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Biomedical subjects

A Goffeau

Publications and source records attributed to A Goffeau.

At least 163 records · Page 9Linked to original sources
PubMed1986

A new mutation for multiple drug resistance and modified plasma membrane ATPase activity in Schizosaccharomyces pombe.

The mutant JV66 was selected from the wild type strain of S. pombe 972h- ade7-413 by its ability to grow on solid rich medium containing 200 micrograms Dio-9/ml. The single nuclear mutation, designated pma1 gives resistance towards diguanidines and several other positively charged compounds. The pma1 mutation also decreases plasma membrane ATPase activity and confers resistance of ATPase to vanadate. The pma1 locus is localized on chromosome I at 5.3 map units from cyh1-C7 and at about 20.7 map units from the centromere. This new mutation is genetically and phenotypically different from the mutation cyh3 and cyh4 previously described (Johnston and Coddington 1983).

Biological Transport↗
PubMed1986

Three proton-pumping ATPases in yeast.

Yeast cells contain three distinct proton-pumping ATPases associated with the mitochondrial, vacuolar or plasma membranes. This review discusses their respective structure and function.

Adenosine Triphosphatases↗
PubMed1985

Essential arginyl residues in the H+-translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe.

The H+-translocating adenosine-5'-triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3-butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3-butanedione. The extent of inactivation is half maximal at 10 mM 2,3-butanedione for an incubation of 30 min at 30 degrees C at pH 7.0. Under the same conditions, the time-dependence of inactivation is biphasic in a semi-logarithmic plot with half-lives of 10.9 min and 65.9 min. Incubation with 2,3-butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity. Magnesium adenosine 5'-triphosphate (MgATP) and magnesium adenosine 5'-diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3-butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.

Arginine↗
PubMed1985

The RNA polymerase I initiation site and the external transcribed spacer of the fission yeast Schizosaccharomyces pombe ribosomal RNA genes.

A 5.45-kb fragment containing the 5' end of the ribosomal RNA transcriptional unit from the fission yeast Schizosaccharomyces pombe was cloned in the yeast-E. coli shuttle vector YEp13. The transcription start point was mapped by R looping and S1 nuclease protection. The sequence of the entire external transcribed spacer (ETS) and its flanking regions was determined. Comparison of the sequence around the transcription start point with those of four budding yeasts (Saccharomycetoideae) reveals a consensus sequence from position -9 to -4 from the start. This sequence is likely to be an important element of the promoter for yeast RNA polymerase I (Pol.I). Comparison of all known Pol.I promoter sequences reveals a strong bias for nucleotides (nt) at several positions between -16 and +10. These nt may have a critical role in the transcription initiation process. The S. pombe ETS, which comprises 1355 bp, is significantly longer than those of the budding yeasts and lacks any significant sequence homology with the Saccharomyces cerevisiae ETS. R-loop analysis reveals a putative processing site within the ETS of S. pombe.

Base Sequence↗
PubMed1984

Isolation of the structural genes for the alpha and beta subunits of the mitochondrial ATPase from the fission yeast Schizosaccharomyces pombe.

The structural genes for the two major subunits of the mitochondrial ATPase were isolated among genomic clones from the yeast Schizosaccharomyces pombe by transformation and complementation of mutants unable to grow on glycerol and lacking either the alpha or the beta subunits. The plasmid pMa1 containing a 2.3-kilobase genomic insert transformed the mutant A23-13 lacking a detectable alpha subunit. The transformant grew on glycerol and contained an alpha subunit of normal electrophoretic mobility. The plasmid pMa2 containing a 5.4-kilobase genomic insert transformed the mutant B59-1 lacking the beta subunit. The transformant grew on glycerol and contained a beta subunit of normal mobility. The structural gene for the beta ATPase subunit for the fission yeast S. pombe was localized within the pMa2 insert by hybridization to a probe containing the beta ATPase gene from the budding yeast Saccharomyces cerevisiae (Saltzgaber, J., Kunapuli, S., and Douglas, M. G. (1983) J. Biol. Chem. 258, 11465-11470). The mRNAs which hybridized to pMa1 and pMa2 were translated by a reticulocyte lysate into polypeptides of Mr = 59,000 and 54,000, respectively. These genes products reacted with an anti-F1-ATPase serum and therefore correspond most probably to precursors of the alpha and beta subunits.

Animals↗
PubMed1984

Independent loci for the structural genes of the yeast mitochondrial alpha and beta ATPase subunits.

In the yeast Schizosaccharomyces pombe, the structural gene mutations A23-13 (alpha-) and B59-1 (beta-) which totally prevent the expression of either the alpha or the beta subunits of the mitochondrial ATPase, were shown by classical genetic mapping studies to be both located on chromosome I but genetically unlinked. It is concluded that the structural genes ATP1 and ATP2 for the alpha and beta subunits of the mitochondrial ATPase are not organized in a cluster. By both meiotic recombination frequency analysis and gene transfer studies, three single nuclear mutations affecting to different extents the electrophoretic mobility of the beta polypeptide were located on the chromosome I very close to the mutation B59-1 (beta-). Two mutations involved a defective ATPase activity and the inability to grow on glycerol (gly). One of these mutants E5-23 (beta") exhibited a beta subunit of slightly reduced electrophoretic mobility. The other mutation F1-10 (beta) was associated with a beta subunit of normal electrophoretic mobility. The plasmid pMa2 (Boutry, M., Vassarotti, A., Ghislain, M., Douglas, M., Goffeau, A. (1984) J. Biol. Chem. 259, 2840-2844) containing the structural gene for the beta subunit complemented the mutants E5-23 (beta") and F1-10 (beta) as well as B59-1 (beta-). These three mutations are therefore likely to affect the beta structural gene itself or a very contiguous gene contained in the 5.4-kilobase genomic insert of pMa2. The mutation F1-10 (beta) was mapped between E5-23 (beta") and B59-1 (beta-) by analysis of the meiotic recombination frequencies. Another mutation F25-28-11 (beta') was responsible for an appreciable decrease of electrophoretic mobility of the beta subunit which, however, did not affect either the ATPase activity or the ability to grow on glycerol (GLY). This mutant transformed by pMa2 was able to express the structural gene for the wild type beta subunit and the resulting transformants synthesized and assembled both the beta and beta' subunits. It is concluded that the mutation F25-28-11 (beta') also affects the structural gene for the beta subunit and does not affect genes controlling the processing machinery.

Ascomycota↗
PubMed1983

The alpha subunit of a plant mitochondrial F1-ATPase is translated in mitochondria.

The mitochondrial F1-ATPase from bean (Vicia faba L.) was solubilized by a chloroform treatment of mitochondrial membranes and purified by centrifugation on a glycerol gradient. The active fraction contained 5 subunits: alpha (Mr = 52,000), beta (Mr = 51,000), gamma (Mr = 34,000), delta (Mr = 23,800), and epsilon (Mr = 22,900). Purified coupled mitochondria were incubated in the presence of [ 35S ]methionine and malate to allow mitochondrial translation to occur. The largest labeled polypeptide (Mr = 52,000) was present in the chloroform extract, co-sedimented with the F1-ATPase on glycerol gradient and co-migrated with the alpha subunit upon two-dimensional electrophoresis. The results indicate that the alpha subunit of bean mitochondrial ATPase is translated on mitoribosomes, in contrast to the situation in other organisms.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

Phosphorylated intermediate of a transport ATPase and activity of protein kinase in membranes from corn roots.

A maize-root microsomal fraction was enriched in ATPase by treatment with Triton X-100. This activity, which reached 1.2-2.0/mumol Pi x min-1 x mg protein-1, was specific for ATP, very slightly stimulated by K+, inhibited by orthovanadate and diethylstilbestrol, resistant to oligomycin and azide, and had a Km of 1.2 mM MgATP. Incubation of the microsomal fraction with [gamma 32-P]ATP followed by electrophoresis in acid conditions revealed the presence of several phosphoproteins. The phosphorylation of a 110000-Mr polypeptide reached the steady-state level in less than 5 s and rapidly turned over the phosphate group. The phosphorylation level was an hyperbolic function of the [ATP] with a Km of 0.6 mM, suggesting that the rate of Pi production was proportional to the phosphoprotein concentration. The extent of phosphoprotein was decreased by vanadate and diethylstilbestrol. The phosphorylation level was 30% decreased by 50 mM K+ or Na+ while the ATPase activity was slightly stimulated (12% and 5%, respectively). The polypeptide could not be phosphorylated in reverse by Pi. This phosphorylated intermediate from maize-root microsomes exhibits molecular properties characteristic of transport ATPases such as the yeast plasma membrane H+-translocating ATPase. This similarity indicates existence of a transport ATPase in plant plasma membranes. Three other plant microsomal polypeptides (Mr = 52000, 17000 and 16000) and a low molecular weight component (Mr less than 1000) were phosphorylated much more slowly, were not undergoing a rapid turnover and were not hydrolysed by hydroxylamine. These phosphoproteins and the Mr less than 1000 phosphorylated component were inhibited by vanadate and diethylstilbestrol. These properties are similar to those of the protein kinase activity recently described in yeast plasma membranes.

Adenosine Triphosphatases↗
PubMed1983

Modified plasma-membrane ATPase in mutants of Saccharomyces cerevisiae.

Mutations affecting the plasma membrane ATPase of Saccharomyces cerevisiae were obtained by selecting mutants resistant to Dio-9. In a plasma-membrane-enriched fraction of the mutant MG2130, the ATPase activity was resistant to vanadate (50% inhibition by 26 microM in the mutant compared to 1.3 microM in the parental strain). Several catalytic properties of the membrane-bound ATPase were modified by 60-120% in the mutant which had a higher Km for MgATP and was more heatstable, less sensitive to mercurials, and more stimulated by monovalent cations than the parental type. A single mutation is responsible for the phenotypes of four independent allelic mutants. Resistance to Dio-9 in vivo and resistance to vanadate in vitro segregated together in three tetrads issued from a cross between the wild type and mutant. The mutation is semi-dominant as shown by expression of the mutant phenotype in a heterozygous diploid resulting from the cross between the wild type and mutant. It is concluded that the pma locus, affected by these mutations, is the structural gene either for the 100000-Mr subunit of plasma membrane ATPase or for a protein which tightly controls the conformation of the plasma-membrane ATPase within the membrane.

Adenosine Triphosphatases↗
PubMed1983

Intramitochondrial ATP and cell functions: yeast cells depleted of intramitochondrial ATP lose the ability to grow and multiply.

Cells of the yeast Saccharomyces cerevisiae could be depleted of their intramitochondrial ATP bu culturing on glucose in the presence of antimycin A, which prevents production of ATP in mitochondria, along with bongkrekic acid, which prevents transport of ATP from the cytosol into mitochondria. Alternatively, the depletion could be achieved by culturing respiration-deficient mutants in the presence of bongkrekic acid. The depleted cells of the respiration-deficient mutant did not grow on glucose in a synthetic medium and growth for a few generations was made possible by adding peptone, yeast extract or some amino acids into the medium. The depleted cells did not differ from control cells in their content of amino acids, proteins, nucleic acids and major phospholipids and had preserved the ability to carry on protein and nucleic acid syntheses and to mate to other cells. No conspicuous cytological differences were found between the control and depleted cells. After culturing in a semi-synthetic medium in the presence of bongkrekic acid the cells of the respiration-deficient mutant exhibited almost no cytochrome c in their spectra and their azide-sensitive ATPase activity was drastically reduced. The results suggest that intramitochondrial syntheses of some low-molecular compounds as well as import and/or assembly of some cytoplasmically synthesized mitochondrial proteins into mitochondria may be impaired in cells lacking intramitochondrial ATP and this may be responsible for their inability to grow and multiply.

Adenosine Triphosphate↗
PubMed1982

Exchange of oxygen between phosphate and water catalyzed by the plasma membrane ATPase from the yeast Schizosaccharomyces pombe.

The ATPase of the plasma membrane isolated from the yeast Schizosaccharomyces pombe catalyses a medium Pi in equilibrium H2O exchange in the presence of Mg2+ and in the absence of ATP and ADP. (formula, see text) The Pi in the E.Pi species tumbles in the active site so that each of its oxygens has an equal probability of exchange with water. The partition coefficient (Pc = k2/k2 + k-1) is 0.45. The total rate of oxygen exchange, Vex, representing the rate of incorporation of water oxygens occurring during hydrolysis of E--P into E.Pi (Vex = k-2[E--P]) is dependent on the [Pi] with an apparent Km of 177 mM, reflecting the very low affinity of the enzyme for Pi. The maximal exchange rate is 6.7 micrograms atoms of oxygen X min-1 X mg-1 of protein. The individual kinetic constants are evaluated: k2 = 3.4 X 10(3) min-1, k-2 = 5.50 X 10(5) min-1 and k-1 = 4.11 X 10(3) min-1. Under conditions of uncoupled transport, the hydrolysis of E--P is exergonic as [E.Pi]/[E--P] = k-2/k2 = 164. During hydrolysis of ATP, the rate of medium Pi in equilibrium H2O exchange activity as well as the extent of phosphorylation of the enzyme from Pi are markedly stimulated: 7.9 and 5.3 times, respectively, whereas the Pc is not modified. These data are most simply interpretated by the existence of two isomeric forms of the enzyme; one is specific for binding ATP and the other for binding Pi. The Pc for intermediate Pi in equilibrium H2O exchange, when the E--P species is formed from cleavage of [gamma-18O]ATP, is the same as for medium exchange, indicating that the same exchange pathway operates under both conditions. Varying the [ATP] had very little effect on the Pc, indicating little or no cooperativity between different catalytic sites under the conditions used in this study.

Adenosine Triphosphatases↗
PubMed1982

Alterations of the alpha or beta subunits of the mitochondrial ATPase in yeast mutants.

Among 979 non-glycerol growers of the yeast Schizosaccharomyces pombe, 40 strains were found to be deficient in the mitochondrial ATPase activity. Three of them exhibited an alteration in either the alpha or beta subunits of the F1ATPase. The alpha subunit was not immunodetected in the A23/13 mutant. The beta subunit was not immuno-detected in the B59/1 mutant. The existence of these two mutants shows that the alpha and beta subunits can be present independently of each other in the inner mitochondrial membrane. The beta subunit of the mutant F25/28 had a slower electrophoretic mobility than that of the wild-type beta subunit. This phenotype indicates abnormal processing or specific modification of the beta subunit. All mutants showed reduced activities of the NADH-cytochrome c reductase and of the cytochrome oxidase and a decreased synthesis of cytochrome aa3 and cytochrome b. This pleiotropic phenotype appears to result from specific modifications in the mitochondrial protein synthesis. The mitochondrial synthesis of four polypeptides (three cytochrome oxidase and one cytochrome b subunits) was markedly decreased or absent while three new polypeptides (Mr = 54000, 20000 and 15000) were detected in all the mutants analysed. This observation suggests that a functional F1ATPase is necessary for the correct synthesis and/or assembly of the mitochondrially made components of the cytochrome oxidase and cytochrome b complexes.

Adenosine Triphosphatases↗
PubMed1982

Reduction of respiratory-chain cytochrome b by lactate in Saccharomyces cerevisiae.

Cytochrome b of yeast mitochondria can be reduced by a part of the electrons resulting from the oxidation of lactate enantiomers. 1. The respiration of D-lactate and L-lactate is 30-40% inhibited by antimycin A. 2. Reduction of cytochrome b is observed in submitochondrial particles in the presence of low concentration of D-lactate and L-lactate (half-optimal concentration of 4.7 mM and 2.4 mM respectively) in the presence of different bc1 inhibitors. 3. Reduction of cytochrome b and c1 occurs in purified complex III of yeast in the presence of L-lactate and added L-lactate: NAD+ oxidoreductase. 4. In the particles obtained from yeast grown in lactate the oxidation of L-lactate involves the reduction of a pigment absorbing at 558 nm.

Chemical Phenomena↗