In eukaryotic cells, transport between several membrane-bound organelles is mediated by vesicles that bud from one membrane and fuse selectively with another. This study focuses on molecules that catalyze site-specific fusion of transport vesicles derived from the endoplasmic reticulum (ER) with the Golgi complex in Saccharomyces cerevisiae. Many of the essential components of this fusion event have been identified through genetic approaches, however the molecular details of site-specific vesicle fusion remain obscure. The long-term goal of my investigation is to reconstitute this reaction with defined protein and lipid fractions for the elucidation of catalytic mechanisms. The underlying mechanisms of vesicle fusion appear to be fundamentally conserved. Therefore these studies are basic for illuminating endocrine and exocrine secretion as well as neurotransmission. Our studies use an in vitro assay that measures the fusion of ER- derived transport vesicles with the Golgi complex. We have reproduced this event with isolated membranes and purified soluble molecules. The reaction proceeds in two biochemically distinct steps; first, vesicles are """"""""tethered"""""""" to the acceptor, and second, a distinct set of proteins catalyze stable docking and bilayer fusion. The objectives of this study are as follows: First, develop methods to detergent solubilize ER-derived vesicles and reconstitute fusion competent proteoliposomes, thereby facilitating an enzymological analysis of membrane bound proteins. Second, determine the protein-protein interactions that functionally tether vesicles to the Golgi compartment. Third, establish reassociation assays with purified fusion factors and isolated membranes. Fourth, identify proteins contained on ER-derived transport vesicles and determine their roles in ER to Golgi transport. Combining these biochemical approaches with a model genetic organism provides my laboratory a unique opportunity to dissect the underlying mechanisms of vesicle fusion.
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