The overall goal of this research is to develop a mechanistic understanding of TGN and vacuole membrane protein sorting and assembly in the yeast Saccharomyces crevisiae. Yeast has proved to be an excellent model system, both for identifying the proteins regulating membrane traffic in eukaryotic cells and for investigating the molecular mechanisms by which these proteins function. Genetic analyses have revealed that a yeast trans-Golgi network (TGN) membrane protein is retained by retrieval, and yielded a large collection of GRD genes involved in the retention and retrieval of yeast TGN membrane proteins. Using deletion and site-directed mutagenesis, the static retention signal on the yeast TGN membrane protein dipeptidyl aminopeptidase A (DPAPA) will be identified and characterized. The very large collection of Grd proteins that has been recently identified will be used to investigate the molecular basis for static retention in the TGN and retrieval of TGN membrane proteins from the prevacuolar compartment. Assays will be developed to determine functions for the individual Grd proteins in static retention or the retrieval step. Genetic analysis has also revealed a large collection of yeast genes encoding not only subunits of the yeast vacuolar proton-translocating ATPase (V-ATPase), but also three genes encoding proteins that are not subunits of the enzyme but instead are required for assembly of the V-ATPase. Using both genetic and biochemical approaches, the V- ATPase assembly factors will be characterized to determine whether they form a stable complex in the ER and whether this complex depends on the continued synthesis of substrates (the v-ATPase subunits) for the assembly reaction. The individual roles for the three V-ATPase assembly factors will be investigated by cross-linking, co- immunoprecipitation and density gradient centrifugation approaches to determine their mechanistic involvement in V-ATPase assembly. To develop a detailed molecular understanding of the assembly pathway of the V-ATPase complex, the kinetics and degree of association between the assembly factors and the V-ATPase integral membrane subunits will be monitored early in the assembly reaction. Additional functions for the assembly factors will be investigated such as escorting the V- ATPase into ER derived vesicles and chaperoning the complex to the cis-Golgi.
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