STRUCTURAL AND FUNCTIONAL ANALYSIS OF P115

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The long-term goal of the work described in this proposal is to further our understanding of the mechanism of vesicular transport in the eucaryotic cell. This is the process by which the cell specifically moves proteins and lipids from one cellular compartment to another. Understanding this complex, multistep process is important because it is crucial for the generation and maintenance of cellular compartments, and hence for cellular viability. The work will focus on functional and structural analyses of a novel transport factor, termed p115, which was recently isolated. p115 is a homo-oligomeric peripheral membrane protein that may act early in vesicular transport through the Golgi apparatus. The specific aims of this proposal are as follows. The point at which P115 acts during transport will be rigorously determined through kinetic analyses in conjunction with morphological analysis of Golgi stacks after transport with and without P115. A "vesicle budding assay" will be developed and used to directly assess p115's possible function in transport vesicle formation. Determination of whether P115 is required at multiple transport steps, besides cis to medial transport through the Golgi, will be undertaken via subcellular localization studies and by determining whether P115 is required in in vitro transport assays for several other transport steps. Putative proteins that interact with P115, both membrane associated and soluble, will be identified through biochemical and molecular genetic techniques, and purified if possible. A mammalian P115 cDNA will be cloned and sequenced. Efforts to identify a P115 homolog in Saccharomyces cerevisiae through biochemical, immunological, and molecular genetic techniques will be undertaken. In a yeast homolog exists, the gene will be cloned and sequenced, and used to initiate a genetic analysis of P115 function in yeast. Finally, P115 structure will be studied via electron microscopy and domain analysis through limited proteolysis. The results of studies of this new transport factor should help illuminate the molecular mechanisms that eucaryotic cells use to effect vesicular traffic.