The overall goal of this project is to elucidate the mechanism of transport vesicle budding and targeting. We will take advantage of the very recent purification of Golgi-derived (""""""""non-clathrin"""""""") coated vesicles in combination with existing cell-free systems to offer molecular insights into the mechanisms of vesicular movement between membrane-bound compartments. These coated vesicles, which transport proteins between the cisternae of the Golgi stack, can be accumulated during cell-free incubations of isolated Golgi membranes, and then purified by density gradient centrifugation following salt extraction. Sufficient quantities can be isolated to enable the individual polypeptides to be characterized by microsequencing, antibodies to be produced, and the genes cloned. Many of these proteins should be needed for vesicle budding and targeting. Antibodies prepared against them will be used as reagents to probe for function in cell-free systems. Specifically, we plan to purify Golgi-derived coated vesicles in bulk as source material for microsequencing. Anti-peptide and then polyclonal antibodies will be produced based on this microsequence information. We will use these antibodies to localize the coated vesicle polypeptides in coated vesicles fractions and in cells at the electron microscope level. Probes based on the amino acid sequences will be used to clone the genes encoding the major polypeptides. We hope to elucidate the physical arrangement of the major polypeptides in membranes and in coats. A number of functional studies are planned in addition to these structural studies. These include determining which subunits of coated vesicles originate from cytosol and which from Golgi membranes, and whether any coated vesicle component are fatty acylated during budding, since fatty acyl-CoA is required for the process. Antibodies to individual polypeptide components will be employed as specific inhibitors with which to ascertain the function preformed by each component. We hope to purify cytosol-derived components from cytosol in their native, transport-competent forms, and to use these as reagents to help develop simplified assays (partial reactions) corresponding to sub-steps in vesicle budding and targeting.
