The Conserved G/F Motif of the DnaJ Chaperone Is Necessary for the Activation of the Substrate Binding Properties of the DnaK Chaperone

doi:10.1074/jbc.270.5.2139

The Conserved G/F Motif of the DnaJ Chaperone Is Necessary for the Activation of the Substrate Binding Properties of the DnaK Chaperone (*)

From the (1)Department of Cellular, Viral and Molecular Biology, University of Utah Medical Center, Salt Lake City, Utah 84132, the
(2)Département de Biochimie Médicale, Centre Médical Universitaire, 1, rue Michel-Servet, 1211 Genève 4, Switzerland, and the
(3)Division of Biophysics, Department of Molecular Biology, University of Gdansk, Kladki 24, Gdansk 80-822, Poland

^§ To whom correspondence should be addressed:
Dept. of Biochemistry, Stanford University, Stanford, CA 94305.
Tel.: 415-723-5685; Fax: 415-723-6783; dwall{at}cmgm.stanford.edu

Abstract

The universally conserved DnaK and DnaJ molecular chaperone proteins bind in a coordinate manner to protein substrates to prevent aggregation, to disaggregate proteins, or to regulate proper protein function. To further examine their synergistic mechanism of action, we constructed and characterized two DnaJ deletion proteins. One has an 11-amino-acid internal deletion that spans amino acid residues 77-87 (DnaJΔ77-87) and the other amino acids 77-107 (DnaJΔ77-107). The DnaJΔ77- 87 mutant protein, was normal in all respects analyzed. The DnaJΔ77-107 mutant protein has its entire G/F (Gly/Phe) motif deleted. This motif is found in most, but not all DnaJ family members. In vivo, DnaJΔ77-107 supported bacteriophage growth, albeit at reduced levels, demonstrating that at least some protein function was retained. However, DnaJΔ77-107 did not exhibit other wild type properties, such as proper down-regulation of the heat-shock response, and had an overall poisoning effect of cell growth. The purified DnaJΔ77-107 protein was shown to physically interact and stimulate DnaK's ATPase activity at wild type levels, unlike the previously characterized DnaJ259 point mutant (DnaJH33Q). Moreover, both DnaJΔ77-107 and DnaJ259 bound to substrate proteins, such as , at similar affinities as DnaJ. However, DnaJΔ77-107 was found to be largely defective in activating the ATP-dependent substrate binding mode of DnaK. In vivo, the ability of the mutant DnaJ proteins to down-regulate the heat-shock response was correlated only with their in vitro ability to activate DnaK to bind , in an ATP-dependent manner, and not with their ability to bind . We conclude, that although the G/F motif of DnaJ does not directly participate in the stimulation of DnaK's ATPase activity, nevertheless, it is involved in an important manner in modulating DnaK's substrate binding activity.

Footnotes

↵* This work was supported by National Institutes of Health Grant GM23917, Swiss National Foundation Grant 31-31129.91, the Canton of Geneva, and Grant P303 042 06 from the Polish State Committee for Scientific Research. 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.
↵¹K. Liberek, D. Wall, and C. Georgopoulos, manuscript in preparation.
² The abbreviations used are:

ELISA

enzyme-linked immunosorbent assay

PBS

phosphate-buffered saline

BSA

bovine serum albumin.
↵³A. Wawrzynow and M. Zylicz, unpublished data.
↵⁴R. McMacken, personal communication.
↵⁵D. Wall and C. Georgopoulos, unpublished data.
- Received July 19, 1994.
- Revision received November 9, 1994.