We propose to study the process of formation of the carrier vesicles that mediate the transport of proteins from the trans Golgi network (TGN) to the cell surface of epithelial cells 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 will use a cell-free system we have developed that recreates in vitro the generation of post Golgi vesicles from a purified Golgi fraction obtained from virus-infected MDCK cells. With this system, the formation of post Golgi vesicles can be effected in two sequential phases of: I) ArfGTP dependent coat assembly/bud formation and, 2) vesicle scission. The scission phase requires a PKC-like molecule in the TGN membrane, but not its phosphorylating activity, as well as several cytosolic proteins, including a membrane scission promoting activity (MSPA) present in a high molecular weight complex that contains an NEM-sensitive component, which we have tentatively identified as the phosphatidylinositol transfer protein (PITP). Employing a combined biochemical, genetic, and electron microscopic approach we will: l) Identify the coat and membrane components of TGN-derived vesicles by analysis of the coats of purified vesicles produced in the presence of GTPlambdaS, which prevents vesicle uncoating, and by assembling the vesicles with purified cytosolic subfractions containing coat subunits. 2) Purify and elucidate the regulation of a cytosolic NEM-sensitive membrane scission promoting factor (MSPA) that we have identified, which is required for vesicle generation and, when separated from other components, causes the uncontrolled vesiculation of uncoated TGN membranes. 3) Test a model in which vesicle scission is dependent on a regulatory mechanism that involves the action of a membrane-associated PKC-like molecule that, in concert with Arf-GTP, a cytosolic PITP that contributes PtdIns(4,5)P2, and possibly RhoA, leads to the activation of a phospholipase D (PLD), which, in TGN membranes, would be the final effector of vesicle scission. In these studies, we will identify the PKC-like molecule that we have implicated in vesicle generation and determine whether it interacts directly with and activates a Golgi associated PLD. We will also clarify the role of PITP and identify the phosphoinositides that it contributes to the scission process.
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