title: DnaJ-promoted binding of DnaK to multiple sites on σ32 in the presence of ATP.
abstract: The Escherichia coli DnaK chaperone system is a canonical heat shock protein 70 (Hsp70) chaperone system comprising Hsp70, Hsp40, and a nucleotide exchange factor. Although Hsp40 is known to facilitate the effective binding of Hsp70 to substrates, the role of Hsp40 in Hsp70-substrate interactions has not yet been fully elucidated. Using the E. coli heat shock transcription factor σ(32) as a substrate in the DnaK chaperone system, we here provide new insight into the Hsp70-substrate interaction. When DnaK-σ(32) complexes formed under various conditions were analyzed by gel filtration, several DnaK-σ(32) complexes with different molecular masses were detected. The results indicated that multiple DnaK molecules simultaneously bind to σ(32), even though it has been suggested that DnaK interacts with σ(32) at a molar ratio of 1:1. Two σ(32) mutants, L201D σ(32) and I54A σ(32), which have reduced affinities for DnaK and DnaJ (Hsp40), respectively, were used to further characterize DnaK-σ(32) complex formation. Pulldown assays demonstrated that the affinity of I54A σ(32) for DnaK was similar to that of wild-type σ(32) in the absence of DnaJ, whereas L201D σ(32) exhibited an extremely low affinity for DnaK. However, in the presence of ATP and DnaJ, the yield of DnaK eluted with L201D σ(32) was much higher than that eluted with I54A σ(32). These results indicate that there are multiple DnaK binding sites on σ(32) and that DnaJ strongly promotes DnaK binding to any site in the presence of ATP, regardless of the intrinsic affinity of DnaK for the site.
authors.affiliation: School of Natural System, College of Science and Engineering, Kanazawa University, Kanazawa, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Noguchi
authors.forename: Ayami
authors.initials: A
authors.lastname: Ikeda
authors.forename: Moeka
authors.initials: M
authors.lastname: Mezaki
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Fukumori
authors.forename: Masaaki
authors.initials: M
authors.lastname: Kanemori
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D012808:Sigma Factor / Q000737:chemistry / 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; D012808:Sigma Factor; C069774:heat-shock sigma factor 32; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli