treatments extract the mitochondria and strip the plasma membrane. From 1 kg commercial baker's yeast, 200 mg of plasma membrane proteins are isolated in 2--3 days. Plasma-membrane-bound ATPase of specific activity of 10--13 mumol Pi x min-1 x mg protein-1 is obtained with a yield estimated to 60%. Dodecylsulfate/polyacrylamide gel electrophoresis shows three predominant polypeptides of Mr = 95000, 70000 and 56000 in the purified membrane fraction. The major polypeptide of Mr = 95000 identified as the ATPase subunit is phosphorylated by millimolar concentrations of ATP. The phosphorylated intermediate reaches the steady-state level in less than 100 ms and turns over very rapidly. It is hydrolyzed by hydroxylamine. Its formation is prevented by the ATPase inhibitors vanadate and Dio-9, a plasma-membrane ATPase inhibitor of unknown structure. At least four other membrane proteins are phosphorylated with much slower kinetics, presumably through the action of protein-kinase(s).

Adenosine Triphosphatases↗
PubMed1981

Spectral properties of cytochrome b-561 and cytochrome b-565 in mucidin-resistant mutants of Saccharomyces cerevisiae.

The oxidation of NADH in submitochondrial particles isolated from MUC1, MUC2 and MUC3 mucidin-resistant mutants of Saccharomyces cerevisiae is specifically resistant to mucidin. Extra reduction of cytochrome b-565 induced by mucidin is demonstrated in all tested mucidin-resistant mutants. Red shift of cytochrome b-561 is induced by mucidin in two independent MUC3 mutants. In MUC1 and MUC2 mutants, the red shift is not induced by mucidin, while that promoted by antimycin A and 2-n-heptyl-4-hydroxyquinoline N-oxide are normal. It is concluded that the extra reduction of cytochrome b-565 and the red shift of cytochrome b-561 elicited by mucidin can be largely dissociated from the overall inhibition of the electron flow by distinct mucidin-resistant mutations in different exons of the split mitochondrial gene of cytochrome b.

Anti-Bacterial Agents↗
PubMed1981

Modification of the spectral properties of cytochrome b in mutants of Saccharomyces cerevisiae resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Mapping at two distinct genetic loci of the split mitochondrial gene of cytochrome b.

The effects of five inhibitors of the cytochrome bc1 complex: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), 2-n-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuron-resistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b. 1. The oxidation of NADH and of cytochrome b-561 exhibits a similar resistance to diuron and HpHOQnO in Diu 1 and Diu 2 mutants. 2. No extra reduction of cytochrome b-561 and cytochrome b-565 is observed in the presence of diuron and HpHOQnO. 3. Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b-561 induced by concentrations of HpHOQno 2 -- 3-times higher than those required in the parental strains. 4. The spectral and respiratory effects of antimycin A, funiculosin and mucidin and generally similar in the diuron-resistant mutants and in their parental strains. However a cross-resistance between diuron and antimycin A is indicated in one Diu 2 mutant. 5. From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in the central region of the apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b-565 and b-561 as well as the flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b-561 is only slightly affected by the diu-1 and diu-2 mutations.

Antimycin A↗
PubMed1980

Phospholipid reactivation of the purified plasma membrane ATPase of yeast.

The plasma membrane ATPase of the yeast Schizosaccharomyces pombe solubilized by lysolecithin and purified by centrifugation through a sucrose gradient is essentially inactive. The phospholipid distribution in the sucrose gradient indicates that inactivation of the ATPase may result from the partial delipidation occurring during purification. Taking into account the 100,000 daltons of the ATPase polypeptide, it is concluded that 74 mol of phospholipids are bound per mol of purified ATP monomer. The ATPase so purified is reactivated simply by mixing the enzyme with preformed lipid micelles or vesicles. Lysolecithins reactivate the enzyme at concentrations around the critical micellar concentration. Gel exclusion chromatography indicates that the enzyme binds reversibly to the lysolecithin micelles. On the other hand, lecithins of varying chain length and unsaturation reactivate the enzyme to different extents and with different efficiencies. In addition, from binding studies, it is observed that each saturated lecithin combines equally well with the ATPase. Using other diacylphospholipids no specificity for the polar head group is observed. Moreover, cardiolipin microvesicles is shown to bind all the protein but not to restore the enzyme activity. From lipid-reactivation titration curves. Arrhenius plots, and physical data of the phospholipids, it is concluded that the major parameter which governs the optimal reactivation of ATPase is the ability of the phospholipids to form amphipathic structures (micelles or vesicles) of sufficient fluidity and hydrophobicity. From these results, a coherent description can be provided for the protein-lipid interactions occurring during solubilization, purification, and the lipid reactivation of the yeast plasma membrane ATPase.

Adenosine Triphosphatases↗
PubMed1980

The purified plasma membrane ATPase of the yeast Schizosaccharomyces pombe forms a phosphorylated intermediate.

An acid slab gel electrophoresis method of high-resolving power allows detection of a phosphorylated form in the purified ATPase of the yeast Schizosaccharomyces pombe and identification of this catalytic intermediate among the different phosphopeptides of a plasma membrane preparation. At a maximum steady state rate of MgATP hydrolysis by the membrane-bound ATPase, 20 to 40% of the ATPase subunits of 100,000 daltons are in a phosphorylated form, while only 0.8% of the subunits of the purified ATPase are phosphorylated under the same conditions. The phosphorylated intermediate reaches the steady state level in less than 2 s and rapidly turns over. The phosphorylated substance is cleaved by hydroxylamine and is relatively stable in acids but is readily hydrolyzed in alkaline or in acid alcoholic media. These results suggest that the intermediate is an acylphosphate. The phosphorylation reaction has an apparent Km value of 3.0 mM MgATP for the plasma membrane-bound ATPase and 0.6 mM MgATP for the purified ATPase. Plasma membranes contain several other minor phosphorylated components whose kinetic behavior is typical of phosphorylation by protein kinase. Artifactual production of two forms of the ATPase by phenylmethanesulfonyl fluoride-sensitive proteases liberated during cell disruption is also demonstrated.

Adenosine Triphosphatases↗
PubMed1979

Genetic control of enhanced mutability of mitochondrial DNA and gamma-ray sensitivity in Saccharomyces cerevisiae.

Five nuclear mutants enhancing the spontaneous mutation rate of mtDNA have been isolated in Saccharomyces cerevisiae. These mutators fall into five complementation groups and are located at five genetic loci different from rad50 to rad57 loci. Three mutants (gam1, gam2, and gam4), insensitive or weakly sensitive to gamma-rays, exhibit increased frequency of spontaneous production of mutants with large deletions of the mtDNA (p-) and of all tested mitochondrial drug-resistant mutants. Two other mutants (gam3 and gam5), highly sensitive to gamma-rays, increase only the mutation rate of particular alleles of the mtDNA. The mutant gam5 enhances only the production of p- and erythromycin-resistant clones. The mutant gam3 exhibits an enhanced rate of oligomycin-resistant clones as well as a collateral increase of nuclear mutability. The existence of gam3 and gam5 mutants indicates that at least two common steps control both nuclear DNA repair and the mutability of particular alleles of the mtDNA. However, the general spontaneous mutability of the mtDNA includes at least three steps not involved in the repair of nuclear DNA, as revealed by the gam1, gam2, and gam4 mutations.

Chromosome Mapping↗