In eukaryotic cell, intracellular transport between membrane-bound compartments is mediated by vesicles that bud from one membrane and fuse selectively with another. This proposal focuses on site-specific fusion of ER-derived transport vesicles with the Golgi complex in Saccharomyces cerevisiae. Although some of the essential components of this vesicle fusion event have been identified, the molecular details associated with this process remain obscure. Thus, the long-term goal of this investigation is to use a combined genetic and biochemical approach to identify and characterized all of the factors required for this vesicle fusion reaction/ A more complete understanding of the regulation and mechanisms that underlie intracellular vesicle fusion could assist in the prevention and treatment of several human diseases. In vitro synthesis of ER-derived transport vesicles has been reconstituted with yeast membranes and three purified protein fractions. Vesicles formed under these conditions are competent for fusion with the Golgi apparatus and are distinct from the donor membrane fraction. A novel procedure has been devised to obtain highly purified ER-derived transport vesicles in sufficient quantities to examine their molecular composition. The targeting and fusion of these purified vesicles requires both a crude cytosol and an acceptor membrane fraction. The availability of purified vesicles and of a functional fusion assay in a model genetic organism provides a unique opportunity to investigate intracellular membrane fusion.
The specific aims of this proposal are to characterize the vesicle proteins and soluble factors required for site-specific fusion of ER-derived vesicles with the Golgi apparatus. These purified vesicles contain a distinct set of polypeptides that are tightly associated with the membrane. Tom determine the roles of these membrane-associated proteins in specific steps of ER to Golgi transport, affinity-purified antibodies that neutralize the function of individual vesicle proteins will be used in the cell-free assay. Vesicle proteins implicated in targeting or fusion will be isolated, their polypeptide sequences determined and the encoding genes cloned. The isolation and characterization of soluble factors required for vesicle fusion with the Golgi complex will be achieved through the fractionation of the crude cytosol required to drive this cell-free fusion reaction. Initial efforts will focus on Ypt1p and Slyp, two proteins that are required for vesicle fusion and for which mutant alleles are available. Biochemical complementation assays that employ membranes and cytosol from ypt1 and sly1 mutant strains may provide a means of purification of these activities from a wild-type cytosol.
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