In eukaryotic cells, transport between several membrane-bound organelles is mediated by vesicles that bud from one membrane and fuse selectively with another. This proposal focuses on the molecules that catalyze vesicular transport between the endoplasmic reticulum (ER) and Golgi complex in Saccharomyces cerevisiae. Many essential components of this transport pathway have been identified though genetic and biochemical approaches; however the underlying molecular mechanisms remain unclear. Our studies combine molecular genetic approaches with in vitro assays that measure protein transport from the ER to the Golgi complex. This transport reaction proceeds through the biochemically distinct stages of COPII-dependent vesicle budding, Us1p-dependent vesicle tethering and SNARE protein-dependent membrane fusion. We have reproduced these stages with isolated membranes and purified soluble molecules. The long-term goal of my investigation is to reconstitute distinct sub-reactions in ER/Golgi transport with defined protein and lipid fractions for the elucidation of catalytic mechanisms. The objectives of this proposal are to investigate the mechanisms by which the COPII coat sorts specific secretory cargo into vesicles, determine the molecular contacts that functionally tether vesicles to acceptor membranes and devise new assays that monitor sub-reactions in the membrane fusion stage. Many of these fundamental mechanisms in intracellular transport are conserved for multiple trafficking pathways within a species and from yeast to mammals. Therefore, these studies are basic for illuminating endocrine and exocrine secretion and will be of broad importance to medicine.
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