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H Cherest

Publications and source records attributed to H Cherest.

At least 19 recordsLinked to original sources
PubMed2000

Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae.

One-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate. The major forms of folates in vivo are polyglutamylated derivatives. In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase. We report here the characterization and molecular analysis of the two glutamate-adding enzymes of Saccharomyces cerevisiae. We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by the YMR113w gene that we propose to rename FOL3. Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity. We show also that folylpolyglutamate synthetase, which catalyzes the extension of the glutamate chains of the folate coenzymes, is encoded by the MET7 gene. Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA. We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons. Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase.

Amino Acid Sequence↗
PubMed1997

Molecular characterization of two high affinity sulfate transporters in Saccharomyces cerevisiae.

Strains resistant to the toxic analogues of sulfate, selenate and chromate have been isolated. Their genetic analysis allowed us to identify four genes. One, called MET28, encodes a transcriptional factor. The three other genes, called SUL1, SUL2 and SUL3, encode proteins involved in sulfate transport. The sequence of Sul1p and Sul2p indicate that they are integral membrane proteins exhibiting, respectively, 11 and 10 transmembrane domains. Moreover, Sul1p and Sul2p share a high degree of similarity. Sulfate transport kinetic studies made with parental and mutant strains show that, as expected from genetic results, Saccharomyces cerevisiae has two high affinity sulfate transport systems. Sul3p has been shown to be involved in the transcriptional regulation of the SUL2 gene.

Amino Acid Sequence↗
PubMed1996

A heteromeric complex containing the centromere binding factor 1 and two basic leucine zipper factors, Met4 and Met28, mediates the transcription activation of yeast sulfur metabolism.

Transcription activation of sulfur metabolism in yeast is dependent on two DNA binding factors, the centromere binding factor 1 (Cbf1) and Met4. While the role of Met4 was clearly established by showing that it acts as a transcription activator, the precise function in transcription of the multi-functional factor Cbf1 remains more elusive. We report here the identification of a new transcription factor Met28 which participates in the regulation of sulfur metabolism. Cloning and sequencing of MET28 revealed that it encodes a new member of the basic leucine zipper DNA binding factor family. We also demonstrate that Met28 possesses no intrinsic transcription activation capabilities. Studies of the DNA binding characteristics of Met28 led us to identify in gel mobility assays a heteromeric complex containing Cbf1, Met4 and Met28. We further demonstrated that the presence of Cbf1 and Met4 stimulates the binding of Met28 to DNA. 'Two-hybrid' studies allowed us to carry out preliminary investigations on the binary protein-protein interactions involved in the formation of the Cbf1-Met4-Met28 complex. Our results give evidence that the leucine zippers of Met4 and Met28, along with the basic helix-loop-helix domain of Cbf1, provide the protein surfaces mediating these interactions. All these results suggest that the multi-functional factor Cbf1 functions in transcription activation by tethering specific activating factors to the DNA.

Amino Acid Sequence↗
PubMed1995

Met30p, a yeast transcriptional inhibitor that responds to S-adenosylmethionine, is an essential protein with WD40 repeats.

A specific repression mechanism regulates the biosynthesis of sulfur amino acids in Saccharomyces cerevisiae. When the intracellular S-adenosylmethionine (AdoMet) concentration increases, transcription of the sulfur genes is repressed. Using a specific reporter system, we have isolated mutations impairing the AdoMet-mediated transcriptional regulation of the sulfur network. These mutations identified a new gene, MET30, and were shown to also affect the regulation of the methyl cycle. The MET30 gene was isolated and sequenced. Sequence analysis reveals that Met30p contains five copies of the WD40 motif within its carboxy-terminal part, like the yeast transcriptional repressors Hir1p and Tup1p. We identified one target of Met30p as Met4p, a transcriptional activator regulating the sulfate assimilation pathway. By the two-hybrid method, we showed that Met30p interacts with Met4p and identified a region of Met4p involved in this interaction. Further analysis reveals that expression of Met30p is essential for cell viability.

Amino Acid Sequence↗
PubMed1994

Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the alpha subunit of sulfite reductase and specify potential binding sites for FAD and NADPH.

The yeast assimilatory sulfate reductase is a complex enzyme that is responsible for conversion of sulfite into sulfide. To obtain information on the nature of this enzyme, we isolated and sequenced the MET10 gene of Saccharomyces cerevisiae and a divergent MET10 allele from Saccharomyces carlsbergensis. The polypeptides deduced from the identically sized open reading frames (1,035 amino acids) of both MET10 genes have molecular masses of around 115 kDa and are 88% identical to each other. The transcript of S. cerevisiae MET10 has a size comparable to that of the open reading frame and is transcriptionally repressed by methionine in a way similar to that seen for other MET genes of S. cerevisiae. Distinct homology was found between the putative MET10-encoded polypeptide and flavin-interacting parts of the sulfite reductase flavoprotein subunit (encoded by cysJ) from Escherichia coli and several other flavoproteins. A significant N-terminal homology to pyruvate flavodoxin oxidoreductase (encoded by nifJ) from Klebsiella pneumoniae, together with a lack of obvious flavin mononucleotide-binding motifs in the MET10 deduced amino acid sequence, suggests that the yeast assimilatory sulfite reductase is a distinct type of sulfite reductase.

Amino Acid Sequence↗
PubMed1993

Cysteine biosynthesis in Saccharomyces cerevisiae occurs through the transsulfuration pathway which has been built up by enzyme recruitment.

The transsulfuration pathways allow the interconversion of homocysteine and cysteine with the intermediary formation of cystathionine. The various organisms studied up to now incorporate reduced sulfur into a three- or a four-carbon chain and use differently the transsulfuration pathways to synthesize sulfur amino acids. In enteric bacteria, the synthesis of cysteine is the first step of organic sulfur metabolism and homocysteine is derived from cysteine. Fungi are capable of incorporating reduced sulfur into a four-carbon chain, and they possess two operating transsulfuration pathways. By contrast, synthesis of cysteine from homocysteine is the only existing transsulfuration pathway in mammals. In Saccharomyces cerevisiae, genetic, phenotypic, and enzymatic study of mutants has allowed us to demonstrate that homocysteine is the first sulfur amino acid to be synthesized and cysteine is derived only from homocysteine (H. Cherest and Y. Surdin-Kerjan, Genetics 130:51-58, 1992). We report here the cloning of genes STR4 and STR1, encoding cystathionine beta-synthase and cystathionine gamma-lyase, respectively. The only phenotypic consequence of the inactivation of STR1 or STR4 is cysteine auxotrophy. The sequencing of gene STR4 has allowed us to compare all of the known sequences of transsulfuration enzymes and enzymes catalyzing the incorporation of reduced sulfur in carbon chains. These comparisons reveal a partition into two families based on sequence motifs. This partition mainly correlates with similarities in the catalytic mechanisms of these enzymes.

Alleles↗
PubMed1992

Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway.

We have identified a mutation in a gene of Saccharomyces cerevisiae, STR1, that leads to a strict nutritional requirement for cysteine. The str1-1 mutation decreases to an undetectable level the cystathionine gamma-lyase activity. This enzyme catalyzes one of the two reactions involved in the transsulfuration pathway that yields cysteine from homocysteine with the intermediary formation of cystathionine. The phenotype induced by this mutation implies that, in S. cerevisiae, the sulfur atom of sulfide resulting from the reductive assimilation of sulfate is incorporated into a four carbon backbone yielding homocysteine, which, in turn, is the precursor of the biosynthesis of both cysteine and methionine. This also reveals that the direct synthesis of cysteine by incorporation of the sulfur atom into a three carbon backbone as found in Escherichia coli does not occur in S. cerevisiae. The study of the meiotic progeny of diploid strains heterozygous at the STR1 locus has shown that the str1-1 mutation undergoes a particularly high frequency of meiotic gene conversion.

Acetyltransferases↗
PubMed1991

Identification of the structural gene for glucose-6-phosphate dehydrogenase in yeast. Inactivation leads to a nutritional requirement for organic sulfur.

Cloning of the MET19 gene revealed that it encodes the glucose-6-phosphate dehydrogenase from yeast. Sequence analysis showed a high degree of similarity between the yeast and the human enzymes. The cloned gene has allowed the construction of a glucose-6-phosphate dehydrogenase null mutant. The only phenotype of such a strain is an absolute requirement for an organic sulfur source, i.e. methionine, S-adenosylmethionine (AdoMet), cysteine, glutathione or homocysteine. The phenotype of this null mutant raises some new questions about the exact functions of the pentose phosphate pathway which was usually considered as the main cellular source of NADPH. Moreover, results reported here show that an increase of the AdoMet pool represses the transcription of the glucose-6-phosphate dehydrogenase gene. This regulation acts on the glucose-6-phosphate dehydrogenase biosynthesis but does not affect the synthesis of 6-phosphogluconate dehydrogenase. That AdoMet controls a part of the pentose phosphate pathway sheds new light on mechanisms regulating the relative fluxes of carbon utilization through the pentose phosphate pathway and glycolysis.

Amino Acid Sequence↗
PubMed1989

Elements involved in S-adenosylmethionine-mediated regulation of the Saccharomyces cerevisiae MET25 gene.

In Saccharomyces cerevisiae, the MET25 gene encodes O-acetylhomoserine sulfhydrylase. Synthesis of this enzyme is repressed by the presence of S-adenosylmethionine (AdoMet) in the growth medium. We identified cis elements required for MET25 expression by analyzing small deletions in the MET25 promoter region. The results revealed a regulatory region, acting as an upstream activation site, that activated transcription of MET25 in the absence of methionine or AdoMet. We found that, for the most part, repression of MET25 expression was due to a lack of activation at this site, reinforced by an independent repression mechanism. The activation region contained a repeated dyad sequence that is also found in the promoter regions of other unlinked but coordinately regulated genes (MET3, MET2, and SAM2). We show that the presence of the two dyads is necessary for maximal gene expression. Moreover, we demonstrate that in addition to this transcriptional regulation, a posttranscriptional regulation, probably targeted at the 5' region of mRNA, is involved in MET25 expression.

Base Sequence↗
PubMed1987

The Saccharomyces cerevisiae MET3 gene: nucleotide sequence and relationship of the 5' non-coding region to that of MET25.

In Saccharomyces cerevisiae, the expression of several genes implicated in methionine biosynthesis is co-regulated by a specific negative control. To elucidate the molecular basis of this regulation, we have cloned two of these genes, MET3 and MET25. The sequence of MET25 has already been determined (Kerjan et al. 1986). Here, we report the nucleotide sequence of the MET3 gene along with its 5' and 3' flanking regions. Plasmids bearing different deletions upstream of the transcribed region of MET3 were constructed. They were introduced into yeast cells and tested for their ability to complement met3 mutations and to respond to regulation by exogenous methionine. The regulatory region was located within a 100 bp region. The sequence of this regulatory region was compared with that of MET25. A short common sequence which occurs 250-280 bp upstream of the translation initiation codon of the gene was found. This sequence is a good candidate for the cis-acting regulatory element.

Base Sequence↗
PubMed1987

Molecular genetics of met 17 and met 25 mutants of Saccharomyces cerevisiae: intragenic complementation between mutations of a single structural gene.

We cloned the MET 17 gene of Saccharomyces cerevisiae by functional complementation after transformation of a yeast met 17 mutant. Restriction mapping and nucleotide sequencing of the MET 17 clones revealed that these were from the same genomic region as clones isolated previously and shown to contain the MET 25 gene encoding the enzyme O-acetylhomoserine, O-acetylserine sulphydrylase (OAH-OAS sulphydrylase). Transformation studies with MET 25 clones showed that the MET 17 and MET 25 functions were both endoced in a single transcription unit. We conclude that met 17 and met 25 are both mutations in the structural gene for the OAH-OAS sulphydrylase subunit and that each affects a different functional domain of the enzyme allowing subunit complementation in the met 17 X met 25 diploid. Enzyme assays indicated that the diploid, although not requiring methionine, had a low OAH-OAS sulphydrylase activity (10% of wild type). This is consistent with MET 17 and MET 25 being the same gene. We found that both met 17 and met 25 mutants were devoid of 3' phospho-adenosine 5' phospho-sulphite (PAPS) reductase activity and that this activity was fully restored in the met 17 X met 25 diploid. The possible interactions between OAH-OAS sulphydrylase and PAPS reductase are discussed.

Carbon-Oxygen Lyases↗
PubMed1986

Nucleotide sequence of the Saccharomyces cerevisiae MET25 gene.

To elucidate further the molecular basis of the specific regulatory mechanism modulating the expression of the genes implicated in methionine metabolism, we have cloned and characterized two genes, MET3 and MET25, and shown that the regulation of their expression is transcriptional. The sequence of the cloned yeast MET25 gene which encodes the O-acetyl homoserine - O-acetyl serine (OAH-OAS) sulfhydrylase is reported here along with its 5' and 3' flanking regions. The amino acid composition predicted from the DNA sequence is in good agreement with that determined by hydrolysis of the purified enzyme. In the 5' flanking region the signal for general amino acid control was not found, corroborating our previous finding that the synthesis of OAH-OAS sulfhydrylase is not submitted to general control. The transcription start points have been determined. The 5' and 3' flanking regions of the MET25 gene suggest initiation and termination signals similar to those associated with other yeast genes.

Amino Acid Sequence↗
PubMed1985

The expression of the MET25 gene of Saccharomyces cerevisiae is regulated transcriptionally.

The MET25 gene of Saccharomyces cerevisiae was cloned by functional complementation after transformation of a yeast met25 mutant. Subcloning of the DNA fragment bearing MET25 located the gene on a 2.3 kb region. The gene was formally identified by integration at the chromosomal MET25 locus. The cloned MET25 gene was used as a probe to measure the MET25 messenger RNA in a wild-type strain grown under conditions which promoted or failed to promote repression of MET25 expression. It was found that, under repression conditions, MET25 messenger RNA was reduced tenfold when compared with non-repression conditions. This suggests that the expression of MET25 is regulated transcriptionally. The direction of transcription, the size of the transcript and the position of the transcribed part of the gene were determined. Deletion mapping of the regulatory region was carried out. Deleted plasmids were introduced back into yeast cells and tested for their ability to complement met25 mutations and to promote regulation of expression of the MET25 gene by exogenous methionine. By this method the regulatory region was found to be confined to a 130 bp region.

Cloning, Molecular↗
PubMed1985

Transcriptional regulation of the MET3 gene of Saccharomyces cerevisiae.

The MET3 gene, coding for ATP sulfurylase (ATPS), an enzyme implicated in methionine biosynthesis in Saccharomyces cerevisiae, was cloned by functional complementation, after transformation, of a yeast met3 mutant strain. The cloned MET3 gene was used as a probe to measure the specific MET3 messenger RNA in a wild-type strain grown under conditions which promote or fail to promote repression of ATPS synthesis. It was found that the level of MET3 messenger RNA is reduced ten-fold when the strain is grown under conditions where ATPS synthesis is repressed, suggesting that the MET3 expression is regulated transcriptionally. The direction of transcription and the size of the transcript have been determined.

Chromosome Mapping↗
PubMed1981

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae.

The lysA gene of Escherichia coli has been cloned from a lambda transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.

Bacterial Proteins↗
PubMed1981

The two methionine adenosyl transferases in Saccharomyces cerevisiae: evidence for the existence of dimeric enzymes.

In Saccharomyces cerevisiae either of the two genes SAM1 and SAM2 is able to produce a functional methionine adenosyl transferase (MATI and MATII). In a wild-type strain, MATI and MATII are present in dimeric forms: MATI-MATI, MATII-MATII and perhaps MATI-MATII. A hypothesis is presented to explain the possible role of these different forms of methionine adenosyl transferase in S. cerevisiae.

Chromatography, Gel↗
PubMed1978

S-adenosyl methionine requiring mutants in Saccharomyces cerevisiae: evidences for the existence of two methionine adenosyl transferases.

Mutants requiring S-adenosyl methionine (SAM) for growth have been selected in Saccharomyces cerevisiae. Two classes of mutants have been found. One class corresponds to the simultaneous occurrence of mutations at two unlinked loci SAM1 and SAM2 and presents a strict SAM requirement for growth on any medium. The second class corresponds to special single mutations in the gene SAM2 which lead to a residual growth on minimal medium but to normal growth on SAM supplemented medium or on a complex medium like YPGA not containing any SAM. These genetic data can be taken as an indication that Saccharomyces cerevisiae possesses two isoenzymatic methionine adenosyl transferases (MAT). In addition, SAM1 and SAM2 loci have been identified respectively with the ETH-10 and ETH2 loci previously described. Biochemical evidences corroborate the genetic results. Two MAT activities can be dissociated in a wild type extract (MATI and MATII) by DEAE cellulose chromatography. Mutations at the SAM1 locus lead to the absence or to the modification of MATII whereas mutations at the SAM2 locus lead to the absence or to the modification of MATI. Moreover, some of our results seem to show that MATI and MATII are associated in vivo.

Bicarbonates↗