RESUMEN
Our aim in this work was to further characterize the complexity and specificity of the three different isoforms (Tpk1, Tpk2 and Tpk3) of the catalytic and regulatory (Bcy1) subunits of PKA in Saccharomyces cerevisiae. We thus analyzed the subcellular localization of the PKA subunits in living cells by using strains carrying GFP (green fluorescent protein) fused to each PKA subunit. During exponential growth on glucose, both Bcy1 and Tpk2 localized in the nucleus, whereas Tpk1 and Tpk3 showed a mixed pattern of nucleo-cytoplasmic localization. During exponential growth on glycerol and during stationary phase, the PKA subunits showed mostly cytoplasmic localization, with the peculiarity that Tpk2 and Tpk3 but not Bcy1, were found associated to P-bodies and EGP bodies. Tpk3 was accumulated into P-bodies during glucose deprivation and hyper osmotic stress. Deletion of Tpk3 altered the kinetics of P-body formation. Analysis of protein expression showed that the relative expression pattern of each Tpk changes from low levels under fermentative metabolism to higher levels during stationary phase. The increase in Tpk levels produced an imbalance with Bcy1 levels. Our data suggest that the signaling specificity through PKA in yeast could be mediated by a particular subcellular localization of each isoform of Tpk.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Gránulos Citoplasmáticos/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/enzimología , Medios de Cultivo/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Citoplasma/enzimología , Gránulos Citoplasmáticos/enzimología , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimologíaRESUMEN
In the yeast Saccharomyces cerevisiae, the zinc finger transcription factor Msn2p is a central component of the general stress response. It is activated in response to a wide variety of environmental changes, including physicochemical stresses as well as nutritional starvation, and induces the expression of a large set of genes required for cellular adaptation. The transcriptional activity of Msn2p in response to stresses is transient, and must therefore be strictly controlled. It is mainly regulated by reversible translocation from the cytoplasm to the nucleus upon the onset of stress, under the control of the cAMP-APK and the TOR pathways. In this report, we describe a new level of control: heat shock-induced degradation of Msn2p by the 26S proteasome. This degradation occurs in the nucleus and is further enhanced when Msn2p is fully active. Moreover, we show that the cyclin-dependent protein kinase Srb10p, a component of the transcription machinery, plays a role in the enhanced degradation of Msn2p upon heat shock. These findings provide new insights into the mechanisms by which Msn2p is transiently activated in response to stress.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Respuesta al Choque Térmico , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN , Hidrólisis , Proteínas de Saccharomyces cerevisiaeRESUMEN
DNA-lacZ fusion libraries of yeast Saccharomyces cerevisiae were used to select genes coordinately regulated by the Ras-cAMP-cAPK signalling pathway. Sixteen new genes (AGP1, APE2, APE3, FPS1, GUT2, MDH2, PLB2, PYK2, RNR3, SUR1, UGA1, YHR033w, YBR006w, YHR143w, YMR086w and YOR173w) were found to be repressed by cAMP. Most of these genes encode for metabolic enzymes and are induced by nutritional limitations. These common properties suggest a role of this pathway in the metabolic adjustment of the cell to nutritional variations. The induction of 10 of these genes is reduced in the msn2,msn4 double mutant, which emphasizes the role of the Msn2/4p transcriptional activators in mediating the Ras-cAMP-cAPK signalling pathway. The Msn2p/Msn4p-independent expression of the six other genes suggests the existence of other regulatory systems under the control of this pathway.
Asunto(s)
AMP Cíclico/fisiología , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Selección Genética , Northern Blotting , AMP Cíclico/genética , Cartilla de ADN/química , ADN de Hongos/química , Biblioteca de Genes , Glucosa/análisis , Glucógeno/análisis , Operón Lac , Reacción en Cadena de la Polimerasa , ARN de Hongos/química , ARN de Hongos/aislamiento & purificación , Saccharomyces cerevisiae/metabolismo , Análisis de Secuencia de ADN , Transducción de Señal , Transformación Genética , Trehalosa/análisis , beta-Galactosidasa/análisisRESUMEN
The heat shock transcription factor Hsf1p and the stress-responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild-type strains using [35S]-methionine labelling and two-dimensional gel electrophoresis. Among 52 proteins induced by a shift from 25 degrees C to 38 degrees C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p. The two sets of proteins overlapped only slightly. Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s). The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon. Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Calor , Regulón , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/genética , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Electroforesis en Gel Bidimensional , Metionina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas ras/metabolismoRESUMEN
The multicopy suppressors of the snf1 defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive cis element (CCCCT) in response to various stresses. This cis element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the msn2 msn4 double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition. Thirty-nine gene products (including those of ALD3, GDH3, GLK1, GPP2, HSP104, HXK1, PGM2, SOD2, SSA3, SSA4, TKL2, TPS1, and YBR149W) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including ACH1, ADH2, ALD6, ATP2, GPD1, ICL1, and KGD2). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.
Asunto(s)
AMP Cíclico/farmacología , Proteínas de Unión al ADN/fisiología , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Factores de Transcripción/fisiología , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Mutación , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
The MBR1 gene was isolated as a multicopy suppressor of the phenotype on glycerol medium of a Saccharomyces cerevisiae strain mutant for the Hap2/3/4/5 transactivator complex. In this paper, we show that Mbr1p is a limiting factor for growth on glycerol medium under the following sub-optimal culture conditions: in late growth phase, at low temperature, at high external pH or in the presence of 1,10-phenanthroline. Moreover, deletion of MBR1 protects cells against stress, whilst overexpression of this gene has the opposite effect. MBR1 expression is induced in the late growth phase and is negatively controlled by the cAMP-dependent protein kinase A (PKA). Both activation of PKA or overexpression of SOK1 or SCH9-two genes isolated as multicopy suppressors of a PKA null mutant-suppress the mbr1 growth defect. Our results indicate that Mbr1p is not an essential element of any one of these pathways. Deletion of SAC1, a gene implicated in vesicular transport, in association with MBR1 deletion, causes synthetic lethality. A possible role of Mbr1p in intracellular trafficking is discussed.
Asunto(s)
Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Proteínas de la Membrana , Proteínas de Transporte de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Bacterianas/fisiología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas Fúngicas/fisiología , Eliminación de Gen , Fenotipo , Proteínas Quinasas/fisiología , Saccharomyces cerevisiae/crecimiento & desarrollo , Transducción de Señal , Activación TranscripcionalRESUMEN
The SDC25 gene of Saccharomyces cerevisiae is homologous to CDC25. Its 3' domain encodes a guanine nucleotide exchange factor (GEF) for Ras. Nevertheless, the GEF encoded by CDC24 is determinant for the Ras/cAMP pathway activation in growth. We demonstrate that the SDC25 gene product is a functional GEF for Ras: the complete SDC25 gene functionally replaces CDC25 when overexpressed or when transcribed under CDC25 transcriptional control at the CDC25 locus. Chimeric proteins between Sdc25p and Cdc25p are also functional GEFs for Ras. We also show that the two genes are differentially regulated: SDC25 is not transcribed at a detectable level in growth conditions when glucose is the carbon source. It is transcribed at the end of growth when nutrients are depleted and in cells grown on nonfermentable carbon sources. In contrast, CDC25 accumulation is slightly reduced when glucose is replaced by a nonfermentable carbon source.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , ras-GRF1 , Secuencia de Bases , Northern Blotting , Proteínas de Ciclo Celular/fisiología , Proteínas Fúngicas/fisiología , Proteínas de Unión al GTP/fisiología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Fenotipo , Factores de Transcripción/fisiología , Transcripción Genética , Proteínas de Unión al GTP rapRESUMEN
Two isofunctional ras genes are present in the yeast Saccharomyces cerevisiae. Albeit their targets differ between mammals and yeast, they have conserved their regulators. The study of their positive regulators, guanine nucleotide exchange factors, have provided routes to the discovery of their regulatory elements in mammals. Ras are signal transducing proteins involved in the activation of the adenylate cyclase in yeast. They are activated by Cdc25p which has been shown to contain a Guanine Exchange Factor domain (GEF). SDC25, a gene partially homologous to CDC25, also contains a GEF domain but seems to be under a different regulation. It has been used to demonstrate the first guanine exchange activity on ras in vitro and was shown to be active by gene transfer in mammalian cells. Both Cdc25p and Sdc25p are associated to membrane and contain SH3 domains which are supposed to bind still unidentified proteins. Cdc25p is an unstable protein which contains a cyclin destruction box. Therefore activating effect on ras could be regulated by its level of expression. We have contributed to the isolation of a mammalian CDC25 homolog and we are analysing by directed mutagenesis key positions for ras activation of the human homolog HGRF55. That was performed by complementation analysis of yeast mutants as well as by use of two hybrid system. These approaches led us to the discovery of residues involved in ras interaction.
Asunto(s)
Canales de Cloruro , Genes ras/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animales , Proteínas de Ciclo Celular/genética , AMP Cíclico/genética , Células Eucariotas/química , Proteínas de Unión al GTP/genética , Proteínas de la Membrana/genética , Fosfoproteínas Fosfatasas/genética , Transducción de Señal , Proteínas de Unión al GTP rap , ras-GRF1RESUMEN
Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Genes Fúngicos , Genes Sintéticos , Nucleótidos de Guanina/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas ras , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Fúngicas/química , Proteínas Fúngicas/clasificación , Proteínas Fúngicas/fisiología , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/fisiología , Factores de Intercambio de Guanina Nucleótido , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/fisiología , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rap , ras-GRF1RESUMEN
The CDC25 gene from S. cerevisiae encodes an activator of Ras proteins. The C-terminal part of a structurally-related protein encoded by the SDC25 gene is characterised by a Ras-guanine nucleotide exchange activity in vitro whereas the C-terminal part of CDC25 gives no detectable exchange activity. A chimera between the 3' regions of these two genes was constructed by homeologous recombination. This chimeric gene suppresses cdc25 mutations. When expressed in E. coli, the chimeric product is detectable by antibodies directed against the carboxy-terminal CDC25 peptide and has an exchange-factor activity on the Ras2 protein. Therefore, the carboxy-terminal parts of both the CDC25 and the SDC25 gene products are structurally and functionally similar. The CDC25 part of the chimeric protein contains an intrinsic guanine exchange factor which does not require an additional cofactor.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas ras , ras-GRF1 , Secuencia de Bases , Clonación Molecular , ADN , Escherichia coli , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Factores de Intercambio de Guanina Nucleótido , Datos de Secuencia Molecular , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Homología de Secuencia de Ácido Nucleico , Supresión Genética , Proteínas de Unión al GTP rap , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
The ccs1-1 mutation of Saccharomyces cerevisiae, which has been previously described, is associated with an increase in cytochrome content, in respiration, and in ATP synthesis. In addition, this mutation leads to the same phenotype as cells de-regulated in the cAMP pathway. From a yeast genomic library, we have isolated a DNA fragment in a recombinant plasmid pCD1 which complements the ccs1-1 mutation. Homologous integration of this DNA in the genome occurs at the CCS1 locus. An 11 kb of the DNA insert is necessary for complementation. Sequencing part of the fragment identifies CCS1 as the IRA2 gene. The IRA2 gene is known to encode an attenuator of RAS gene product activity which stimulates the GTPase activity of the RAS proteins. This result underlines the involvement of cAMP-dependent phosphorylation in mitochondrial function. We present the sequence of 1 kb DNA upstream of the putative ATG of the IRA2/CCS1 gene product which is devoid of an ORF and could contain several regulatory sites.
Asunto(s)
AMP Cíclico/metabolismo , Genes Fúngicos/genética , Mitocondrias/fisiología , Saccharomyces cerevisiae/genética , Secuencia de Bases , Clonación Molecular , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura AbiertaRESUMEN
In the cellular slime mould Dictyostelium discoideum the two enzymatic activities of the pyrimidine pathway, orotidine-5'-phosphate decarboxylase (EC 4.1.1.23; OMPdecase) and orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase), are encoded by a single gene (DdPYR5-6). As in higher eukaryotes the bifunctional enzyme is referred to as UMP synthase. Here we present a method that allows efficient generation and selection of mutants lacking UMP synthase. D. discoideum cells are transformed with either of two different types of plasmids. One plasmid type contains no sequences homologous to the UMP synthase gene whereas the other type contains at least parts of this gene. UMP synthase- mutants, which were positively selected for in the presence of 5-fluoroorotic acid (5-FOA), were obtained with both plasmids. However, mutation rates were at least one order of magnitude higher if plasmids containing various portions of the UMP synthase gene were used as opposed to plasmids that lack any homology to the UMP synthase locus. Several mutant strains were extensively characterized. These strains lack OMPdecase activity and exhibit in addition to 5-FOA resistance a ura- phenotype. All mutants carry UMP synthase loci with deletions of various extents but integration of transforming plasmids was not detected. This efficient generation of 5-FOA resistance is part of a proposed complex selection scheme which allows multiple rounds of transformation of D. discoideum.
Asunto(s)
Dictyostelium/genética , Complejos Multienzimáticos/genética , Mutación , Orotato Fosforribosiltransferasa/genética , Ácido Orótico/análogos & derivados , Orotidina-5'-Fosfato Descarboxilasa/genética , Southern Blotting , Dictyostelium/efectos de los fármacos , Dictyostelium/enzimología , Dictyostelium/aislamiento & purificación , Farmacorresistencia Microbiana/genética , Genes Fúngicos , Genotipo , Cinética , Complejos Multienzimáticos/metabolismo , Orotato Fosforribosiltransferasa/metabolismo , Ácido Orótico/farmacología , Orotidina-5'-Fosfato Descarboxilasa/metabolismo , Fenotipo , Plásmidos , Mapeo Restrictivo , Transformación GenéticaRESUMEN
The evidence presented here indicates that the domain containing the COOH-terminal part of the Saccharomyces cerevisiae SCD25 gene product (C-domain), which is homologous to the COOH-terminal part of CDC25 protein, can elicit activation of mammalian ras proteins in CHO cells. Transfection of expression vectors carrying the C-domain of SCD25, but not of CDC25, promotes the GTP-bound form of ras proteins as determined by analysis of the guanine nucleotides bound to ras proteins immunoprecipitated by Y13-259 mAb, and enhances transcription of a HIV-LTR-CAT construct. This is the first demonstration of the activation of ras proteins by transfection of a single heterologous gene.
Asunto(s)
Genes Fúngicos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Saccharomyces cerevisiae/genética , Animales , Línea Celular , Cricetinae , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , TransfecciónRESUMEN
In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas ras , ras-GRF1 , Secuencia de Bases , Northern Blotting , Clonación Molecular , Análisis Mutacional de ADN , ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica , Genes Supresores , Datos de Secuencia Molecular , Fenotipo , ARN de Hongos/genética , ARN Mensajero/genética , Mapeo Restrictivo , Proteínas de Unión al GTP rapRESUMEN
In the yeast Saccharomyces cerevisiae, addition of glucose to cells grown under glucose-derepressed conditions induces a transient rise in the intracellular level of cAMP. This modulation requires functional elements of the cAMP-producing pathway, adenylate cyclase, ras proteins and the product of CDC25 gene. To determine whether or not the CDC25 gene product is a transducing element in the signal-transmission pathway leading from glucose to ras adenylate cyclase we have made use of the mutated allele RAS2Ile152 whose gene product uncouples the product of CDC25 from adenylate cyclase, but does not promotes other secondary phenotypes. The transient increase in cAMP is lost in cells lacking a functional CDC25 gene product, although they produce a normal amount of cAMP with the RAS2Ile152 gene. This result demonstrates the requirement of CDC25 for mediation of glucose signal transmission. The fact that cells grow normally on glucose in the absence of glucose-induced cAMP signaling confirms that this signaling pathway is not essential for growth on glucose. To further analyze the role of the CDC25 gene product we have made use of truncated versions of the gene. The results show that the C-terminal part of the gene alone is able to mediate glucose-induced activation of the RAS adenylate cyclase pathway.
Asunto(s)
Proteínas de Ciclo Celular , AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Glucosa/farmacología , Saccharomyces cerevisiae/fisiología , Transducción de Señal , ras-GRF1 , Adenilil Ciclasas/metabolismo , Genotipo , Cinética , Mapeo Restrictivo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Transducción de Señal/efectos de los fármacosRESUMEN
In Saccharomyces cerevisiae, the product of the CDC25 gene controls the RAS-mediated production of adenosine 3',5'-monophosphate (cAMP). In vivo the carboxyl-terminal third of the CDC25 gene product is sufficient for the activation of adenylate cyclase. The 3'-terminal part of SCD25, a gene of S. cerevisiae structurally related to CDC25, can suppress the requirement for CDC25. Partially purified preparations of the carboxy-terminal domain of the SCD25 gene product enhanced the exchange rate of guanosine diphosphate (GDP) to guanosine triphosphate (GTP) of pure RAS2 protein by stimulating the release of GDP. This protein fragment had a similar effect on the human c-H-ras-encoded p21 protein. Thus, the SCD25 carboxyl-terminal domain can enhance the regeneration of the active form of RAS proteins.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacología , Nucleótidos de Guanina/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Fragmentos de Péptidos/farmacología , Proteínas de Saccharomyces cerevisiae , Proteínas ras , ras-GRF1 , Escherichia coli/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Cinética , Plásmidos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/genética , TransfecciónRESUMEN
In Saccharomyces cerevisiae, the product of the CDC25 gene is required for progression in the cell division cycle. It is necessary for cAMP production. It has been suggested that the CDC25 gene product acts through Ras proteins. We report the cloning of a DNA fragment from a new gene able to suppress the thermosensitive phenotype of the cdc25-5 mutation. It is unable to suppress the defect of a mutant of the adenylate cyclase gene or of the ras1, ras2ts double mutant. This DNA fragment prevents the drop in cAMP level in cdc25-5 mutant cells shifted to restrictive temperature. The complementing part of this fragment contains a truncated open reading frame (ORF) corresponding to the 3' end of a gene we named SCD25. The 584-amino acid sequence deduced from this ORF shares 45% identity with the 592-aa C-terminal part of the CDC25 ORF which is sufficient for complementation of cdc25 mutations. Some of the common sequences between these two genes are also partially homologous with the amino acid sequence of LTE1, another gene of S. cerevisiae. The capacity of the SCD25 fragment to suppress a cdc25 mutation and its homology to the C-terminal part of the CDC25 led us to propose that the CDC25 and the SCD25 C-terminal fragments each encode a protein domain which is capable in itself to support a similar biochemical function.
Asunto(s)
Proteínas de Ciclo Celular , ADN de Hongos/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Supresión Genética , ras-GRF1 , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , AMP Cíclico/genética , AMP Cíclico/metabolismo , ADN de Hongos/aislamiento & purificación , ADN de Hongos/fisiología , Escherichia coli/genética , Datos de Secuencia Molecular , Fenotipo , Mapeo Restrictivo , Saccharomyces cerevisiae/crecimiento & desarrollo , Homología de Secuencia de Ácido Nucleico , Temperatura , Transformación GenéticaRESUMEN
Developmental variations in the expression of two genes of the de novo pyrimidine biosynthetic pathway have been examined in Dictyostelium discoideum. One gene, DdPYR4, encodes the dihydroorotate dehydrogenase (EC 1.3.3.1); the other, DdPYR5-6, encodes the UMP synthase which in D. discoideum is a bifunctional enzyme harboring both the orotate phosphoribosyl transferase activity (EC 2.4.2.10) and the OMP decarboxylase activity (EC 4.1.1.23). The relative amount of mRNA for both genes has been estimated by hybridization with the previously cloned DNAs and compared with the amount of actin mRNA. The level of both mRNAs is dramatically reduced after 4 h of development and remains at a low level later in development. In contrast to these variations, the specific activity of the enzymes encoded by these genes during development is similar to that measured during exponential growth. These results lead us to propose that DdPYR4 and DdPYR5-6 genes encode for relatively stable proteins and that their synthesis is reduced to maintain a constant level of enzymes in non-growing cells. This mode of regulation could apply to a large number of housekeeping genes.
Asunto(s)
Carboxiliasas/genética , Dictyostelium/genética , Dihidroorotato Oxidasa/genética , Regulación de la Expresión Génica , Genes Fúngicos , Orotato Fosforribosiltransferasa/genética , Orotidina-5'-Fosfato Descarboxilasa/genética , Oxidorreductasas/genética , Pentosiltransferasa/genética , Animales , Técnicas In Vitro , ARN Mensajero/análisisRESUMEN
We have identified a mutation called rcal (for rescue by cAMP) which allows adenylate cyclase-deficient mutants to divide in the presence of cAMP. We took advantage of this rcal mutation to study the effect of externally added cAMP on the onset of the resting state when cells are starved for ammonium. We measured the resistance of the cells to zymolyase treatment as a parameter of the resting state. We observed that the onset of the resting state is reversibly blocked by cAMP. This inhibitory effect of cAMP is discussed together with the cAMP control of the start. This leads us to propose a model in which the cAMP level, controlled by the availability of nutrients, should trigger the choice between the entry of the cell into the resting state and the initiation of a new division cycle.
Asunto(s)
Ciclo Celular , AMP Cíclico/fisiología , Compuestos de Amonio Cuaternario/metabolismo , Saccharomyces cerevisiae/citología , Alelos , Interfase , Mutación , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismoRESUMEN
The cell division cycle of the yeast Saccharomyces cerevisiae is triggered at the stage called 'START'. Many results strongly suggest that adenylate cyclase is an essential element of the control of START. We report here results arguing for a positive control of the cAMP level by the CDC25 gene, another gene of START. Firstly, cdc25 cells can be rescued by extracellular cAMP. Secondly, the cellular cAMP content drops when thermosensitive cdc25 mutant cells are shifted to restrictive temperature. We report the molecular cloning of the CDC25 gene by complementation of cdc25 mutant cells. The identity of the cloned gene was confirmed by site-specific gene re-integration experiments and segregation analysis: the isolated fragment is shown to integrate into the cdc25 gene locus. When transferred in cdc25 mutant cells this DNA prevents the drop of the cAMP level at restrictive temperature. This gene is transcribed in a 5200-nucleotides mRNA. We have determined the nucleotide sequence of a 5548-bp DNA fragment which shows an uninterrupted open reading frame (ORF) coding for a 1587-amino acid polypeptide chain. Only the C-terminal part of the ORF appears to be essential for the complementation of the cdc25-5 allele, suggesting a multidomain protein.