Genetic and biochemical characterization of mutations affecting the carboxy-terminal domain of the Escherichia coli molecular chaperone DnaJ

Mol Microbiol. 1998 Oct;30(2):329-40. doi: 10.1046/j.1365-2958.1998.01067.x.

Abstract

DnaJ is a universally conserved heat shock protein involved in protein folding. DnaJ contains four conserved domains. The N-terminal 'J-domain' has been shown to be responsible for the recruitment of its specific DnaK partner protein. The 'Gly/Phe'- and 'Cys-rich' domains have been implicated in stabilizing interactions with DnaK. DnaJ is also able to interact independently with unfolded or native polypeptides. Very little is known regarding such binding/chaperone abilities, but it has been suggested that the least conserved carboxy-terminal domain could contribute to these properties. To gain insight into the biological activity of this fourth domain, we deleted two relatively conserved patches of amino acid residues, a 'G-rich' cluster and a 'G-D-L-Y-V' motif, resulting in the DnaJDelta[230-238] and DnaJDelta[242-246] mutant proteins respectively. Both mutant proteins are partially defective in stimulating the ATPase activity of DnaK and in preventing aggregation of firefly luciferase in vitro. Both mutants have lost the ability to regulate the sigma32-dependent heat shock response, as shown in vivo using a heat shock transcriptional fusion. Furthermore, and unlike wild-type DnaJ, DnaJDelta[242-246] is unable to assist the DnaK-dependent refolding of denatured luciferase. In agreement with these results, we found that DnaJDelta[242-246] is unable to restore either the temperature-sensitive phenotype or the motility defect of a dnaJ null mutation. Substitution of amino acids [242-246] by five alanines leads to similar phenotypic defects, suggesting that altering the 'G-D-L-Y-V' motif leads to partial loss of DnaJ activity. Our data clearly support a role in the intrinsic chaperone/substrate binding ability of the carboxy-terminal domain of DnaJ.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacteriophage lambda / pathogenicity
  • Cell Division / genetics
  • Conserved Sequence
  • Down-Regulation
  • Escherichia coli / genetics*
  • Escherichia coli / physiology
  • Escherichia coli / virology
  • Escherichia coli Proteins*
  • HSP40 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins / metabolism
  • Heat-Shock Proteins / genetics*
  • Heat-Shock Proteins / metabolism*
  • Heat-Shock Response
  • Luciferases / metabolism
  • Molecular Sequence Data
  • Mutation*
  • Sequence Deletion
  • Sigma Factor*
  • Transcription Factors / metabolism

Substances

  • DnaJ protein, E coli
  • Escherichia coli Proteins
  • HSP40 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Sigma Factor
  • Transcription Factors
  • heat-shock sigma factor 32
  • Luciferases
  • dnaK protein, E coli