RESUMEN
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
Asunto(s)
Núcleo Celular/genética , Proteínas Fúngicas/genética , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alelos , Transporte Biológico , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Dosificación de Gen , Regulación Fúngica de la Expresión Génica , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Fusión de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Chaperonas Moleculares , Mutación , Membrana Nuclear/genética , Proteínas Nucleares/metabolismo , Canales de Translocación SEC , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Supresión GenéticaRESUMEN
Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote. To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2. First, we examined the complete array of single and double mutants. In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants. The exception was rvs161Delta fus2Delta, suggesting that Rvs161p and Fus2p act in concert. Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p. Second, electron microscopic analysis was used to define the mutant defects in cell fusion. In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone. The vesicles were associated with regions of cell wall thinning. Analysis of Fus- zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering. In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning. These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Proteínas Fúngicas/fisiología , Proteínas de Unión al GTP , Proteínas de la Membrana/fisiología , Proteínas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Complejo del Señalosoma COP9 , Membrana Celular , Pared Celular , Proteínas del Citoesqueleto/genética , Proteínas Fúngicas/genética , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/genética , Mutagénesis , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologíaRESUMEN
FUS7 was previously identified by a mutation that causes a defect in cell fusion in a screen for bilateral mating defects. Here we show that FUS7 is allelic to RVS161/END6, a gene implicated in a variety of processes including viability after starvation, endocytosis, and actin cytoskeletal organization. Two lines of evidence indicate that RVS161/END6's endocytic function is not required for cell fusion. First, several other endocytic mutants showed no cell fusion defects. Second, we isolated five function-specific alleles of RVS161/FUS7 that were defective for endocytosis, but not mating, and three alleles that were defective for cell fusion but not endocytosis. The organization of the actin cytoskeleton was normal in the cell fusion mutants, indicating that Rvs161p's function in cell fusion is independent of actin organization. The three to fourfold induction of RVS161 by mating pheromone and the localization of Rvs161p-GFP to the cell fusion zone suggested that Rvs161p plays a direct role in cell fusion. The phenotypes of double mutants, the coprecipitation of Rvs161p and Fus2p, and the fact that the stability of Fus2p was strongly dependent on Rvs161p's mating function lead to the conclusion that Rvs161p is required to interact with Fus2p for efficient cell fusion.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Actinas/metabolismo , Alelos , Proteínas del Citoesqueleto/genética , Endocitosis , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Eliminación de Gen , Expresión Génica , Factor de Apareamiento , Fusión de Membrana , Proteínas de la Membrana/genética , Péptidos/fisiología , Saccharomyces cerevisiae/metabolismoRESUMEN
KAR5 is required for membrane fusion during karyogamy, the process of nuclear fusion during yeast mating. To investigate the molecular mechanism of nuclear fusion, we cloned and characterized the KAR5 gene and its product. KAR5 is a nonessential gene, and deletion mutations produce a bilateral defect in the homotypic fusion of yeast nuclei. KAR5 encodes a novel protein that shares similarity with a protein in Schizosaccharomyces pombe that may play a similar role in nuclear fusion. Kar5p is induced as part of the pheromone response pathway, suggesting that this protein uniquely plays a specific role during mating in nuclear membrane fusion. Kar5p is a membrane protein with its soluble domain entirely contained within the lumen of the endoplasmic reticulum. In pheromone-treated cells, Kar5p was localized to the vicinity of the spindle pole body, the initial site of fusion between haploid nuclei during karyogamy. We propose that Kar5p is required for the completion of nuclear membrane fusion and may play a role in the organization of the membrane fusion complex.
Asunto(s)
Fusión de Membrana , Proteínas de la Membrana/genética , Membrana Nuclear/fisiología , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Compartimento Celular , Polaridad Celular , Clonación Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Factor de Apareamiento , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Péptidos/farmacología , Feromonas/farmacología , Estructura Secundaria de Proteína , Mapeo Restrictivo , Saccharomyces cerevisiae/efectos de los fármacos , Análisis de Secuencia de ADN , Huso Acromático/químicaRESUMEN
During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory. Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion. Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent. Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain. FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor. Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype. First, overexpression of a-factor in the fus mutants suppressed the Fus- defect. Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans. Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating. We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion.
Asunto(s)
Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Alelos , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Insulisina/genética , Metaloendopeptidasas , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación/fisiología , Fenotipo , Feromonas/genética , Feromonas/metabolismo , Plásmidos , Reproducción , Saccharomyces cerevisiae/ultraestructura , Transferasas/genética , Cigoto/metabolismoRESUMEN
The ilv-leu operon of Bacillus subtilis is regulated in part by transcription attenuation. The cis-acting elements required for regulation by leucine lie within a 683-bp fragment of DNA from the region upstream of ilvB, the first gene of the operon. This fragment contains the ilv-leu promoter and 482 bp of the ilv-leu leader region. Spontaneous mutations that lead to increased expression of the operon were shown to lie in an imperfect inverted repeat encoding the terminator stem within the leader region. Mutations within the inverted repeat of the terminator destroyed most of the leucine-mediated repression. The remaining leucine-mediated repression probably resulted from a decrease in transcription initiation. A systematic analysis of other deletions within the ilv-leu leader region identified a 40-bp region required for the derepression that occurred during leucine limitation. This region lies within a potential RNA stem-and-loop structure that is probably required for leucine-dependent control. Deletion analysis also suggested that alternate secondary structures proximal to the terminator are involved in allowing transcription to proceed beyond the terminator. Additional experiments suggested that attenuation of the ilv-leu operon is not dependent on coupling translation to transcription of the leader region. Our data support a model proposed by Grundy and Henkin (F. J. Grundy and T. M. Henkin, Cell 74:475-482, 1993) in which uncharged tRNA acts as a positive regulatory factor to increase gene expression during amino acid limitation.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Leucina/farmacología , Operón , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Bases , Sistema Libre de Células , Análisis Mutacional de ADN , Operón Lac , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia , Regiones Terminadoras Genéticas , Transcripción GenéticaRESUMEN
The persistence of Salmonella enteritidis temperature-sensitive (ts) mutants of different phenotypes in Peyer's patches (PP) and the spleen, and their immunogenicity after intragastric (i.g.) and peroral (p.o.) administration to mice was investigated. After p.o. administration the ts mutant C/2/2 colonized PP, but was not recovered from the spleen. After i.g. administration the ts mutant E/1/3 colonized both the spleen and PP for at least 2 weeks. Mutant C/2/2 persisted in PP up to 8 days but was not found in the spleen. Mutant H/2/26, although it poorly colonized the PP, was recovered from the spleen up to day 15 after i.g. administration. Immunization with E/1/3 by either the i.g. or the p.o. routes protected mice from challenge with 100 LD50 of the virulent wild-type (wt) strain. Immunization with either C/2/2 or H/2/26 did not confer protection. The three ts mutants induced the production of local IgA after i.g. administration regardless of their protective capacity.