ABSTRACT
Clathrin and clathrin adaptors on clathrin-coated pits exchange with cytosolic clathrin and clathrin adaptors in vivo. This exchange might require the molecular chaperone Hsc70 and J-domain-protein auxilin, which, with ATP, uncoat clathrin-coated vesicles both in vivo and in vitro. We find that, although Hsc70 and ATP alone could not uncoat clathrin-coated pits, further addition of auxilin caused rapid uncoating of clathrin but not AP2 and epsin. By contrast, cytosol uncoats clathrin, AP2 and epsin from pits in permeabilized cells, and, concomitantly, these proteins in the cytosol rebind to the same pits, establishing that, like in vivo, these proteins exchange in permeabilized cells. Dissociation and exchange of clathrin in permeabilized cells can be prevented by inhibiting Hsc70 activity. The presence of clathrin-exchange in the permeabilized system substantiates our in vivo observations, and is consistent with the view that Hsc70 and auxilin are involved in the clathrin-exchange that occurs as clathrin-coated pits invaginate in vivo.
