The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone.
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 (DnaJ delta 77-87) and the other amino acids 77-107 (DnaJ delta 77-107). The DnaJ delta 77-87 mutant protein, was normal in all respects analyzed. The DnaJ delta 77-107 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, DnaJ delta 77-107 supported bacteriophage lambda growth, albeit at reduced levels, demonstrating that at least some protein function was retained. However, DnaJ delta 77-107 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 DnaJ delta 77-107 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 DnaJ delta 77-107 and DnaJ259 bound to substrate proteins, such as sigma 32, at similar affinities as DnaJ+. However, DnaJ delta 77-107 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 sigma 32, in an ATP-dependent manner, and not with their ability to bind sigma 32. 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.
Department of Cellular, Viral and Molecular Biology, University of Utah Medical Center, Salt Lake City 84132.
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Wall
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Zylicz
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Georgopoulos
D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism; D013329:Structure-Activity Relationship
D017931:DNA Primers; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D012808:Sigma Factor; C063842:dnaK protein, E coli