ATP hydrolysis is not required for the dissociation of a substance P.BiP complex.
BiP is a member of the hsp70 family of proteins that is present in the endoplasmic reticulum where it functions as a molecular chaperone. Rapid quantitative assays have been used to study the effect of mutating BiP residue 229, located in the ATP binding site, from threonine to glycine. Although binding of ATP to the mutant BiP was not affected, the mutant protein possessed 10-20% of the wild-type BiP ATPase activity. Binding to a model peptide substrate, substance P (Brot et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12120-12124), was twofold higher with mutant BiP at 4 degrees C than with wild-type BiP, and was ATP dependent. Under these conditions the substance P that was bound to mutant BiP, but not the wild-type, could be released by higher levels of ATP (5-10 microM), and the ratio of substance P released to ATP hydrolyzed was greater than 10. These results suggest that stoichiometric ATP hydrolysis is not required for release of a chaperone from its substrate.
Vidal
V
V
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110-1199, USA.
Qiu
N H
NH
Redfield
B
B
Carlino
A
A
Brot
N
N
Weissbach
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013373:Substance P / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D013373:Substance P; D000255:Adenosine Triphosphate