RESUMEN
The earliest known step in yeast spindle pole body (SPB) duplication requires Cdc31p and Kar1p, two physically interacting SPB components, and Dsk2p and Rad23p, a pair of ubiquitin-like proteins. Components of the PKC1 pathway were found to interact with these SPB duplication genes in two independent genetic screens. Initially, SLG1 and PKC1 were obtained as high-copy suppressors of dsk2Delta rad23Delta and a mutation in MPK1 was synthetically lethal with kar1-Delta17. Subsequently, we demonstrated extensive genetic interactions between the PKC1 pathway and the SPB duplication mutants that affect Cdc31p function. The genetic interactions are unlikely to be related to the cell-wall integrity function of the PKC1 pathway because the SPB mutants did not exhibit cell-wall defects. Overexpression of multiple PKC1 pathway components suppressed the G2/M arrest of the SPB duplication mutants and mutations in MPK1 exacerbated the cell cycle arrest of kar1-Delta17, suggesting a role for the PKC1 pathway in SPB duplication. We also found that mutations in SPC110, which encodes a major SPB component, showed genetic interactions with both CDC31 and the PKC1 pathway. In support of the model that the PKC1 pathway regulates SPB duplication, one of the phosphorylated forms of Spc110p was absent in pkc1 and mpk1Delta mutants.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Proteínas Quinasas Activadas por Mitógenos , Proteína Quinasa C , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiología , Huso Acromático/genética , Western Blotting , Proteínas de Unión a Calmodulina , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/metabolismo , Fase G2 , Regulación Fúngica de la Expresión Génica , Prueba de Complementación Genética , Proteínas de la Membrana/genética , Mitosis , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Fosforilación , Saccharomyces cerevisiae/genética , Temperatura , Factores de Tiempo , Ubiquitinas/genéticaRESUMEN
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.