40] Adaptor protein 1-dependent clathrin coat assembly on synthetic liposomes and Golgi membranes

Abstract

To understand the AP-1 clathrin coat assembly process more completely, this chapter presents the in vitro assay that uses protein-free liposomes, ARF1, GTP, and cytosol or soluble clathrin coat proteins to reconstitute AP-l-dependent clathrin-coated vesicle formation. This simple assay allows for the dissection of the contributions of membrane and soluble cytosolic components to coat assembly that is not possible in a reconstituted system using purified Golgi membranes. Specific phospholipid(s), particularly phosphatidylserine, a cytosolic factor(s), and ARF. GTP are the minimal components required for AP-l-mediated clathrin coat assembly. From study data it is suggested that AP-1 and clathrin can be recruited onto protein-free liposomes with an efficiency that is comparable to that obtained with purified Golgi-enriched membranes.