A Conserved HPD Sequence of the J-domain Is Necessary for YDJ1 Stimulation of Hsp70 ATPase Activity at a Site Distinct from Substrate Binding

doi:10.1074/jbc.271.16.9347

A Conserved HPD Sequence of the J-domain Is Necessary for YDJ1 Stimulation of Hsp70 ATPase Activity at a Site Distinct from Substrate Binding (*)

From the Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

^¶ To whom correspondence should be addressed:
Sigma Diagnostics, 545 S. Ewing, St. Louis, MO 63103.
Tel.: 800-521-8956; Fax: 314-531-2586.

↵^§ Present address: Neurobiology Dept., Swiss Federal Institute of Technology, CH8093 Zürich, Switzerland.

Abstract

The 46-kDa protein YDJ1 is one of several known yeast homologues of the Escherichia coli DnaJ protein. Like all J homologues, it shares homology with the highly conserved NH-terminal “J-domain” of DnaJ. A component of the DnaK (Hsp70) chaperone machinery that mediates protein folding, DnaJ is necessary for survival at elevated temperatures. It stimulates ATP hydrolysis by DnaK and effects the release of DnaK-bound polypeptides. Previous genetic and biochemical studies indicate that the J-domain is necessary for these functions. Using peptides corresponding to J-domain sequence, we show that a peptide containing the highly conserved His-Pro-Asp sequence at positions 34-36 in the J-domain competes off YDJ1 stimulation of Hsp70 ATPase activity. Inhibitory concentrations of peptide do not prevent binding of folding substrates, therefore YDJ1 must interact with Hsp70 at a site distinct from that for substrate binding. This interaction is critical for Hsp70 activity, since a mutant YDJ1 protein harboring a H34Q change (ydj1Q34) stimulates neither Hsp70 ATPase nor substrate release. The importance of the proper function of this region of the protein is supported by the poor growth and temperature-sensitive phenotype of yeast expressing ydj1Q34.

Footnotes

↵* This work was supported by National Institutes of Health Grant 5-RO1-AG11527-01-03 and American Heart Association Grant 92006620 (to M. G. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Hsp(s)

heat shock protein(s)

Fmoc

N-(9-fluorenyl)methoxycarbonyl

HPLC

high performance liquid chromatography

DTT

dithiothreitol

MOPS

3-(N-morpholino)propanesulfonic acid

PCR

polymerase chain reaction

CMLA

carboxymethyllactalbumin.
↵²J. Thissen, personal communication.
↵³J. Tsai, L. Estey, and M. G. Douglas, submitted for publication.
↵⁴S. Campbell-Burk, personal communication.
- Received October 17, 1995.
- Revision received February 7, 1996.