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¹ Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115
² Le Centre Hospitalier Universitaire de Quebec, pavillon CHUL, Rhumatologie et Immunology, Quebec, Canada G1V4G2
³ Laboratory of Cellular Oncology, Division of Basic Sciences, National Cancer Institute, Bethesda, MD 20892
⁴ Department of Medicine, Division of Gastroenterology, Duke University Medical Center, Durham, NC 27710

Address correspondence to Victor W. Hsu, Brigham and Women's Hospital, One Jimmy Fund Way, Smith 538 Boston, MA 02115. Tel.: (617) 525-1103. Fax: (617) 525-1104. E-mail: vhsu{at}rics.bwh.harvard.edu

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

Key Words: GAP; COPI; ARF1; vesicular transport; Golgi complex

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