When Hsc70 uncoats clathrin-coated vesicles in an auxilin- and ATP dependen
t reaction, a single round of rapid uncoating occurs followed by very slow
steady-state uncoating. We now show that this biphasic time course occurs b
ecause Hsc70 sequentially forms two types of complex with the dissociated c
lathrin triskelions. The first round of clathrin uncoating is driven by for
mation of a pre steady-state assembly protein (AP)-clathrin-Hsc70-ADP compl
ex. Then, following exchange of ADP with ATP, a steady-state AP-clathrin-Hs
c70-ATP complex forms that ties up Hsc70, preventing further uncoating, Thi
s steady-state complex forms only during uncoating in the presence of APs;
in the absence of APs, Hsc70 rapidly dissociates from the uncoated clathrin
and continues to carry out uncoating, Whether it is complexed with ATP or
ADP, the steady-state complex has very different properties from the pre-st
eady-state complex in that it cannot be immunoprecipitated by anti-clathrin
antibodies and is readily dissociated by fast protein liquid chromatograph
y. Remarkably, when the steady-state complex is incubated with uncoated ves
icle membranes in ATP, the pre steady state complex reforms, suggesting tha
t the clathrin triskelions in the steady-state complex rebind to the membra
nes and are again uncoated by Hsc70. We propose that Hsc70 not only uncoats
clathrin but also chaperones it to prevent it from inappropriately polymer
izing in the cell cytosol and primes it to reform clathrin coated pits.