RESUMEN
Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated. In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule. In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana. The function of each enzyme was demonstrated by complementation of yeast mutants deficient in DHFS or FPGS activity, and by measuring in vitro glutamate incorporation into dihydrofolate or tetrahydrofolate. DHFS is present exclusively in the mitochondria, making this compartment the sole site of synthesis of dihydrofolate in the plant cell. In contrast, FPGS is present as distinct isoforms in the mitochondria, the cytosol, and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes. The compartmentation of FPGS isoforms is in agreement with the predominance of gamma-glutamyl-conjugated tetrahydrofolate derivatives and the presence of serine hydroxymethyltransferase and C1-tetrahydrofolate interconverting enzymes in the cytosol, the mitochondria, and the plastids. Thus, the combination of FPGS with these folate-mediated reactions can supply each compartment with the polyglutamylated folate coenzymes required for the reactions of C1 metabolism. Also, the multicompartmentation of FPGS in the plant cell suggests that the transported forms of folate are unconjugated.
Asunto(s)
Arabidopsis/metabolismo , Isoenzimas/metabolismo , Péptido Sintasas/metabolismo , Tetrahidrofolatos/biosíntesis , Arabidopsis/enzimología , Arabidopsis/genética , ADN Complementario , Prueba de Complementación Genética , Datos de Secuencia Molecular , Péptido Sintasas/genética , Filogenia , Saccharomyces cerevisiae/genética , Fracciones Subcelulares/enzimologíaRESUMEN
Living organisms encounter various growth conditions in their habitats, raising the question of whether ecological fluctuations could alter biological macromolecules. The advent of complete genome sequences and the characterization of whole metabolic pathways allowed us to search for such ecological imprints. Significant correlations between atomic composition and metabolic function were found in sulfur- and carbon-assimilatory enzymes, which appear depleted in sulfur and carbon, respectively, in both the bacterium Escherichia coli and the eukaryote Saccharomyces cerevisiae. In addition to genetic instructions, genomic data thus also provide paleontological records of environmental nutrient availability and of metabolic costs.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/química , Evolución Molecular , Proteínas Fúngicas/química , Saccharomyces cerevisiae/química , Animales , Proteínas Bacterianas/genética , Carbono/análisis , Ecología , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas Fúngicas/genética , Humanos , Nitrógeno/análisis , Ratas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Azufre/análisisRESUMEN
The intracellular ratio between methionine and its activated form S-adenosylmethionine (AdoMet) is of crucial importance for the one-carbon metabolism. AdoMet recycling into methionine was believed to be largely achieved through the methyl and the thiomethyladenosine cycles. We show here that in yeast, AdoMet recycling actually occurs mainly through the direct AdoMet-dependent remethylation of homocysteine. Compelling evidences supporting this result were obtained owing to the identification and functional characterization of two new genes, SAM4 and MHT1, that encode the yeast AdoMet-homocysteine methyltransferase and S-methylmethionine-homocysteine methyltransferase, respectively. Homologs of the Sam4 and Mht1 proteins exist in other eucaryotes, indicating that such enzymes would be universal and not restricted to the bacterial or fungal kingdoms. New pathways for AdoMet or S-methylmethionine-dependent methionine synthesis are presented.
Asunto(s)
Proteínas de la Membrana , Metionina/biosíntesis , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae , Levaduras/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/fisiología , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Desoxiadenosinas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación de la Expresión Génica , Homocisteína S-Metiltransferasa/fisiología , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae/fisiología , Tionucleósidos/metabolismo , Vitamina B 12/farmacología , Vitamina U/metabolismoRESUMEN
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.
Asunto(s)
ADN Mitocondrial/genética , Ácido Fólico/metabolismo , Metionina/biosíntesis , Péptido Sintasas/metabolismo , Ácido Poliglutámico/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Clonación Molecular , Genoma Fúngico , Datos de Secuencia Molecular , Péptido Sintasas/química , Péptido Sintasas/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Progression through the cell cycle requires the coordination of basal metabolism with the cell cycle and growth machinery. Repression of the sulfur gene network is mediated by the ubiquitin ligase SCF(Met30), which targets the transcription factor Met4p for degradation. Met30p is an essential protein in yeast. We have found that a met4Deltamet30Delta double mutant is viable, suggesting that the essential function of Met30p is to control Met4p. In support of this hypothesis, a Met4p mutant unable to activate transcription does not cause inviability in a met30Delta strain. Also, overexpression of an unregulated Met4p mutant is lethal in wild-type cells. Under non-permissive conditions, conditional met30Delta strains arrest as large, unbudded cells with 1N DNA content, at or shortly after the pheromone arrest point. met30Delta conditional mutants fail to accumulate CLN1 and CLN2, but not CLN3 mRNAs, even when CLN1 and CLN2 are expressed from strong heterologous promoters. One or more genes under the regulation of Met4p may delay the progression from G(1) into S phase through specific regulation of critical G(1) phase mRNAs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Fase G1/fisiología , Ligasas/metabolismo , Proteínas Represoras , Fase S/fisiología , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Complejos de Ubiquitina-Proteína Ligasa , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , División Celular , Ciclinas/genética , Proteínas F-Box , Fase G1/genética , Genes Fúngicos , Modelos Biológicos , Mutación , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fase S/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supresión Genética , Ubiquitina-Proteína LigasasRESUMEN
We report here the characterization and the molecular analysis of the two high affinity permeases that mediate the transport of S-adenosylmethionine (AdoMet) and S-methylmethionine (SMM) across the plasma membrane of yeast cells. Mutant cells unable to use AdoMet as a sulfur source were first isolated and demonstrated to lack high affinity AdoMet transport capacities. Functional complementation cloning allowed us to identify the corresponding gene (SAM3), which encodes an integral membrane protein comprising 12 putative membrane spanning regions and belonging to the amino acid permease family. Among amino acid permease members, the closest relative of Sam3p is encoded by the YLL061w open reading frame. Disruption of YLL061w was shown to specifically lead to cells unable to use SMM as a sulfur source. Accordingly, transport assays demonstrated that YLL061w disruption mutation impaired the high affinity SMM permease, and YLL061w was therefore renamed MMP1. Further study of sam3Delta and mmp1Delta mutant cells showed that in addition to high affinity permeases, both sulfonium compounds are transported into yeast cells by low affinity transport systems that appear to be carrier-facilitated diffusion.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Proteínas de Escherichia coli , Proteínas de Transporte de Membrana/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Compuestos de Sulfonio/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Clonación Molecular , Difusión , Proteínas Fúngicas , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Sulfur amino acid biosynthesis in Saccharomyces cerevisiae involves a large number of enzymes required for the de novo biosynthesis of methionine and cysteine and the recycling of organic sulfur metabolites. This review summarizes the details of these processes and analyzes the molecular data which have been acquired in this metabolic area. Sulfur biochemistry appears not to be unique through terrestrial life, and S. cerevisiae is one of the species of sulfate-assimilatory organisms possessing a larger set of enzymes for sulfur metabolism. The review also deals with several enzyme deficiencies that lead to a nutritional requirement for organic sulfur, although they do not correspond to defects within the biosynthetic pathway. In S. cerevisiae, the sulfur amino acid biosynthetic pathway is tightly controlled: in response to an increase in the amount of intracellular S-adenosylmethionine (AdoMet), transcription of the coregulated genes is turned off. The second part of the review is devoted to the molecular mechanisms underlying this regulation. The coordinated response to AdoMet requires two cis-acting promoter elements. One centers on the sequence TCACGTG, which also constitutes a component of all S. cerevisiae centromeres. Situated upstream of the sulfur genes, this element is the binding site of a transcription activation complex consisting of a basic helix-loop-helix factor, Cbf1p, and two basic leucine zipper factors, Met4p and Met28p. Molecular studies have unraveled the specific functions for each subunit of the Cbf1p-Met4p-Met28p complex as well as the modalities of its assembly on the DNA. The Cbf1p-Met4p-Met28p complex contains only one transcription activation module, the Met4p subunit. Detailed mutational analysis of Met4p has elucidated its functional organization. In addition to its activation and bZIP domains, Met4p contains two regulatory domains, called the inhibitory region and the auxiliary domain. When the level of intracellular AdoMet increases, the transcription activation function of Met4 is prevented by Met30p, which binds to the Met4 inhibitory region. In addition to the Cbf1p-Met4p-Met28p complex, transcriptional regulation involves two zinc finger-containing proteins, Met31p and Met32p. The AdoMet-mediated control of the sulfur amino acid pathway illustrates the molecular strategies used by eucaryotic cells to couple gene expression to metabolic changes.
Asunto(s)
Aminoácidos Sulfúricos/metabolismo , Saccharomyces cerevisiae/metabolismo , Aminoácidos Sulfúricos/genética , Cisteína/metabolismo , Regulación Fúngica de la Expresión Génica , Metionina/metabolismo , Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Sulfur amino acid metabolism in Saccharomyces cerevisiae is regulated by the level of intracellular S-adenosylmethionine (AdoMet). Two cis-acting elements have been previously identified within the 5' upstream regions of the structural genes of the sulfur network. The first contains the CACGTG motif and is the target of the transcription activation complex Cbflp-Met4p-Met28p. We report here the identification of two new factors, Met31p and Met32p, that recognize the second cis-acting element. Met31p was isolated through the use of the one-hybrid method, while Met32p was identified during the analysis of the yeast methionine transport system. Met31p and Met32p are highly related zinc finger-containing proteins. Both LexA-Met31p and LexA-Met32p fusion proteins activate the transcription of a LexAop-containing promoter in a Met4p-dependent manner. Northern blot analyses of cells that do not express either Met31p and/or Met32p suggest that the function of the two proteins during the transcriptional regulation of the sulfur network varies from one gene to the other. While the expression of both the MET3 and MET14 genes was shown to strictly depend upon the presence of either Met31p or Met32p, the transcription of the MET25 gene is constitutive in cells lacking both Met31p and Met32p. These results therefore emphasise the diversity of the mechanisms allowing regulation of the expression of the methionine biosynthetic genes.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Regulación Fúngica de la Expresión Génica , Metionina/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Mapeo Cromosómico , Clonación Molecular , Secuencia de Consenso , Proteínas de Unión al ADN/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , Dedos de ZincRESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Proteínas de Transporte de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas de Transporte de Anión , Clonación Molecular , Regulación Fúngica de la Expresión Génica , Metionina/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Sulfatos/metabolismoRESUMEN
Siroheme is a uroporphyrinogen III-derivative used by sulfite reductase as a prosthetic group. We investigated in Saccharomyces cerevisiae the possible involvement in siroheme biosynthesis of three genes, MET1, MET8 and MET20. The MET1 gene from S. cerevisiae was cloned and shown to be the same gene as MET20. Sequence similitudes as well as complementation studies indicate that Met1p and Met8p are both involved in siroheme biosynthesis. In addition, we show formally that S. cerevisiae does not need vitamin B12 for growth.
Asunto(s)
Ferroquelatasa , Proteínas Fúngicas/metabolismo , Hemo/análogos & derivados , Metiltransferasas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Hemo/biosíntesis , Datos de Secuencia Molecular , Fenotipo , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
The screening of mutants resistant to the oxidized analogues of methionine (methionine sulphoxide and ethionine sulphoxide) allowed the characterisation of a yeast mutant strain lacking the high affinity methionine permease and defining a new locus that was called MUP1. The study of MUP1 mutants showed that methionine is transported into yeast cells by three different permeases, a high affinity and two low affinity permeases. The MUP1 gene was cloned and was shown to encode an integral membrane protein with 13 putative membrane-spanning regions. Database comparisons revealed that the yeast genome contains an ORF whose product is highly similar to the MUP1 protein. This protein is shown here to encode very low affinity methionine permease and the corresponding gene was thus called MUP3. It has previously been suggested that the amino acid permeases from yeast all belong to a single family of highly similar proteins. The two methionine permeases encoded by genes MUP1 and MUP3 are only distantly related to this family and thus define a new family of amino acid transporters.
Asunto(s)
Proteínas de Transporte de Membrana/genética , Metionina/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos , Mapeo Cromosómico , Secuencia Conservada , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Fenotipo , Proteínas/genética , Proteínas/metabolismo , Mapeo Restrictivo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Leucina Zippers/fisiología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Azufre/metabolismo , Transactivadores/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , Línea Celular , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Liasas de Carbono-Oxígeno , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Complejos Multienzimáticos , Proteínas Represoras/biosíntesis , S-Adenosilmetionina/farmacología , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Complejos de Ubiquitina-Proteína Ligasa , Secuencia de Aminoácidos , Secuencia de Bases , Cisteína Sintasa , Proteínas F-Box , Genes Fúngicos , Prueba de Complementación Genética , Genotipo , Liasas/metabolismo , Datos de Secuencia Molecular , Oxidorreductasas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Represoras/genética , Mapeo Restrictivo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Sulfato Adenililtransferasa/metabolismoRESUMEN
Saccharomyces cerevisiae general regulatory factor CP1 (encoded by the gene CEP1) is required for optimal chromosome segregation and methionine prototrophy. MET16-CYC1-lacZ reporter constructs were used to show that MET16 5'-flanking DNA contains a CP1-dependent upstream activation sequence (UAS). Activity of the UAS required an intact CP1-binding site, and the effects of cis-acting mutations on CP1 binding and UAS activity correlated. In most respects, MET16-CYC1-lacZ reporter gene expression mirrored that of chromosomal MET16; however, the endogenous gene was found to be activated in response to amino acid starvation (general control). The latter mechanism was both GCN4 and CP1 dependent. MET25 was also found to be activated by GCN4, albeit weakly. More importantly, MET25 transcription was strongly CP1 dependent in gcn4 backgrounds. The modulation of MET gene expression by GCN4 can explain discrepancies in the literature regarding CP1 dependence of MET gene transcription. Lastly, micrococcal nuclease digestion and indirect end labeling were used to analyze the chromatin structure of the MET16 locus in wild-type and cep1 cells. The results indicated that CP1 plays no major role in configuring chromatin structure in this region, although localized CP1-specific differences in nuclease sensitivity were detected.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Metionina/biosíntesis , Oxidorreductasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transcripción Genética , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Cromatina/ultraestructura , Cromosomas Fúngicos/genética , Cisteína Sintasa/biosíntesis , Cisteína Sintasa/genética , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Datos de Secuencia Molecular , Mutación , Oxidorreductasas/biosíntesis , Regiones Promotoras Genéticas/genética , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/enzimologíaRESUMEN
In order to isolate new mutations impairing transcriptional regulation of sulfur metabolism in Saccharomyces cerevisiae, we used a potent genetic screen based on a gene fusion expressing XylE (from Pseudomonas putida) under the control of the promoter region of MET25. This selection yielded strains mutated in various different genes. We describe in this paper the properties of one of them, MET27. Mutation or disruption of MET27 leads to a methionine requirement and affects S-adenosylmethionine (AdoMet)-mediated transcriptional control of genes involved in sulfur metabolism. The cloning and sequencing of MET27 showed that it is identical to VPS33. Disruptions or mutations of gene VPS33 are well known to impair the biogenesis and inheritance of the vacuolar compartment. However, the methionine requirement of vps33 mutants has not been reported previously. We show here, moreover, that other vps mutants of class C (no apparent vacuoles) also require methionine for growth. Northern blotting experiments revealed that the met27-1 mutation delayed derepression of the transcription of genes involved in sulfur metabolism. By contrast, this delay was not observed in a met27 disrupted strain. Physiological and morphological analyses of met27-1 and met27 disrupted strains showed that these results could be explained by alterations in the ability of the vacuole to transport or store AdoMet, the physiological effector of the transcriptional regulation of sulfur metabolism.
Asunto(s)
Aminoácidos Sulfúricos/biosíntesis , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Saccharomyces cerevisiae/metabolismo , Azufre/metabolismo , Vacuolas/fisiología , Aminoácidos Sulfúricos/genética , Clonación Molecular , Eliminación de Gen , Homeostasis , Metionina/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/genética , Transactivadores/genética , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Cistationina betasintasa/genética , Cistationina gamma-Liasa/genética , Cisteína/biosíntesis , Saccharomyces cerevisiae/enzimología , Alelos , Animales , Secuencia de Bases , Southern Blotting , Catálisis , Clonación Molecular , Cistationina betasintasa/metabolismo , Cistationina gamma-Liasa/metabolismo , ADN de Hongos , Genes Fúngicos , Datos de Secuencia Molecular , Fenotipo , Ratas , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Azufre/metabolismoRESUMEN
The progressive salinization of irrigated land poses a threat to the future of agriculture in arid regions. The identification of crucial metabolic steps in salt tolerance is important for the understanding of stress physiology and may provide the tools for its genetic engineering. In the yeast Saccharomyces cerevisiae we have isolated a gene, HAL2, which upon increase in gene dosage improves growth under NaCl and LiCl stresses. The HAL2 protein is homologous to inositol phosphatases, enzymes known to be inhibited by lithium salts. Complementation analysis demonstrated that HAL2 is identical to MET22, a gene involved in methionine biosynthesis. Accordingly, methionine supplementation improves the tolerance of yeast to NaCl and LiCl. These results demonstrate an unsuspected interplay between methionine biosynthesis and salt tolerance.
Asunto(s)
Adaptación Fisiológica , Cloruros/farmacología , Proteínas Fúngicas/genética , Litio/farmacología , Metionina/biosíntesis , Nucleotidasas/genética , Monoéster Fosfórico Hidrolasas/genética , Saccharomyces cerevisiae/fisiología , Cloruro de Sodio/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , ADN de Hongos , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Cloruro de Litio , Datos de Secuencia Molecular , Nucleotidasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The Saccharomyces cerevisiae HOM6 gene, encoding homoserine dehydrogenase (EC 1.1.1.3) was cloned and its nucleotide sequence determined. The yeast homoserine dehydrogenase shows extensive homology to the homoserine dehydrogenase domains of the two aspartokinase-homoserine dehydrogenases from Escherichia coli as well as to the homoserine dehydrogenases from Gram positive bacteria. Sequence alignment reveals that the yeast enzyme is the smallest homoserine dehydrogenase known, owing to the absence of a C-terminal domain endowed with the L-threonine allosteric response in Gram positive bacteria. Accordingly, the S. cerevisiae enzyme appears to be a naturally occurring feedback resistant homoserine dehydrogenase. Our results indicate that homoserine dehydrogenase was originally an unregulated enzyme and that feedback control acquisition occurred twice during evolution after the divergence between Gram positive and Gram negative bacteria.
Asunto(s)
Evolución Biológica , Escherichia coli/genética , Genes Fúngicos , Homoserina Deshidrogenasa/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Escherichia coli/enzimología , Genes Bacterianos , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
The assimilation of sulphate in Saccharomyces cerevisiae, comprising the reduction of sulphate to sulphide and the incorporation of the sulphur atom into a four-carbon chain, requires the integrity of 13 different genes. To date, the functions of nine of these genes are still not clearly established. A set of strains, each bearing a mutation in one MET gene, was studied. Phenotypic studies and enzyme determinations showed that the products of at least five genes are needed for the synthesis of an enzymically active sulphite reductase. These genes are MET1, MET5, MET8, MET10 and MET20. Wild-type strains of S. cerevisiae can use organic metabolites such as homocysteine, cysteine, methionine and S-adenosylmethionine as sulphur sources. They are also able to use inorganic sulphur sources such as sulphate, sulphite, sulphide or thiosulphate. Here we show that both of the two sulphur atoms of thiosulphate are used by S. cerevisiae. Thiosulphate is cleaved into sulphite and sulphide prior to utilization by the sulphate assimilation pathway, as the metabolism of one sulphur atom from thiosulphate requires the presence of an active sulphite reductase.
Asunto(s)
Saccharomyces cerevisiae/metabolismo , Sulfatos/metabolismo , Cisteína/metabolismo , Genes Fúngicos , Homocisteína/metabolismo , Metionina/metabolismo , Mutación , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Sulfuros/metabolismo , Sulfitos/análisis , Sulfitos/metabolismo , Tiosulfatos/metabolismoRESUMEN
Inactivation of the centromere-binding factor 1 (CBF1) gene results in yeast strains that require methionine for growth. This auxotrophy is due to the inability of such strains to concentrate and assimilate sulfate from the medium. Northern (RNA) blot experiments reveal that the CBF1 protein is required for full induction of MET25 and MET16 gene transcription. However, we show that induction of the sulfate assimilation pathway is not achieved solely by CBF1. This induction also requires the integrity of a positive trans-acting factor, encoded by the MET4 gene. The MET4 gene was cloned, and its sequence reveals that it encodes a protein related to the family of the bZIP transcriptional activators. Evidence that MET4 is a transcriptional activator was provided by demonstrating that DNA-bound LexA-MET4 fusion proteins stimulate expression of a nearby promoter. The use of LexA-MET4 fusion proteins also reveals that the leucine zipper of MET4 is required for the recognition of the MET25 promoter. Moreover, an 18-bp fragment of the MET25 5' upstream region was found to confer S-adenosylmethionine-dependent regulation of a fusion gene. This regulation was shown to depend on both MET4 and CBF1. The obtained results suggest that the binding of CBF1 to its cognate sequences increases the ability of MET4 to stimulate transcription of the MET genes.