The in vitro generation of post-Golgi vesicles carrying viral envelope glycoproteins requires an ARF-like GTP-binding protein and a protein kinase C associated with the Golgi apparatus

We have developed a system that recreates in vitro the generation of post-Golgi vesicles from an isolated Golgi fraction prepared from vesicular stomatitis virus- or influenza virus infected Madin-Darby canine kidney or HepG2 cells. In this system, vesicle generation is temperature and ATP-dependent and requires a supply of cytosolic proteins, including an N-ethylmaleimide-sensitive factor distinct from NSF, Cytosolic proteins obtained from yeast were as effective as mammalian cytosolic proteins in supporting vesicle formation and had the same requirements, The vesicles produced (50-80 nm in diameter) are depleted of the trans Golgi marker sialyltransferase, contain the viral glycoprotein molecules with their cytoplasmic tails exposed, and do not show an easily recognizable protein coat, Vesicle generation was inhibited by brefeldin A, which indicates that it requires the activation of an Arf-like GTP-binding protein that promotes assembly of a vesicle coat, Vesicles formed in the presence of the nonhydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate retained a nonclathrin protein coat resembling that of COP-coated vesicles, and sedimented more rapidly in a sucrose gradient than the uncoated ones generated in its absence, This indicates that GTP hydrolysis is not required for vesicle generation but that it is for vesicle uncoating, The activity of a Golgi-associated protein kinase C (PKC) was found to be necessary for the release of post-Golgi vesicles, as indicated by the capacity of a variety of inhibitors and antibodies to PKC to suppress it, as well as by the stimulatory effect of the PRC activator 12-O-tetradecanoylphorbol-13-acetate.