Sec16p potentiates the action of COPII proteins to bud transport vesicles.
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.
Supek
Frantisek
F
Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California-Berkeley, Berkeley, CA 94720, USA.
Madden
David T
DT
Hamamoto
Susan
S
Orci
Lelio
L
Schekman
Randy
R
D001692:Biological Transport; D022181:COP-Coated Vesicles / Q000378:metabolism* / Q000648:ultrastructure*; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000032:analysis; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000502:physiology*; D033921:Vesicular Transport Proteins
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D008081:Liposomes; D008565:Membrane Proteins; C096367:SEC16 protein, S cerevisiae; C079037:SEC23 protein, S cerevisiae; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020559:Monomeric GTP-Binding Proteins; C077395:SAR1 protein, S cerevisiae