Functional reconstitution of COPI coat assembly and disassembly using chemically defined components.

Abstract

Coat protein I (COPI)-coated transport vesicles mediate protein and lipid transport in the early secretory pathway. The basic machinery required for the formation of these transport intermediates has been elucidated based on the reconstitution of COPI-coated vesicle formation from chemically defined liposomes. In this experimental system, the coat components coatomer and GTP-bound ADP-ribosylation factor (ARF), as well as p23 as a membrane-bound receptor for COPI coat proteins, were shown to be both necessary and sufficient to promote COPI-coated vesicle formation. Based on biochemical and ultrastructural analyses, we now demonstrate that the catalytic domain of ARF-GTPase-activating protein (GAP) alone is sufficient to initiate uncoating of liposome-derived COPI-coated vesicles. By contrast, ARF-GAP activity is not required for COPI coat assembly and, therefore, does not seem to represent an essential coat component of COPI vesicles as suggested recently [Yang, J. S., Lee, S. Y., Gao, M., Bourgoin, S., Randazzo, P. A., et al. (2002) J. Cell Biol. 159, 69-78]. Thus, a complete round of COPI coat assembly and disassembly has been reconstituted with purified components defining the core machinery of COPI vesicle biogenesis.