A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase.

Immediately after clathrin-coated pits pinch off from the cell membrane to form clathrin-coated vesicles, clathrin dissociates from the vesicles. In vitro studies suggest that this dissociation is carried out by the uncoating ATPase, a constitutive member of the 70-kDa heat shock family. Aside from the requirement for ATP, nothing is known about the regulation of the uncoating process. We now show that clathrin baskets prepared from highly purified clathrin and AP2, the assembly protein associated with plasma membrane coated vesicles, cannot be uncoated by the bovine brain uncoating ATPase alone. A 100-kDa protein cofactor, which was isolated from coated vesicles, is essential for uncoating by the uncoating ATPase. This cofactor restores normal uncoating when present at a molar ratio of about 1 to 10 to clathrin and uncoating ATPase.