In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein

We describe an in vitro system in which post-Golgi vesicles containing meta bolically labeled, sialylated, vesicular stomatitis virus (VSV)G protein mo lecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isola ted Golgi membrane fraction. This fraction is prepared from VSV-infected Ma din-Darby canine kidney (MDCK) cells in which the S-35-labeled viral envelo pe glycoprotein was allowed to accumulate In the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this syst em are separated from the remnant Golgi membranes by differential centrifug ation or by velocity sedimentation in a sucrose gradient. Vesicle productio n, quantified as the percentage of labeled VSV-G released from the Golgi me mbranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation fac tor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinosit ol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTP gamma S or GMP-PNP, retain a coatomer coat visible in the electron microscope, sedi ment more rapidly in sucrose density gradients than those generated with AT P or GTP, and can be captured with anticoatomer antibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two di stinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are inc ubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degree s C. The scission phase, which leads to vesicle release, takes place when c oated Golgi membranes, recovered after phase I, are incubated at higher tem peratures in the presence of cytosolic proteins, The scission phase does no t take place if protein kinase C inhibitors are added during the first phas e, even though these inhibitors do not prevent membrane coating and bud for mation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP. (C) 2000 Academic Press.