ANALYSIS OF GOLGI MEMBRANE PROTEIN LOCALIZATION IN YEAST

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The sorting and localization of proteins in the secretory and endosomal pathways will be investigated, using the model system of localization of proteins to the TGN in S. cerevisiae. The PI's working model proposes two independent mechanisms that both act to localize proteins to the TGN: selective retrieval from the prevacuolar compartment and a reduced rate of transport out to the TGN. Preliminary data suggest that retrieval involves the interaction of cargo proteins with Vps35p, a prevacuolar peripheral membrane protein, and this hypothesis will be tested in a genetic suppressor approach, as well as in biochemical binding assays. Slow exit from the TGN is proposed to involve the phosphorylation of cytosolic domains of resident TGN proteins, and this will be investigated using biochemical approaches to identify the specific sites of phosphorylation followed by specific site mutagenesis to alter these phosphorylation sites. Finally, several genes have been identified in genetic screens as defective in Golgi retention of proteins, and four of these will be investigated in further detail: grd20, grd21, grd22 and grd23.