Recruitment of Coat Proteins to Peptidoliposomes

Intracellular transport between compartments within the cell is generally mediated by membrane vesicles. Their formation is initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo proteins, and deform the lipid bilayer. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro . To analyze the involvement of membrane proteins, the cytosolically exposed sequences may be coupled chemically to reactive lipids in the membrane. Here we describe the use of such peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1, requires specific lipids, and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin.