Abstract
The Coat Protein I (COPI) complex forms vesicles from Golgi membrane for retrograde transport among the Golgi stacks, and also from the Golgi to the endoplasmic reticulum (ER). We have been elucidating the mechanistic details of COPI vesicle formation through a reconstitution system that involves the incubation of Golgi membrane with purified components. This approach has enabled us recently to gain new insight into how certain lipids are critical for the fission stage of COPI vesicle formation. Lipid geometry has been proposed to act in the formation of transport carriers by promoting membrane curvature. However, evidence for this role has come from studies using simplified membranes, while confirmation in the more physiologic setting of native membranes has been challenging, as such membranes contain a complex composition of lipids and proteins. We have recently refined the COPI reconstitution system to overcome this experimental obstacle. This has led us to identify an unanticipated type of lipid geometry needed for COPI vesicle fission. This chapter describes the approach that we have developed to enable this discovery. The methodologies include: (i) preparation Golgi membrane from cells that are deficient in a particular lipid enzyme activity and (ii) functional rescue of this deficiency by introducing the product of the lipid enzyme, with experiments being performed at the in vitro level to gain mechanistic clarity and at the in vivo level to confirm physiologic relevance.