Auxilin-induced interaction of the molecular chaperone Hsc70 with clathrin baskets.
We previously reported that a 100-kDa cofactor, recently identified as auxilin, is a DnaJ homolog which is required for Hsc70 to uncoat clathrin baskets. In the present study we investigated the effect of auxilin on the interaction of Hsc70 with pure clathrin baskets at pH 6, where no uncoating occurs. In a reaction which required auxilin, the baskets activated the Hsc70 ATPase activity more than 100-fold with an apparent dissociation constant of about 0.2 microM. Maximal ATPase activity occurred at a 1 to 1 molar ratio of auxilin to clathrin triskelion independent of the Hsc70 concentration suggesting that auxilin is primarily complexed with the clathrin baskets. The binding of Hsc70 to baskets also required auxilin, but less auxilin was needed for maximum binding than for maximum ATPase activity showing that auxilin can catalytically induce binding of Hsc70. The binding also required ATP; Hsc70 dissociated from baskets with a 6 min half-life when ATP was hydrolyzed to ADP. In contrast to auxilin, the assembly proteins, AP-2 and AP180, did not support activation of the Hsc70 ATPase activity by clathrin baskets nor did soluble clathrin triskelions at pH 7 significantly activate the ATPase activity with auxilin present. Therefore, the interaction of auxilin, clathrin baskets, and Hsc70-ATP is highly specific with auxilin first binding to a clathrin triskelion in the baskets and then Hsc70-ATP strongly binding to the auxilin-clathrin complex; the auxilin can then migrate to another clathrin triskelion before the ATPase cycle is complete.
Barouch
W
W
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Prasad
K
K
Greene
L
L
Eisenberg
E
E
D033942:Adaptor Proteins, Vesicular Transport; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000494:pharmacology*; D010750:Phosphoproteins / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; C439023:clathrin assembly protein AP180; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases