Martin Gerard (Gerry) Waters - US grants

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.

The overall goal of the work proposed here is to further understanding of protein traffic through the secretory pathway of the eucaryotic cell. This process is crucial for the generation and maintenance of cellular compartment, and hence for viability. GTC (for Golgi Transport Complex) is a novel hetero-oligomeric protein complex that has recently been identified and purified based on its ability to facilitate transport through the Golgi in vitro. A cDNA fore one of the subunits was cloned, and used to generate recombinant protein for antibody production. Use of the antibody in subcellular fractionation and indirect immunofluorescence studies showed that GTC is localized to the Golgi. The specific aims of the work proposed here are designed to elucidate the role of GTC in the secretory pathway. To do this, a high yield GTC purification will be developed employing immunoblotting, rather than GTC activity, as a detection method. Peptide sequence data will be obtained from the subunits in order to clone cDNAs for each of the polypeptides. Recombinant GTC-subunits will be used to generate polyclonal and monoclonal anti-GTC antibodies for structural and functional analyses. The structural studies will entail a rigorous determination of GTC subunit stoichiometry, morphological examination by deep-etch electron microscopy, localization of the subunits within the complex, an attempt to generate functional subcomplexes, and nearest neighbor analyses by chemical cross-linking and perhaps two-hybrid studies. The intracellular localization of GTC will be analyzed in detail using subcellular fractionation, and confocal and immuno-gold microscopy. Potential residence within the Golgi-matrix or on COPI-coated vesicles will be examined biochemically GTC function will be analyzed in vivo, in cells over-expressing or under-expressing GTC subunits to determine at what stage of the early secretory pathway GTC functions. These results will be corroborated through use of semi-intact cell systems that monitor several early transport steps. In an effort to determine whether GTC affects vesicle budding, or membrane docking/fusion the potential GTC requirement in two in vitro assays will be examined; one reconstitutes only vesicle budding, and the other measures only membrane docking/fusion. Lastly, since GTC is tightly associated with the Golgi membrane, experiments to identify and purify a putative GTC receptor will be undertaken. These studies will provide the first insights into the role of this novel protein complex in the process of intracellular protein traffic.