RESUMEN
We recently showed that transport of ergosterol from the ER (endoplasmic reticulum) to the sterol-enriched PM (plasma membrane) in yeast occurs by a non-vesicular (Sec18p-independent) mechanism that results in the equilibration of sterol pools in the two organelles [Baumann, Sullivan, Ohvo-Rekilä, Simonot, Pottekat, Klaassen, Beh and Menon (2005) Biochemistry 44, 5816-5826]. To explore how this occurs, we tested the role of proteins that might act as sterol transporters. We chose to study oxysterol-binding protein homologues (Osh proteins), a family of seven proteins in yeast, all of which contain a putative sterol-binding pocket. Recent structural analyses of one of the Osh proteins [Im, Raychaudhuri, Prinz and Hurley (2005) Nature (London) 437, 154-158] suggested a possible transport cycle in which Osh proteins could act to equilibrate ER and PM pools of sterol. Our results indicate that the transport of newly synthesized ergosterol from the ER to the PM in an OSH deletion mutant lacking all seven Osh proteins is slowed only 5-fold relative to the isogenic wild-type strain. Our results suggest that the Osh proteins are not sterol transporters themselves, but affect sterol transport in vivo indirectly by affecting the ability of the PM to sequester sterols.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteroles/metabolismo , Transporte Biológico Activo , Membrana Celular/química , Retículo Endoplásmico/química , Saccharomyces cerevisiae/química , Esteroles/químicaRESUMEN
The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis. To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants. All 127 combinations and permutations of OSH deletion alleles were constructed. Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal. Thus, the family members shared an essential function. In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis. Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects. Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype. Therefore, each gene performed distinct nonessential functions and contributed to a common essential function. Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels.
Asunto(s)
Receptores de Esteroides/genética , Receptores de Esteroides/fisiología , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , Supervivencia Celular , Clonación Molecular , Ergosterol/biosíntesis , Eliminación de Gen , Genes Reporteros , Genotipo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Fenotipo , Plásmidos/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Esteroles/metabolismo , Factores de TiempoRESUMEN
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
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
The Saccharomyces cerevisiae gene ERD2 is responsible for the retrieval of lumenal resident proteins of the endoplasmic reticulum (ER) lost to the next secretory compartment. Previous studies have suggested that the retrieval of proteins by ERD2 is not essential. Here, we find that ERD2-mediated retrieval is not an essential process only because, on its failure, a second inducible system acts to maintain levels of ER proteins. The second system is controlled by the ER membrane-bound kinase encoded by IRE1. We conclude that IRE1 and ERD2 together maintain normal concentrations of resident proteins within the ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores de Péptidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Compartimento Celular , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos , Genes Letales , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Immunoblotting , Glicoproteínas de Membrana/genética , Mutagénesis , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy.
Asunto(s)
Núcleo Celular/fisiología , Genes Fúngicos , Fusión de Membrana , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Núcleo Celular/ultraestructura , Cruzamientos Genéticos , Diploidia , Técnica del Anticuerpo Fluorescente , Prueba de Complementación Genética , Genotipo , Mutagénesis , Mutagénesis Insercional , Membrana Nuclear/fisiología , Membrana Nuclear/ultraestructura , Saccharomyces cerevisiae/ultraestructuraRESUMEN
We describe an acid phosphatase enzyme (EC 3.1.3.2) that is localized to the intestine of the nematode Caenorhabditis elegans and that should serve as a convenient biochemical marker for gut differentiation. In adult worms, acid phosphatase activity is located along the edge of the gut lumen in the vicinity of the intestinal brush border. All but the anterior six cells of the intestine stain for phosphatase activity; the nonstaining cells all descend from the Ea(l/r)(a/p)a cells. Acid phosphatase activity is low in oocytes and early embryos but increases substantially when embryos reach late morphogenesis stage; this increase corresponds to the appearance of a major band of acid phosphatase activity detectable on isoelectric focusing gels. We designate this band as the product of the pho-1 gene. The pattern of acid phosphatase expression in several embryonic mutants suggests that pho-1 expression in the developing intestine is lineage autonomous. We induced an isoelectric focusing variant in the pho-1 enzyme and used this to map the pho-1 locus about 1.5 map units to the left of center of chromosome II. We purified the pho-1 enzyme to homogeneity (6500-fold purification; 4% recovery of activity); the pho-1 acid phosphatase is a homodimeric glycoprotein with a subunit molecular weight of 55,000 Da. This paper establishes a new experimental system with which to investigate the molecular basis of lineage-specific gene expression during C. elegans development.