P230 IS ASSOCIATED WITH VESICLES BUDDING FROM THE TRANS-GOLGI NETWORK

Transport vesicle formation requires the association of cytosolic prot eins with the membrane, We have previously described a brefeldin-A sen sitive, hydrophilic protein (p230), containing a very high frequency o f heptad repeats, found in the cytosol and associated with Golgi membr anes, We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialy ltransferase as a compartment-specific marker, The role of G protein a ctivators on the binding of p230 to Golgi membranes and in vesicle bio genesis has been investigated, Treatment of streptolysin-O permeabilis ed HeLa cells with either GTP gamma S or AlF4- resulted in accumulatio n of p230 on Golgi membranes, Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesi cles, showed that p230 is predominantly localised to the cytoplasmic s urface of trans-Golgi network-derived budding structures and small coa ted vesicles, p230-labeled vesicles have a thin (similar to 10 nm) ele ctron dense cytoplasmic coat and could be readily distinguished from c lathrin-coated vesicles, Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be dis tinct molecules, appear to be localised on separate populations of ves icles budding from the trans-Golgi network, These results strongly sug gest the presence of distinct populations of non-clathrin coated vesic les derived from the trans-Golgi network, As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a spec ific population of non-clathrin coated vesicles.