Membrane fission is required to dissociate cargo filled transport carriers from the maternal compartment. We are specifically interested in the fission of transport carriers of the trans Golgi network (TGN) to the cell surface pathway. Our working hypothesis is that a special class of proteins are recruited to modulate lipid composition of TGN to generate a localized and transient fission activity. We have found that trimeric G protein subunits GB-gamma generate diacylglycerol (DAG) in the TGN. DAG activates the TGN bound protein kinase Ceta and recruits protein kinase D (PKD). PKCeta phosphorylates to activate PKD. Inactivation of these components causes accumulation of cell surface destined cargo in large tubules attached to the TGN. Their overactivation, on the other hand, vesiculates the TGN. These components fit the criteria expected of components involved in membrane fission. Our new data suggests that GB-gamma dependent DAG production in the TGN is through activation and recruitment of phospholipase (33,(aim#1). DAG generated as a result is metabolized, post-fission, by PKD dependent activation of diacylglycerol kinase(DGK)0 (aim#2). ln addition, our results have revealed a PKD binding protein (called yusukin), which we propose regulates the timing of PKD dependent activation of DGK0. This essentially prevents premature consumption of DAG, which would inhibit fission in the midst of transport carrier formation. The first 3 aims of this proposal describe experiments that will strengthen the proposed involvement of PLCB3, yusukin and DGK0 in PKD dependent fission of TGN to cell surface transport carriers. We have screened the drosophila genome by SiRNA and identified 130 transport components.
Our aim (#4) is to identify from this pool, those specifically involved in PKD dependent membrane fission in mammalian cells.
Aim #5 describes experiments to reconstitute membrane fission in vitro using purified components and rat liver Golgi membranes. Biochemical and morphological procedures will be used to monitor sequential recruitment of proteins, and generation of modified lipids such as DAG and phosphatidylinositol-4-phosphate (PIP) in events leading to membrane fission. Our findings will reveal the mechanism by which important receptors for growth factors, hormones and neuropeptides are transported to the cell surface. Inappropriate delivery of these components is 1 of the major causes of events leading to defects in cell growth and differentiation. ? ? ?
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