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Vol. 9, Issue 8, 2217-2229, August 1998
Department of Cell Biology, The Scripps Research Institute,
La Jolla, California 92037
Clathrin-coated vesicles (CCV) mediate protein sorting and
vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes
(APs), must be released. Previous work has established that
hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in
vitro, and nothing is known about the requirements for this reaction.
We report a novel quantitative assay for the ATP- and cytosol-
dependent release of APs from CCV. As expected, hsc70 is not sufficient
for AP release; however, immunodepletion and reconstitution experiments
establish that it is necessary. Interestingly, 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 identification of the
additional cytosolic factor(s) required for AP release.
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