INHIBITION OF HSC70-CATALYZED CLATHRIN UNCOATING BY HSJ1 PROTEINS

The uncoating of clathrin-coated vesicles can be mediated in vitro by the 'uncoating ATPase' that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of mole cular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinet ics of clathrin release from coated vesicles, we have developed a quan titative, two-site ELISA for clathrin triskelions and demonstrated tha t stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, bu t correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins in terfere with an endogenous DnaJ-like protein that is involved in uncoa ting. Recent evidence suggests that the brain-specific vesicle-associa ted protein auxilin could play such a role. Athough we find no evidenc e for auxilin in our coated vesicle preparation, our results predict t hat an auxilin-like protein will be a general factor in clathrin uncoa ting.