title: Synergistic binding of DnaJ and DnaK chaperones to heat shock transcription factor σ32 ensures its characteristic high metabolic instability: implications for heat shock protein 70 (Hsp70)-Hsp40 mode of function.
abstract: Escherichia coli heat shock transcription factor σ(32) is rapidly degraded by ATP-dependent proteases, such as FtsH and ClpYQ. Although the DnaK chaperone system (DnaK, DnaJ, and GrpE) promotes σ(32) degradation in vivo, the precise mechanism that is involved remains unknown. Our previous results indicated that σ(32) mutants containing amino acid substitution in the N-terminal half of Region 2.1 are markedly stabilized in vivo. Here, we report the further characterization of these mutants by examining purified σ(32) mutants in vitro. Surprisingly, I54A σ(32), a very stable mutant, is more susceptible to ClpYQ and FtsH proteases than wild-type σ(32), indicating that the stability of σ(32) does not always reflect its susceptibility to proteases. Co-precipitation and gel filtration analyses show that purified σ(32) mutants exhibit a reduced affinity for DnaJ, leading to a marked decrease in forming a complex with DnaK in the presence of DnaJ and ATP. Other mutants with modestly increased stability (A50S σ(32) and K51E σ(32)) show an intermediate efficiency of complex formation with DnaK, suggesting that defects in binding to DnaK and DnaJ are well correlated with metabolic stability; effective interaction with DnaK promotes σ(32) degradation in vivo. We argue that the stable and effective interaction of heat shock protein 70 (Hsp70) with a substrate polypeptide may generally require the simultaneous binding of heat shock protein 40 (Hsp40) to distinct sites on the substrate.
authors.affiliation: School of Natural System, College of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Suzuki
authors.forename: Ayami
authors.initials: A
authors.lastname: Ikeda
authors.forename: Sachie
authors.initials: S
authors.lastname: Tsuchimoto
authors.forename: Ko-ichi
authors.initials: K
authors.lastname: Adachi
authors.forename: Aki
authors.initials: A
authors.lastname: Noguchi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Fukumori
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kanemori
mesh_terms: D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D011485:Protein Binding; D055550:Protein Stability; D059748:Proteolysis; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D046912:Multiprotein Complexes; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; C063842:dnaK protein, E coli