Functional interaction of the auxilin J domain with the nucleotide- and substrate-binding modules of Hsc70.
The uncoating of clathrin-coated vesicles requires the DnaJ homologue auxilin for targeting Hsc70 to clathrin coats. This function involves a transient interaction of the auxilin J domain with Hsc70. We have now identified the structural elements of Hsc70 that are responsible for the uncoating activity, and we show that the hitherto accepted view, which implicates the 10-kDa carboxyl-terminal variable domain of Hsc70, is incorrect. A 60-kDa chymotryptic or analogous recombinant fragment of Hsc70, which contains the ATPase- and substrate-binding domains, is sufficient to liberate clathrin from coated vesicles. Consistent with this was the observation that Hsp70 uncoats coated vesicles with the same efficacy as Hsc70 and that DnaK possesses vestigial uncoating activity. Direct binding studies demonstrated that the auxilin J domain undergoes an ATP-dependent reaction only with fragments of Hsc70 that contain both the ATPase- and substrate-binding domains. The individual domains by themselves did not bind to the J domain nor did a recombinant protein that contained the substrate-binding domain attached to the 10-kDa variable domain.
Ungewickell
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Center for Immunology, Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. ungewickell@pathology.wustl.edu
Ungewickell
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Holstein
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D033942:Adaptor Proteins, Vesicular Transport; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002966:Clathrin / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli