This application is concerned with the nature of the carrier vesicles that mediate the transport of proteins from the trans-Golgi network (TGN) to the cell surface of epithelial cells. It proposes to study the process of their formation and to establish the mode of action of the specific proteins, lipids and cofactors that in the cytosol and TGN membranes play essential roles in this process. For these studies we use a cell free system that we developed that recreates in vitro the generation of post-Golgi vesicles from a purified Golgi fraction obtained from virus-infected MDCK cells that contains accumulated sialylated VSV-G protein molecules. For vesicle generation, the Golgi fraction is incubated with cytosolic proteins and a source of nucleoside triphosphates. The vesicles, when produced in the presence of GTPgammaS, contain a coatomer coat. They also contain rab11, which has been previously localized to the TGN and implicated in transport of VSV-G to the plasma membrane through the endosomal compartment The formation of post-Golgi vesicles can be effected in two sequential phases, a first one of Arf-dependent coat assembly/bud formation and a second one of membrane scission. We will identify the cytosolic proteins and lipid that play a key role in the scission of post-Golgi vesicles and determine their role in the process. These include the phospatidylinositol transfer protein (PITP), a scission factor of unknown protein composition that contains bourn lysophospatidic acid (LPA) and is likely to be a lysophosphatidic acid acyltransferase, (possibly BARS-50, the target of the Golgi tubulating agent, BFA), a fatty acid binding protein (FABP) that keeps the scission factor inactive and a postulated factor that restricts cleavage of coatomer-coated membranes to the necks of coated buds. We will test a model in which activated Arf sets the scission machinery in motion at the neck of a coated bud by dissociating the acyltransferase from the FABP that keeps it inactive. We will elucidate the possible role in scission of a phospholipase D and that of a PKC-like molecule that regulates it. We will also identify protein molecules in the coat and membranes of the TGN-derived that contribute to their targetting to an acceptor compartment and to identify the nature of that compartment. Since the vesicles contain rab11, we will determine whether the vesicle can dock and fuse with endosomes.
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