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© The Rockefeller University Press, 0021-9525/1997//505 $5.00
The Journal of Cell Biology, Volume 138, Number 3, , 1997 505-515

Article

ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells



Victor Faúndez, Jim-Tong Horng, and Regis B. Kelly

Department of Biochemistry and Biophysics, The Hormone Research Institute, University of California, San Francisco, California 94143-0534

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. 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 vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

Abbreviations used in this paper: ARF, ADP ribosylation factor; BFA, brefeldin A; COPI and COPII, coat proteins I-II; HMW, high molecular weight; SV, synaptic vesicle; TAg, T Antigen; VAMP, vesicle-associated membrane protein.

Please address all correspondence to Regis B. Kelly, Department of Biochemistry and Biophysics, and the Hormone Research Institute, University of California, San Francisco, CA 94143-0534. Tel.: (415) 476-4095. Fax: (415) 731-3612. e-mail: kelly{at}cgl.ucsf.edu

Funded by National Institutes of Health (NIH) grants NS09878, NS15927, and DA10154 to R.B. Kelly. V. Faundez is the recipient of a NIH Fogarty International Postdoctoral Fellowship.


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