RESUMO
BOS1 and BET1 are required for transport from the ER to the Golgi complex in yeast and genetically interact with each other and a subset of the other genes, whose products function at this stage of the secretory pathway. In a previous study, we reported that BOS1 encodes a putative 27 kDa membrane protein. Here we show that BET1 is structurally similar to the synaptobrevins and identical to the SLY12 gene product. Overexpression of SLY12 compensates for the loss of function of the ras-like GTP-binding protein Ypt1. Both Bos1p and Bet1p are cytoplasmically oriented membrane proteins. Bos1p co-purifies with the ER to Golgi transport vesicles and co-fractionates with Bet1p and the ER membrane.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Genes Fúngicos , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Sequência de Bases , Proteínas de Transporte/isolamento & purificação , Centrifugação com Gradiente de Concentração , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Genes Supressores , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Proteínas Qb-SNARE , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificaçãoAssuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico/genética , Permeabilidade da Membrana Celular , Citosol , Fator de Acasalamento , Mutação , Biossíntese Peptídica , Peptídeos/farmacocinética , Biossíntese de Proteínas , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Esferoplastos , Frações SubcelularesRESUMO
We have used an in vitro assay that reconstitutes transport from the ER to the Golgi complex in yeast to identify a functional vesicular intermediate in transit to the Golgi apparatus. Permeabilized yeast cells, which serve as the donor in this assay, release a homogeneous population of vesicles that are biochemically distinct from the donor ER fraction. The isolated vesicles, containing a post-ER/pre-Golgi form of the marker protein pro-alpha-factor, were able to bind to and fuse with exogenously added Golgi membranes. The ability to isolate fusion competent vesicles provides direct evidence that ER to Golgi membrane transport is mediated by a discrete population of vesicular carriers.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Organelas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Citosol/metabolismo , Citosol/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Cinética , Fator de Acasalamento , Modelos Biológicos , Peso Molecular , Organelas/ultraestrutura , Peptídeos/genética , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Feromônios/genética , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/ultraestruturaRESUMO
Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin-binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I-talin and the 125I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment was about three-fold more effective at competing for 125I-85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin-talin interaction even though the talin-binding domain is localized in the 85 kDa fragment.
