page 1029, Supek et al. illustrate how a peripheral membrane protein organizes a coat protein complex involved in secretory vesicle formation.
The protein in question, yeast Sec16p, is an ER resident required in vivo for COPII-dependent vesicle budding. In vitro, Sec16p is not necessary for budding from liposomes reconstituted with pure cytosolic COPII proteins. However, this in vitro reaction depends on a nonhydrolyzable form of GTP, probably because the COPII coat falls apart when Sar1p (the initiator of coat assembly) hydrolyzes GTP. Until now, the function of Sec16p in liposome budding could not be tested, because the protein was difficult to purify
Supek et al. report conditions that stabilize Sec16p and have purified enough protein for in vitro studies. Microsomal membranes stripped of endogenous Sec16p were stimulated in vesicle budding by the purified. protein, but only in the presence of hydrolyzable GTP. Thus, the in vivo function of Sec16p may be either to slow GTP hydrolysis during budding or to stabilize the COPII coat even after GTP is hydrolyzed by Sar1p.
The authors used liposomes to show that Sec16p recruited Sar1p-GTP and another coat component, Sec23/24p, to membranes. However, this interaction did not inhibit GTP hydrolysis. Thus, the function of Sec16p appears to be to tether the COPII coat on membranes. ▪