In eukaryotic cells, intracellular transport between distinct membrane-bound compartments proceeds through dissociated carriers that bud from one membrane and then fuse selectively with another. This proposal will focus on the molecules and conserved mechanisms that catalyze vesicular transport between the endoplasmic reticulum (ER) and Golgi complex in Saccharomyces cerevisiae. Genetic approaches have identified essential components required for this transport pathway. However, many of the molecular mechanisms underlying this process remain obscure. Our studies combine molecular genetic approaches with in vitro assays that measure protein transport from the ER to early Golgi compartments. This transport reaction proceeds through the biochemically resolvable stages of COPII-dependent cargo selection and vesicle budding, Usolp-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 research program is to elucidate catalytic mechanisms underlying these events though analysis of stage-specific assays and reconstitution experiments with defined protein and lipid fractions. The objectives of this proposal are to: dissect the mechanism by which the sorting adaptor Erv26p functions with the COPII coat to sort secretory proteins into ER-derived vesicles; determine the role of the integral membrane Yip1/Yif1/Yos1 complex in vesicle budding from the ER; investigate the role of specific lipid species in COPII vesicle formation; and analyze the fate of ER derived vesicles in homotypic and heterotypic membrane fusion reactions using newly established SNARE protein cysteine-disulfide cross-linking assays. It is estimated that 20-30% of cellular proteins enter the secretory pathway. Our experimental aims are designed to address fundamental questions on how secretory proteins are selectively exported from the ER and directed to the Golgi complex. This intracellular transport step is essential for virtually all cell growth and function. Therefore, the proposed studies are basic for understanding numerous health related issues and are specifically relevant to understanding cholesterol regulation, Alzheimer's disease and cystic fibrosis. ? ? ?
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