| Abstract
| - To determine the effect of mutations at thenucleotide-binding site of recombinant Hsp70 onits interaction with protein and peptide substrates, point mutationswere made at D10 and K71, two residuesat the active site. The D10S mutation weakened both ATP and ADPbinding, while the K71E mutationweakened only ATP binding. In binding experiments using Hsp70 withno bound nucleotide, the mutatedHsp70s interacted with clathrin and peptide just like the wild-typeHsp70. However, the D10 mutationcompletely abolished the effects of both ATP and ADP on peptide andclathrin binding. The K71 mutationalso abolished the effect of ATP on substrate binding, but ADP, whichstill bound tightly, had its normaleffect on substrate binding. In addition, the D10S and K71Emutants had greatly reduced ability to uncoatclathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0,and undergo polymerization inducedby YDJ1 in the presence of ATP. We conclude, first, thatnucleotides must bind strongly to Hsp70 toaffect substrate binding and, second, that interaction of Hsp70 withDnaJ homologues may also requirea strongly bound ATP.
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