Hsc70 chaperones clathrin and primes it to interact with vesicle membranes.
When Hsc70 uncoats clathrin-coated vesicles in an auxilin- and ATP-dependent reaction, a single round of rapid uncoating occurs followed by very slow steady-state uncoating. We now show that this biphasic time course occurs because Hsc70 sequentially forms two types of complex with the dissociated clathrin triskelions. The first round of clathrin uncoating is driven by formation of a pre-steady-state assembly protein (AP)-clathrin-Hsc70-ADP complex. Then, following exchange of ADP with ATP, a steady-state AP-clathrin-Hsc70-ATP complex forms that ties up Hsc70, preventing further uncoating. This 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-steady-state complex in that it cannot be immunoprecipitated by anti-clathrin antibodies and is readily dissociated by fast protein liquid chromatography. Remarkably, when the steady-state complex is incubated with uncoated vesicle membranes in ATP, the pre-steady-state complex reforms, suggesting that the clathrin triskelions in the steady-state complex rebind to the membranes and are again uncoated by Hsc70. We propose that Hsc70 not only uncoats clathrin but also chaperones it to prevent it from inappropriately polymerizing in the cell cytosol and primes it to reform clathrin-coated pits.
Jiang
R
R
Laboratory of Cell Biology, Section on Cell Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Gao
B
B
Prasad
K
K
Greene
L E
LE
Eisenberg
E
E
D033942:Adaptor Proteins, Vesicular Transport; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D018699:Coated Vesicles / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate