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Author Notes:

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@cgl.ucsf.edu

We thank Dr. F. Brodsky (University of California, San Francisco, CA [UCSF]) for the generous gift of antibodies to clathrin (X-22) and TD.1. Dr. T. Kreis (University of Geneva, Switzerland) kindly provided antibodies to β-COP (EAGE), and Dr. F. Letourneur (Basel Institute for Immunology, Switzerland) provided fusion proteins GST-WBP1-KK and GST-WBP1-SS.

We are also grateful to Drs. L. Nagy (University of Guelph, Ontario, Canada) and J. Roos (UCSF) for their helpful comments in the initial preparation of the manuscript.

Subjects:

Research Funding:

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.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Cell Biology
  • POLYMERIC IMMUNOGLOBULIN RECEPTOR
  • CULTURED HIPPOCAMPAL-NEURONS
  • TRANS-GOLGI NETWORK
  • COP-COATED VESICLES
  • GTP-BINDING PROTEIN
  • BREFELDIN-A
  • PHOSPHOLIPASE-D
  • IN-VITRO
  • ENDOPLASMIC-RETICULUM
  • ORGANELLE STRUCTURE

ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells

Tools:

Journal Title:

Journal of Cell Biology

Volume:

Volume 138, Number 3

Publisher:

, Pages 505-515

Type of Work:

Article | Final Publisher PDF

Abstract:

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.

Copyright information:

© The Rockefeller University Press,

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License (http://creativecommons.org/licenses/by-nc-sa/4.0/).

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