Interaction of nucleotide-free Hsc70 with clathrin and peptide and effect of ATP analogues.
The functions of the 70 kDa heat-shock proteins (hsp70s) are regulated by their bound nucleotide. We previously observed major differences in the effect of bound ATP and ADP on the interaction of hsc70 (constitutive hsp70) with its protein substrates. In the present study, we investigated the interaction of protein substrates with nucleotide-free hsc70 and with hsc70 with bound ATP analogues. We found, first, that nucleotide-free hsc70 appeared to interact differently with different substrates. Specifically, nucleotide-free hsc70 behaved much more like hsc70-ATP than hsc70-ADP in that clathrin very rapidly bound to and dissociated from nucleotide-free hsc70 in contrast to its very slow binding to and dissociation from hsc70-ADP. On the other hand, nucleotide-free hsc70 behaved more like hsc70-ADP than hsc70-ATP in that cytochrome c peptide dissociated very slowly from nucleotide-free hsc70 compared to its rapid dissociation from hsc70-ATP. Second, binding of the ATP analogues AMP-PNP, dATP, and ATP gamma S to nucleotide-free hsc70 had very little further effect on the properties of the nucleotide-free hsc70. Therefore, previously observed effects of ATP analogues may have been due to removal of the bound ADP rather than to the presence of analogues.
Gao
B
B
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Eisenberg
E
E
Greene
L
L
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding
D002352:Carrier Proteins; D002966:Clathrin; D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D009711:Nucleotides; D010455:Peptides; C050632:polypeptide C; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C026600:2'-deoxyadenosine triphosphate