COPII-COATED VESICLE FORMATION RECONSTITUTED WITH PURIFIED COAT PROTEINS AND CHEMICALLY-DEFINED LIPOSOMES

COPII vesicle formation requires only three coat assembly subunits: Sa r1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate i s required for the binding of these proteins to liposomes. The GTP-bou nd form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the bi nding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated bu ds, and coated vesicles of 50-90 nm in diameter. Budding proceeds with out rupture of the donor liposome or vesicle product. These observatio ns suggest that the assembly of the COPII coat on the ER occurs by a s equential binding of coat proteins to specific lipids and that this as sembly promotes the budding of COPII-coated vesicles.