Dominant-interfering hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the in ternalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hs c70, has been implicated in regulating coat disassembly. We find that in ce lls overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inh ibited in vivo, and the majority of unassembled cytosolic clathrin shifts t o an assembled pool that cofractionates with AP1 and AP2. Surprisingly, thi s assembled pool of coat proteins accumulates in the absence of cargo recep tors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 muta nts is a block in transferrin receptor (TfnR) recycling, which cannot be ac counted for by the degree of inhibition of uncoating of endocytic CCVs. The se results suggest that hsc70 participates in multiple transport and/or sor ting events between endosomal compartments. Additionally, the mutant-expres sing cells are defective at internalizing transferrin. In the most potent c ase, the initial rate of uptake is inhibited 10-fold, and TfnR levels doubl e at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates cl athrin dynamics throughout the CCV cycle.