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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.
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