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J Velours

Publications and source records attributed to J Velours.

At least 19 recordsLinked to original sources
PubMed1996

ATP synthase of yeast mitochondria. Isolation of the subunit h and disruption of the ATP14 gene.

A new subunit of the yeast ATP synthase (termed subunit h) has been isolated. Amino acid composition and N-terminal sequencing were determined by chemical methods. These data were in agreement with the sequence of the hypothetical protein L8003.20 whose primary structure was deduced from DNA sequencing of the yeast chromosome XII. The amino acid sequence encoded by ATP14 gene is 32 amino acids longer than the mature protein, which contains 92 amino acids corresponding to a calculated mass of 10,408 Da. The protein is hydrophilic and acidic with a calculated pHi of 4.08. It is not apparently related to any subunit described in other ATP synthases. A null mutant was constructed. The mutation was recessive and the mutant strain was unable to grow on glycerol medium. A high percentage of rho- cells arose spontaneously. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity, but still contained ATPase activity with a catalytic sector dissociated from the membranous components. The mutant mitochondria did not contain subunit h, and the mitochondrially encoded hydrophobic subunit 6 was not present.

Amino Acid Sequence↗
PubMed1996

The inhibition of phosphoenolpyruvate carboxykinase following in vivo chronic phenobarbital treatment in the rat is due to a post-translational event.

Chronic treatment of rats with phenobarbital has been reported to decrease gluconeogenesis in rat hepatocytes by a 50% inhibition of phosphoenolpyruvate (P-pyruvate) carboxykinase activity [Argaud, D., Halimi, S., Catelloni, F. & Leverve, X. (1991) Biochem. J. 280, 663-669]. Contrary to the current knowledge of P-pyruvate carboxykinase regulation, we failed to find a diminution of either P-pyruvate carboxykinase protein (by using a polyclonal antibody) or P-pyruvate carboxykinase mRNA, in the liver of rats treated with phenobarbital for 2 weeks. Kinetic studies of P-pyruvate carboxykinase activity, measured by either carboxylation of P-pyruvate or decarboxylation of oxaloacetate, revealed a decrease in both V(max) and Km after phenobarbital treatment, whereas the nutritional state affected only the V(max), as expected. Assessment of P-pyruvate carboxykinase specificity was confirmed by the full inhibition of the enzyme with its specific inhibitor 3-mercaptopicolinate in the micromolar range. P-Pyruvate carboxykinase, purified either by ammonium sulfate fractionation or by immunoprecipitation, exhibited a similar decrease in affinity after phenobarbital treatment. Although the molecular mass does not appear to be altered, the pH sensitivity to 3-mercaptopicolinate inhibition and the enzyme recovery after immunoprecipitation both seemed to be affected. This leads us to propose that the effect of chronic phenobarbital treatment on P-pyruvate carboxykinase activity is not the result of transcriptional regulation but is exerted at the post-translational level.

Animal Nutritional Physiological Phenomena↗
PubMed1994

Cloning of the yeast ATP3 gene coding for the gamma-subunit of F1 and characterization of atp3 mutants.

Saccharomyces cerevisiae pet mutants, of complementation group G115, are deficient in mitochondrial ATPase and have properties indicative of defective F1. In this study we show that C287/LU1, a mutant belonging to group G115, is complemented by the yeast nuclear ATP3 gene coding for the gamma-subunit of the mitochondrial F1-ATPase. The amino-terminal sequence of the mature gamma-subunit matches the sequence encoded by ATP3 starting with the 34th amino acid confirming the identity of the gene, and earlier evidence indicating that this F1 component is synthesized as a precursor with a long amino-terminal extension. The properties of the mitochondrial ATPase have been studied in C287/LU1 with an Ala273-->Val substitution in the carboxyl-terminal region of the gamma-subunit and in W303 delta ATP3, a mutant lacking the gamma-subunit as a result of a deletion in ATP3. Both strains have negligible ATPase activity but near normal concentrations of the alpha- and beta-subunits of F1. In W303 delta ATP3, the subunits do not form a stable F1 oligomer nor are they firmly associated with F0. This is not true of C287/LU1, which was found to assemble an F1-F0 complex. These data indicate that the yeast gamma-subunit has dual functions, one in catalysis of ATP hydrolysis/synthesis and the second in assembly/stability of F1.

Amino Acid Sequence↗
PubMed1994

Mutation in the hydrophobic domain of ATP synthase subunit 4 (subunit b) of yeast mitochondria disturbs coupling between proton translocation and catalysis.

We introduced mutations to test the function of the hydrophobic sector of subunit 4 from Saccharomyces cerevisiae ATP synthase. Mutations were introduced at the chromosomic locus by homologous transformation of a strain disrupted in the ATP4 gene. The strain carrying the replacement Leu68-Val69-->Arg-Glu did not grow at 37 degrees C owing to a lack of assembly of F1 and Fo sectors at this temperature. The mutant strain grew slowly by oxidative phosphorylation at 28 degrees C with a growth yield 30% lower than the wild type. Analysis of the mutant strain showed a homogeneous population of altered ATP synthase with an energy coupling impairment. The mutant strain was oligomycin-resistant since the I50 value of oligomycin inhibition of ATPase and ATP synthase activities was 2-3-fold higher than that of the wild type, thus showing an alteration of the target to oligomycin. The level of phosphorylation or ATP induced a proton-dissipating pathway through Fo, which was insensitive to oligomycin but was sensitive to dicyclohexylcarbodiimide, thus suggesting an alteration in the regulation of ATP synthase proton permeability by the catalytic sector. From these results, we propose that the dicyclohexylcarbodiimide inhibition site is located upstream of the oligomycin inhibition site when considering the proton flux occurring during ATP synthesis.

Base Sequence↗
PubMed1994

ATP synthase of yeast mitochondria. Isolation of the F1 delta subunit, sequence and disruption of the structural gene.

The delta-subunit was isolated from the purified yeast F1. Partial protein sequences were determined by direct methods. From this information, degenerated primers were constructed. A part of the ATP delta gene was amplified by polymerase chain reaction from yeast genomic DNA. From the amplified DNA sequence, a nondegenerated oligonucleotide probe was constructed to isolate a 2.6-kbp BamHI-EcoRI DNA fragment bearing the whole gene. A 1036-bp DraI fragment was sequenced. A 480-bp open reading frame encoding a 160-amino-acid polypeptide is described. The deduced amino acid sequence is 22 amino acids longer than the mature protein, which is 138 amino acids long with a mass of 14,555 Da. The delta-subunit of Saccharomyces cerevisiae is 21%, 35%, 52% identical and 66%, 61% and 92% similar to the epsilon-subunit of Escherichia coli and the delta-subunits of beef heart and Neurospora crassa, respectively. A null mutant was constructed. The mutation was recessive and dramatically affected mitochondrial DNA stability since the transformed cells were 100% cytoplasmic petite. The double mutant (rho-, ATP delta::URA3) displayed low or no ATPase activity with an unstable catalytic sector, since a polyclonal antibody directed against the beta subunit did not coprecipitate the alpha subunit.

Amino Acid Sequence↗
PubMed1994

Over-expression of the yeast ATP synthase subunit D in Escherichia coli: use of polyclonal antibodies directed against recombinant subunit D.

The yeast ATP synthase subunit d was over-expressed in E. coli and formed inclusion bodies. It was purified by solubilization in urea and slow removal of the urea by stepwise dialysis in the presence of a non-ionic detergent. The resulting soluble subunit d was used to prepare polyclonal antibodies. Blots of yeast mitochondrial proteins were probed with these antibodies. The strain disrupted in ATP4 gene encoding the subunit 4 displayed only 8% of the wild type subunit d. Antibodies against subunit d did not inhibit the wild type ATPase activity.

Antibodies, Fungal↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

ATP synthase of yeast mitochondria. Isolation and disruption of the ATP epsilon gene.

The nuclear gene encoding the subunit epsilon of the catalytic sector F1 of the yeast Saccharomyces cerevisiae ATP synthase was cloned and sequenced. Degenerated oligonucleotide primers were constructed from primary structure data. A part of the ATP epsilon gene was amplified by polymerase chain reaction from yeast genomic DNA. From the amplified DNA sequence a nondegenerated oligonucleotide probe was constructed and used for isolating a 2040-base pair EcoRI fragment bearing the whole gene. A 186-base pair open reading frame encoding a 62-amino acid polypeptide is described. The deduced amino acid sequence was one amino acid longer than the mature protein. A null mutant was constructed. The mutant strain was unable to grow on glycerol medium. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity. The catalytic sector appeared unstable during purification but F0-subunits were still bound to F1. The mutation promoted a highly oligomycin-sensitive uncoupling of the mitochondrial respiration rate.

Amino Acid Sequence↗
PubMed1993

Tumor necrosis factor alpha in human kidney transplant rejection--analysis by in situ hybridization.

Macrophagic infiltration and necrosis of rejected kidney transplants represent two pejorative patterns. It has been assumed that the macrophagic toxicity is mediated partly by secretion of tumor necrosis factor alpha. On the other hand, TNF is also involved in many inflammatory and immunological phenomena. We thus evaluated the expression of TNF mRNA by in situ hybridization in 6 rejected kidney transplants using a radiolabeled TNF-c DNA probe. Then the synthesis of TNF alpha protein was studied by immunohistochemistry using an anti-TNF alpha antibody. In severely rejected kidney grafts, TNF mRNA is expressed in some monomorphic infiltrating cells, mostly located in the deepest part of the cortex and around the tubes. These cells do not bind other probes, such as dopa-decarboxylase DNA or preproenkephalin RNA. They are also recognized by a monoclonal antibody directed against TNF alpha. What is more, this antibody binds with some glomerular endothelial and tubular epithelial cells that do not express TNF mRNA. These cells are likely target cells for TNF. In the normal kidney, there are no cells expressing TNF-alpha mRNA.

Gene Expression↗
PubMed1993

CL307-24, a new antibiotic complex from Saccharopolyspora aurantiaca sp. nov. II. Physico-chemical and biological properties.

CL307-24I, the main component of the CL307-24 complex produced by Saccharopolyspora aurantiaca sp. nov., was found to be a potent inhibitor of yeast mitochondrial ATPase. CL307-24I displayed a high degree of activity towards some coryneform bacteria and also has been shown to possess an insecticidal activity. Its biological and physico-chemical properties clearly distinguish it from previously known ATPase inhibitors.

Adenosine Triphosphatases↗
PubMed1992

The C-terminal region of subunit 4 (subunit b) is essential for assembly of the F0 portion of yeast mitochondrial ATP synthase.

The role of the C-terminal part of yeast ATP synthase subunit 4 (subunit b) in the assembly of the whole enzyme was studied by using nonsense mutants generated by site-directed mutagenesis. The removal of at least the last 10 amino-acid residues promoted mutants which were unable to grow with glycerol or lactate as carbon source. These mutants were devoid of subunit 4 and of another F0 subunit, the mitochondrially encoded subunit 6. The removal of the last eight amino-acid residues promoted a temperature-sensitive mutant (PVY161). At 37 degrees C this strain showed the same phenotype as above. When grown at permissive temperature (30 degrees C) with lactate as carbon source, PVY161 and the wild-type strain both displayed the same generation time and growth yield. Furthermore, the two strains showed identical cellular respiration rates at 30 degrees C and 37 degrees C. However, in vitro the ATP hydrolysis of PVY161 mitochondria exhibited a low sensitivity to F0 inhibitors, while ATP synthesis displayed the same oligomycin sensitivity as wild-type mitochondria. It is concluded that, in this mutant, the assembly of the truncated subunit 4 in PVY161 ATP synthase is thermosensitive and that, once a functional F0 is formed, it is stable. On the other hand, the removal of the last eight amino-acid residues promoted in vitro a proton leak between the site of action of oligomycin and F1.

Adenosine Triphosphatases↗
PubMed1991

ATP synthase of yeast mitochondria. Characterization of subunit d and sequence analysis of the structural gene ATP7.

The subunit analogous to the d-subunit of ATP synthase from bovine heart mitochondria was isolated from the purified yeast enzyme. Partial protein sequences were determined by direct methods. From this information, two oligonucleotide probes were constructed and used for screening a DNA genomic bank of Saccharomyces cerevisiae. The sequence of yeast subunit d was deduced from the DNA sequence of ATP7 gene. Mature yeast subunit d is 173 amino acids long. Its NH2-terminal serine is blocked by an N-acetyl group, and the protein has no processed NH2-terminal sequence other than the removal of the initiator methionine. The protein is predominantly hydrophilic. The amino acid sequence is 22% identical and 44% homologous to bovine subunit d. A null mutant was constructed. The mutant strain was unable to grow on glycerol medium. The mutant mitochondria had no detectable oligomycin-sensitive ATPase activity, and the catalytic sector F1 was loosely bound to the membranous part. The mutant mitochondria did not contain subunit d, and the mitochondrially encoded hydrophobic subunit 6 was not present.

Amino Acid Sequence↗
PubMed1991

Isolation of the ATP synthase subunit 6 and sequence of the mitochondrial ATP6 gene of the yeast Candida parapsilosis.

The mitochondrially translated product called subunit 6 was extracted from the yeast Candida parapsilosis mitochondria using an organic solvent mixture and purified by reverse-phase HPLC. The partial N-terminal sequence of subunit 6 reveals a post-translational cleavage site as in Saccharomyces cerevisiae. The structural mitochondrial gene ATP6 was isolated form a mitochondrial DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13tg130 and M13tg131 phage vectors. The insert contained an open reading frame 738-bp encoding a 246-amino-acid polypeptide. Mature subunit 6 contains 243 amino acid residues and the predicted molecular mass is 26,511 Da. The subunit shows 52% similarity with ATP synthase subunit 6 of the yeast S. cerevisiae. Comparison between protein and DNA sequences shows that the CUN codon family codes for a leucine in C. parapsilosis mitochondria.

Amino Acid Sequence↗
PubMed1991

Isolation and complete amino acid sequence of the mitochondrial ATP synthase epsilon-subunit of the yeast Saccharomyces cerevisiae.

All five subunits of yeast mitochondrial F1-ATPase have been isolated by reverse-phase high performance liquid chromatography. This procedure allows micro-preparative purification of all the subunits with 60% recoveries. The complete amino acid sequence of the epsilon-subunit has been established. This has been achieved by the sequence analysis of subnanomole amounts of the intact molecule and that of peptides derived by enzymatic digestion with endoproteinase Arg-C and by chemical cleavage with hydroxylamine. Yeast ATP synthase epsilon-subunit is composed of 61 residues with a calculated molecular mass of 6612 Da. This polypeptide is rather basic since it contains 7 basic residues and 3 acidic residues. This study shows a slight similarity with the bovine epsilon-subunit ATP synthase since there are 16 identical residues.

Amino Acid Sequence↗
PubMed1990

Direct protein sequencing of wheat mitochondrial ATP synthase subunit 9 confirms RNA editing in plants.

RNA editing, a process that results in the production of RNA molecules having a nucleotide sequence different from that of the initial DNA template, has been demonstrated in several organisms using different biochemical pathways. Very recently RNA editing was described in plant mitochondria following the discovery that the sequence of certain wheat and Oenothera cDNAs is different from the nucleotide sequence of the corresponding genes. The main conversion observed was C to U, leading to amino acid changes in the deduced protein sequence when these modifications occurred in an open reading frame. In this communication we show the first attempt to isolate and sequence a protein encoded by a plant mitochondrial gene. Subunit 9 of the wheat mitochondrial ATP synthase complex was purified to apparent homogeneity and the sequence of the first 32 amino acid residues was determined. We have observed that at position 7 leucine was obtained by protein sequencing, instead of the serine predicted from the previously determined genomic sequence. Also we found phenylalanine at position 28 instead of a leucine residue. Both amino acid conversions, UCA (serine) to UUA (leucine) and CUC (leucine) to UUC (phenylalanine), imply a C to U change. Thus our results seem to confirm, at the protein level, the RNA editing process in plant mitochondria.

Amino Acid Sequence↗
PubMed1989

The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex.

The yeast nuclear gene ATP4, encoding the ATP synthase subunit 4, was disrupted by insertion into the middle of it the selective marker URA3. Transformation of the Saccharomyces cerevisiae strain D273-10B/A/U produced a mutant unable to grow on glycerol medium. The ATP4 gene is unique since subunit 4 was not present in mutant mitochondria; the hypothetical truncated subunit 4 was never detected. ATPase was rendered oligomycin-insensitive and the F1 sector of this mutant appeared loosely bound to the membrane. Analysis of mitochondrially translated hydrophobic subunits of F0 revealed that subunits 8 and 9 were present, unlike subunit 6. This indicated a structural relationship between subunits 4 and 6 during biogenesis of F0. It therefore appears that subunit 4 (also called subunit b in beef heart and Escherichia coli ATP synthases) plays at least a structural role in the assembly of the whole complex. Disruption of the ATP4 gene also had a dramatic effect on the assembly of other mitochondrial complexes. Thus, the cytochrome oxidase activity of the mutant strain was about five times lower than that of the wild type. In addition, a high percentage of spontaneous rho- mutants was detected.

Adenosine Triphosphatases↗