ATP-DEPENDENT AND CYTOSOL-DEPENDENT RELEASE OF ADAPTER PROTEINS FROM CLATHRIN-COATED VESICLES - A DUAL ROLE FOR HSC70

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular t rafficking from the plasma membrane and the trans-Golgi network. Befor e delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be rele ased. Previous work has established that hsc70/the uncoating ATPase me diates clathrin release in vitro without the release of APs. AP releas e has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay f or the ATP- and cytosol- dependent release of APs from CCV. As expecte d, hsc70 is not sufficient for AP release; however, immunodepletion an d reconstitution experiments establish that it is necessary. Interesti ngly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for ident ification of the additional cytosolic factor(s) required for AP releas e.