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Published 16 September 2002. doi:10.1083/jcb.200207053
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© The Rockefeller University Press, 0021-9525/2002/9/1029 $5.00
The Journal of Cell Biology, Volume 158, Number 6, September 16, 2002 1029-1038

Article

Sec16p potentiates the action of COPII proteins to bud transport vesicles



Frantisek Supek1, David T. Madden1, Susan Hamamoto1, Lelio Orci2 and Randy Schekman1

1 Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, CA 94720
2 Department of Morphology, University of Geneva Medical School, 1211 Geneva 4, Switzerland

Address correspondence to Randy Schekman, Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, 229 Stanley Hall, Berkeley, CA 94720-3206. Tel.: (510) 642-5686. Fax: (510) 642-7846. E-mail: schekman{at}uclink4.berkeley.edu

SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major–minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major–minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major–minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP–dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.

Key Words: COPII; Sar1p; Sec16p; vesicle budding; liposomes


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