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S Manon

Publications and source records attributed to S Manon.

34 records · Page 2Linked to original sources
PubMed1995

Stimulation of oxidative phosphorylation by electrophoretic K+ entry associated to electroneutral K+/H+ exchange in yeast mitochondria.

The effect of the addition of KCl, at constant osmolarity, was investigated on oxidative phosphorylation in isolated yeast mitochondria. KCl stimulated both respiration and ATP synthesis rates without changing the ATP/O ratio. KCl did not change the relationships between respiration rates and the protonmotive force. Since the K+/H+ exchange activity was active under these conditions, the stimulatory effect of respiration could be explained by the net proton entry caused by the electrophoretic K+ entry/electroneutral K+/H+ exchange cycle. On the other hand, K+ entry stimulated phosphate accumulation and transport under non-phosphorylating conditions and decreased the kinetic control by phosphate transport under phosphorylating conditions. Additionally, the stimulation of ATP synthesis strongly depended on the activity of phosphate transport. Taken together, these data showed that electrophoretic K(+)-entry and electroneutral K+/H+ exchange occurred in phosphorylating yeast mitochondria but did not promote any uncoupling between respiration and ATP synthesis.

Adenosine Triphosphate↗
PubMed1995

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 F0F1-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+.

Adenosine Triphosphate↗
PubMed1995

Relationship of subunit 8 of yeast ATP synthase and the inner mitochondrial membrane. Subunit 8 variants containing multiple lysine residues in the central hydrophobic domain retain function.

A molecular genetic approach has been used to test the proposition that the central hydrophobic domain of yeast mitochondrial ATP synthase subunit 8 represents a transmembrane stem in contact with the lipid bilayer. The rationale for this approach is the general inability of membrane bilayers to accomodate unshielded charged residues of polypeptide chains. Non-polar residues at several positions within the central hydrophobic domain of subunit 8 were replaced with the positively charged amino acid lysine. This was done in an attempt to disrupt subunit 8 function, and thereby determine the boundaries of the putative transmembrane stem. Each subunit 8 variant was allotopically expressed in vivo as a mitochondrial import precursor encoded by a nuclear gene. It was found that all variants, which included proteins carrying two lysines at various positions in the hydrophobic domain, exhibited the ability to restore growth of subunit-8-deficient cells on the non-fermentable substrate ethanol. This indicated that the function of none of these subunit 8 variants was severely compromised. There was also no detectable change in the proteolipid characteristics of subunit 8, as defined by the chloroform/methanol solubility properties of variant proteins extracted from membranes following import into isolated mitochondria. These data suggest that subunit 8 is located in a hydrophobic niche in the mitochondrial ATP synthase, probably in contact with other protein subunits of the complex. We conclude that the function of subunit 8 does not necessarily require it to be integrated within the inner mitochondrial membrane, in contact with the lipid bilayer. Our findings also suggest that hydropathy plots, indicating hydrophobic domains within polypeptides, cannot reliably be interpreted as transmembrane helices in the absence of independent evidence.

Amino Acid Sequence↗
PubMed1995

Investigations of the inhibitory effect of propranolol, chlorpromazine, quinine, and dicyclohexylcarbodiimide on the swelling of yeast mitochondria in potassium acetate. Evidences for indirect effects mediated by the lipid phase.

The mode of action of propranolol, chlorpromazine, and quinine, three cationic drugs inhibiting swelling of yeast mitochondria in potassium acetate, was investigated by looking at their effect on fluorescent probes of the polar heads and of the nonpolar moiety of the membranes, under inhibitory conditions of swelling. As expected, propranolol and chlorpromazine exhibited specificity for anionic phospholipids since they increased the binding of the anionic probe 1-anilino 8-naphthalenesulfonate (ANS). Although propranolol did not release 1,6-diphenyl-1,3,5-hexatriene (DPH) from the hydrophobic moiety of the membrane, it increased the excimer/monomer fluorescence ratio of 10-(1-pyrene)decanoate, suggesting that it induced a limitation in the movements of the aliphatic chains of phospholipids. Opposite to propranolol, chlorpromazine removed DPH from the membrane, suggesting that it bound essentially to the hydrophobic moiety. However, chloramphenicol, which was also able to remove DPH but did not increase the binding of ANS, did not inhibit swelling. Inhibition by chlorpromazine therefore appeared to be related to its binding to the hydrophobic moiety of anionic phospholipids. Quinine had no effect on membrane properties: at inhibitory concentrations of swelling in potassium acetate, it did not inhibit swelling in ammonium phosphate (mediated by the phosphate/H+ cotransporter), whereas propranolol and chlorpromazine did, suggesting a more specific effect of quinine on (a) protein(s) involved in the K+/H+ exchange. Dicyclohexylcarbodiimide (DCCD), which irreversibly inhibits the swelling in potassium acetate, bound to ethanolamine heads; despite this effect, DCCD had no major consequences on the binding of the probes. Consequently, propranolol and chlorpromazine are of no help for characterizing protein(s) catalyzing the K+/H+ exchange, although their effect on lipids seems to involve limited zones of the inner mitochondrial membrane. Quinine and DCCD, although they also bind to lipids, may inhibit the activity by acting on a limited number of proteins.

Acetates↗
PubMed1995

Decoupling of the bc1 complex in S. cerevisiae; point mutations affecting the cytochrome b gene bring new information about the structural aspect of the proton translocation.

Four mutations in the mitochondrial cytochrome b of S. cerevisiae have been characterized with respect to growth capacities, catalytic properties, ATP/2e- ratio, and transmembrane potential. The respiratory-deficient mutant G137E and the three pseudo-wild type revertants E137 + I147F, E137 + C133S, and E137 + N256K were described previously (Tron and Lemesle-Meunier, 1990; Di Rago et al., 1990a). The mutant G137E is unable to grow on respiratory substrates but its electron transfer activity is partly conserved and totally inhibited by antimycin A. The secondary mutations restore the respiratory growth at variable degree, with a phosphorylation efficiency of 12-42% as regards the parental wild type strain, and result in a slight increase in the various electron transfer activities at the level of the whole respiratory chain. The catalytic efficiency for ubiquinol was slightly (G137E) or not affected (E137 + I147F, E137 + C133S, and E137 + N256K) in these mutants. Mutation G137E induces a decrease in the ATP/2e- ratio (50% of the W.T. value) and transmembrane potential (60% of the W.T. value) at the bc1 level, whereas the energetic capacity of the cytochrome oxidase is conserved. Secondary mutations I147F, C133S, and N256K partly restore the ATP/2e- ratio and the transmembrane potential at the bc1 complex level. The results suggest that a partial decoupling of the bc1 complex is induced by the cytochrome b point mutation G137E. In the framework of the protonmotive Q cycle, this decoupling can be explained by the existence of a proton wire connecting centers P and N in the wild type bc1 complex which may be amplified or uncovered by the G137E mutation when the bc1 complex is functioning.

Amino Acid Sequence↗
PubMed1995

Investigation of the effects of Zn2+ and Cu2+ on the K+ transport in yeast mitochondria. Evidences for the involvement of a Zn(2+)-binding protein in the K+/H+ exchange.

Effect of Zn2+ and Cu2+ were studied on the spontaneous swelling of yeast mitochondria in potassium acetate. (i) Whatever the pH, Cu2+ stimulated the swelling. (ii) Zn2+ inhibited or stimulated the swelling depending both on pH of the medium and of its concentration: it induced an inhibition of the K+/H+ exchange at pH 6.8 and a stimulation at pH 7.7; at pH 7.4, Zn2+ inhibited the swelling at low concentration and stimulated the swelling at higher concentration. (iii) The thiol reagent mersalyl prevented the inhibition by Zn2+ whereas N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoate) prevented the stimulation by Zn2+: this allowed to hypothesize that two distinct Zn(2+)-binding sites were involved in the inhibition and in the stimulation of the swelling of yeast mitochondria suspended in potassium acetate.

Antiporters↗
PubMed1994

In vivo effects of interleukin-10 on contact hypersensitivity and delayed-type hypersensitivity reactions.

Interleukin (IL) 10 is a recently discovered cytokine, originally isolated from T-helper 2 (Th2) cells, which inhibits cytokine production of T-helper 1 (Th1) cells. Because Th1 cells appear to be of importance during the contact hypersensitivity reaction (CHS) we hypothesized that IL-10 might modulate the outcome of CHS in vivo. Intraperitoneal injection of murine recombinant IL-10 (1000 ng) into naive mice 24, 72, or 120 h before sensitization by epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) did not affect ear swelling when ears were challenged 5 d later. However, intraperitoneal injection of IL-10 into already sensitized mice 24 h before challenge resulted in a significant suppression of the ear swelling response, suggesting that under the conditions employed IL-10 is able to block the effector phase, but not the induction phase of CHS in vivo. The suppression could be reversed by the concurrent injection of an IL-10 antibody. Moreover, heat inactivation of native IL-10 resulted in loss of the inhibitory capacity. When mice were sensitized by subcutaneous injection of trinitrophenyl-coupled spleen cells (DTH) instead of epicutaneous application of the hapten (CHS), intraperitoneally-injected IL-10 suppressed the effector phase, but also the induction phase of DTH. IL-10 did not inhibit the toxic ear-swelling response induced by topical application of two irritants tested (croton oil or benzalkonium chloride). The capacity of IL-10 to suppress the effector phase of CHS and DTH supports an important role for this cytokine in the downregulation of type IV immune reactions in vivo. The finding that IL-10 suppresses the induction of DTH, but not of CHS, further suggests that CHS and DTH are related but distinct immune reactions.

Animals↗
PubMed1993

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

We 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. Similarities and differences between these three electrophoretic pathways and the electrophoretic systems described in mammalian and plant mitochondria are discussed.

Adenosine Triphosphate↗
PubMed1993

Effect of anisotropic inhibitors of ATP synthesis in mitochondria of Saccharomyces cerevisiae. Comparison between a wild-type and a mutant strain altered in the stoichiometry of the MT DNA encoded proteolipids of ATP synthase.

The effect of a group of non-usual inhibitors of ATP synthesis was investigated in yeast mitochondria. Tetraphenylphosphonium, tetraphenylarsonium and ethidium decreased the rate of ATP synthesis but did not decrease the ATP/O ratio. They did not inhibit the ATPase activity and the translocase, and did not decrease the proton-motive force. They inhibited phosphate transport under conditions in which it was strictly energy-dependent, but did not under conditions in which it was energy-independent. These inhibitors had a poor effect on ATP synthesis in mitochondria isolated from a nuclear mutant which contained a reduced amount of the subunits 8 and 6 of the Fo part of the ATP synthase as compared to wild-type and which were unable to support any energy-dependent phosphate-transport. Thus, it appeared that the phosphate-dependence of ATP synthesis was altered in the same way by both tetraphenylphosphonium and the mutation.

Adenosine Triphosphate↗
PubMed1992

K+/H+ exchange in yeast mitochondria: sensitivity to inhibitors, solubilization and reconstitution of the activity in proteoliposomes.

The K+/H+ exchange activity of the inner mitochondrial membrane was investigated in the yeast Saccharomyces cerevisiae. Swelling experiments in potassium acetate indicated that the K+/H+ exchange was active without any additional treatment after the mitochondria isolation, such as a Mg2+ depletion. As in mammalian mitochondria, the activity of yeast mitochondria was stimulated by increasing pH and was inhibited by the amphiphilic amines quinine and propranolol and by the carboxyl reagent dicyclohexylcarbodiimide. However, the activity was poorly inhibited by Mg2+ and consequently was only slightly stimulated by the Mg2+/H+ exchanger A23187. On the other hand, Zn2+ was very efficient for inhibiting the exchange and consequently the activity was strongly stimulated by the permeant metal-chelator o-phenanthroline. The [86Rb]Rb+ accumulation in mitochondria and mitoplasts was only partially inhibited by quinine and propranolol suggesting that part of the accumulation monitored under these conditions was due to cation leak through the inner membrane together with adsorption on the membrane. The DCCD-sensitive activity could be reconstituted from mitochondria and from mitoplasts solubilized with Triton X-100; this activity, measured by [86Rb]Rb+ accumulation, was quinine- and propranolol-sensitive. A spectrophotometric method, based on the capacity of negatively charged proteoliposomes to swell, was then developed in order to continuously follow the reconstituted activity.

Hydrogen↗
PubMed1989

Modifications of the relative proteolipid composition in the ATP synthase of a respiratory competent mutant of Saccharomyces cerevisiae.

A comparative study of the proteolipid composition of the F0-sector of the ATP synthase of wild-type strain of Saccharomyces cerevisiae and of nuclear mutants, modified at the level of the oxidative phosphorylation due to an enhanced proton permeability of the inner membrane, was carried out. Analysis of the crude proteolipid extract by electrophoresis and high liquid performance chromatography showed some differences at the level of mitochondrial DNA encoded proteolipids. Subunit 6 and in particular subunit 8 were present in reduced amounts, whereas subunit 9 was present in equal amounts in both types of strain. However, the phosphate binding affinity of subunit 8 was the same in wild-type and mutant strains. The fact that subunit 6 and subunit 8 are cotranscripted on a single mRNA led to the problem of the regulation of the mitochondrial synthesis of these two proteins by a nuclear gene.

Chromatography, High Pressure Liquid↗
PubMed1989

Inhibition of the phosphate-stimulated cytochrome c oxidase activity by thiophosphate.

Yeast and mammalian cytochrome c oxidase activity is inhibited by thiophosphate. This inhibition was observed when using either whole mitochondria or the isolated or reconstituted enzyme. The kinetics of the reduction reaction enabled us to demonstrate that thiophosphate acted on the electron transfer between hemes a and a3. With whole mitochondria, phosphate alone stimulated respiration. The inhibition induced by thiophosphate was suppressed by phosphate only in mitochondria, but not when the isolated enzyme was used. The possibility of a kinetic regulation is discussed.

Animals↗
PubMed1989

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.

Candida↗
PubMed1988

In vivo and in vitro evidence for a proton leakage through the inner mitochondrial membrane in a mutant of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae were isolated which supported three mutations: two unlinked chromosomic mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial mutation conferring paromomycin sensitivity. When studied on isolated mitochondria, these mutants exhibited low phosphorylation efficiency and great proton permeability of their inner mitochondrial membrane. Experiments were carried out on whole cells: determination of growth rates, cellular yields and cellular respiration, either in the presence of triethyltin, an ATP synthase inhibitor, or in the presence of uncoupler, demonstrating that the proton leakage is actually a physiological phenomenon linked to the cold-sensitive phenotype. Experiments performed on isolated mitochondria confirmed the existence of such a proton leakage.

Biological Transport, Active↗
PubMed1988

Modifications of oxidative phosphorylations in mitochondria isolated from a mutant of Saccharomyces cerevisiae. Possible alterations of the phosphate transport.

Mutants of Saccharomyces cerevisiae were isolated which supported two unlinked nuclear mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial one conferring paromomycin sensitivity. Mitochondria isolated from such a mutant exhibited modifications of several phosphate-requiring functions: (a) kinetic parameters of the phosphate dependence of ATP synthesis were modified; (b) in the absence of phosphate the inner mitochondrial membrane exhibited a high proton leakage; (c) mutant mitochondria always exhibited a poor respiratory control and required tenfold more phosphate to reach a maximal state 3 of respiration; (d) phosphate transport, as measured by swelling experiments, was mersalyl-insensitive and, consequently, state 3 of the respiration and ATP synthesis remained less mersalyl-sensitive than in wild-type mitochondria. Analysis of the mitochondrial metabolism of diploid and segregant strains indicates that these modifications are related to the cryosensitive phenotype; however, at present, a cooperative effect of the mitochondrial mutation cannot be eliminated. It is proposed that the phosphate carrier itself or a regulatory element was modified.

Adenosine Triphosphate↗