Uncoupling of peptide-stimulated ATPase and clathrin-uncoating activity in deletion mutant of hsc70.
The recombinant 60-kDa fragment of rat hsc70 has been overexpressed in Escherichia coli. The recombinant protein is not capable of disassembling clathrin from coated vesicles. However, the affinity for peptides and the peptide-stimulated ATPase activity of the intact protein are retained in the 60-kDa fragment. The dissociation constants of peptide P3a (the recognition sequence of clathrin light chain LCa by hsc70) and S peptide of ribonuclease for 60-kDa protein are 13 and 7 microM, respectively. The maximal velocities of stimulated ATPase activity by peptides P3a and GT4 are 0.25 and 0.31 nmol/h/microgram of protein, respectively, and the EC50 values (the concentration of peptides that brought about half-maximum hydrolysis) for peptides P3a and GT4 are 0.56 and 0.30 mM, respectively. These results indicate that peptide-stimulated ATPase activity of hsc70 is not sufficient for clathrin uncoating. We suggest that other activities or cellular components as yet unidentified associated with the C-terminal 10-kDa fragment of hsc70 are required for clathrin uncoating.
Tsai
M Y
MY
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Wang
C
C
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000494:pharmacology*; D010957:Plasmids; D051381:Rats; D017384:Sequence Deletion
D002352:Carrier Proteins; D002966:Clathrin; D017931:DNA Primers; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D010455:Peptides; D000251:Adenosine Triphosphatases