Successive and synergistic action of the Hsp70 and Hsp100 chaperones in protein disaggregation.
Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.
Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.
Szymon
S
Zietkiewicz
Joanna
J
Krzewska
Krzysztof
K
Liberek
D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism / Q000502:physiology*; D002845:Chromatography; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism / Q000502:physiology*; D019791:Guanidine / Q000737:chemistry; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000378:metabolism / Q000502:physiology*; D006358:Hot Temperature; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D012542:Scattering, Radiation; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000255:Adenosine Triphosphate; D008156:Luciferases; D049071:Endopeptidase Clp; C069775:ClpB protein, E coli; D019791:Guanidine