Sec16p is organized in domains and localized on the cytosolic surface of the ER in a manner that suggests a role in recruiting each of the proteins involved in forming a COPII coat. Kaiser's lab dissected Sec16p into domains that interact with three of the four proteins that comprise the bulk of the coat (Espenshade et al., 1995
; Gimeno et al., 1996
; Shaywitz et al., 1997
). In addition, genetic evidence suggests a functional interaction between SEC16
and Sar1p, the GTP-binding protein responsible for initiating coat assembly (Nakano and Muramatsu, 1989
; Gimeno et al., 1995
; Saito et al., 1999
). However, the tight peripheral association of Sec16p with the ER membrane and the extreme proteolytic sensitivity of the protein have made a biochemical characterization of the role of the protein difficult. Until now, our studies relied on ER membranes that provide Sec16p, and on the cytosolic proteins required for transport vesicle budding, the subunits of COPII. By adjusting conditions of regulated induction of SEC16
expression and by the use of an NH2
-terminal MBP chimera, we were able to develop a single step procedure to isolate pure, intact MBP–Sec16p. Nonetheless, the protein is not abundant and the yield of pure protein is low (~200 μg/700 g cells).
Membranes stripped of Sec16p respond to COPII proteins and bud transport vesicles containing gpαf and Sec22p nearly normally, provided the nonhydrolyzable GTP analogue, GMP-PNP, is included in the incubation. However, in the presence of GTP, a limiting concentration of COPII proteins produces a threefold stimulation by Sec16p. Previously, we demonstrated that Sar1p is shed and COPII subunits are less stably bound to vesicles formed in the presence of GTP, whereas the coat is retained on vesicles formed in the presence of GMP-PNP (Barlowe et al., 1994
). A dynamic assay of coat assembly reveals a dramatic difference in coat stability in reactions containing GTP or GMP-PNP (Antonny et al., 2001
). Thus, the effect of Sec16p implies some role in the coordination of GTP hydrolysis and vesicle budding or cargo protein capture into vesicles. Either Sec16p suppresses GTP hydrolysis by Sar1p during a crucial phase of cargo protein selection or completion of vesicle budding, or the COPII coat subunits are stabilized on the surface of a forming vesicle in contact with Sec16p after GTP hydrolysis is completed.
We evaluated the recruitment of COPII proteins by Sec16p using a liposome binding assay (Matsuoka et al., 1998
). A liposome formulation containing a mixture of neutral and acidic phospholipids (major–minor mix) binds Sec16p in a Sar1p- and nucleoside triphosphate–independent process, which contrasts the recruitment of COPII proteins, which requires Sar1p and GTP or GMP-PNP (Antonny et al., 2001
). Sec16p bound to major–minor mix liposomes recruits Sec23/24p or Sec13/31p without Sar1p and nucleotide. However, the addition of Sec16p to a complete COPII incubation containing GMP-PNP facilitates the assembly of the coat and the formation of coated vesicles.
Although Sec16p facilitated coat assembly and vesicle budding in liposome reactions containing GMP-PNP, the reaction with native ER membranes was stimulated by Sec16p more dramatically in the presence of GTP. Unlike liposomes, the ER membrane is populated by membrane proteins that may assist in the recruitment of COPII subunits. We showed previously that Sar1p, GMP-PNP, and Sec23/24p form a complex with the cytosolic domain of SNARE proteins required to address COPII vesicles to the cis-Golgi (Springer and Schekman, 1998
). This complex is not detected with GTP in place of GMP-PNP, probably because the SNAREs do not suppress the GAP activity of Sec23p and rapid GTP hydrolysis by Sar1p renders the complex unstable. Thus, if the principal role of Sec16p on the ER membrane is to stabilize COPII subunits, this function may be replaced by SNAREs or other proteins in incubations that contain GMP-PNP. However, Sec16p may play a unique and stimulatory role if it retains COPII proteins on the membrane after Sar1p hydrolyzes GTP.
To investigate this possibility, we exploited the observation that Sec16p binds neutral liposomes (PC/PE) only in the presence of Sar1p-GTP (or GMP-PNP). In contrast to the instability of a Sar1-GTP–Sec23/24p complex that forms on major–minor mix liposomes (Antonny et al., 2001
), or on PC/PE liposomes (unpublished data), Sec16p stabilizes this association on PC/PE liposomes. This stabilization appears not to be the result of an inhibition by Sec16p of GTP hydrolysis by Sar1p and Sec23/24p. Furthermore, a productive complex including Sec16p forms in the presence of GDP and BeFx
, an analogue that complexes the GTP binding site of Sar1p only when it is bound to Sec23/24p (Antonny et al., 2001
We propose that Sec16p organizes an initial step in the assembly of the COPII coat. Sec16p may be bound to the ER membrane in the vicinity of Sec12p, the nucleotide exchange catalyst of Sar1p (d'Enfert et al., 1991
; Barlowe and Schekman, 1993
). Sar1p-GTP would form and bind Sec16p, recruiting Sec23/24p to make a coat initiation complex. During the lifetime of Sar1p-GTP, cargo molecules destined for transport may engage the complex and be captured in advance of GTP hydrolysis (Kuehn et al., 1998
; Springer et al., 1999
). Although GTP hydrolysis is not required in protein sorting mediated by the COPII coat, it is an essential aspect of coat disassembly. Thus, GTP hydrolysis accelerated by the action of the Sec23 GAP on Sar1p (Yoshihisa et al., 1993
), or further enhanced by the polymerization of the coat induced by Sec13/31p (Antonny et al., 2001
), is required to produce an uncoated vesicle exposing SNARE proteins to a target membrane. Sec16p may act as a tether to prevent premature discharge of coat subunits before the completion of vesicle fission from the ER membrane.