ADP-RIBOSYLATION FACTOR-1 IS REQUIRED FOR SYNAPTIC VESICLE BUDDING INPC12 CELLS

Carrier vesicle generation from donor membranes typically progresses t hrough a GTP-dependent recruitment of coats to membranes, Here we expl ore the role of ADP ribosylation factor (ARF) 1, one of the GTP-bindin g proteins that recruit coats, in the production of neuroendocrine syn aptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) stron gly and reversibly inhibited SV formation in vivo in three different P C12 cell lines expressing vesicle-associated membrane protein-T Antige n derivatives, Other membrane traffic events remained unaffected by th e drug, and the BFA effects were not mimicked by drugs known to interf ere with formation of other classes of vesicles, The involvement of AR F proteins in the budding of SVs was addressed in a cell-free reconsti tution system (Desnos, C., I,. Clift-O'Grady, and R,B, Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vi tro incubation in the presence of the mutant ARFs, the labeled precurs or membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formati on in the presence of a high molecular weight, ARF-depleted fraction f rom brain cytosol was significantly enhanced by the addition of recomb inant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.