Heat-inactivated proteins are rescued by the DnaK⋅J-GrpE set and ClpB chaperones

Abstract

Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated in vitro. In a eubacterium Thermus thermophilus, DnaK and DnaJ exist as a stable trigonal ring complex (TDnaK⅐J complex) and the dnaK gene cluster contains a clpB gene. When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the TDnaK⅐J complex could suppress the aggregation of proteins in an ATP-and TGrpE-dependent manner. Subsequent incubation of these heated preparations at moderate temperature after addition of TClpB resulted in the efficient reactivation of the proteins. Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the TDnaK⅐J complex, TGrpE, TClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that TGroEL͞ES could not substitute TClpB.

Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

“…In eukaryotes, this cooperation helps yeast cells to recover from severe heat stress (8 -10), and in vitro experiments have shown that HSP104-HSP70 cooperation facilitates reactivation of the proteins that had been chemically denatured and aggregated (3). In prokaryotes, as we have demonstrated for chaperones from a thermophilic eubacteria, Thermus thermophilus, heatdamaged proteins were rescued by the cooperative chaperone functions of ClpB (bacterial HSP104), DnaK (bacterial HSP70), DnaJ, and GrpE (4,11). ClpB forms a homo-hexameric complex (580 kDa) as an active species and needs ATP hydrolysis for the function (12)(13)(14)(15)(16)(17).…”

“…3-D-threo-isopropylmalate dehydrogenase from T. thermophilus (TIPMDH) was expressed in E. coli and purified as described (25). K⅐J complex, TGrpE, and TClpB were expressed in E. coli and purified as described (4,20,21). Additional gel filtration by G3000SWXL (Tosoh) was performed for K⅐J to remove trace amounts of free TDnaK and TDnaJ.…”

“…TDafA is a small protein necessary to assemble TDnaK and TDnaJ into K⅐J (20); TGrpE (22 kDa) is isolated as a homodimer (21)(22)(23). The stable K⅐J has been reported only for DnaK and DnaJ from T. thermophilus, but the chaperone function of K⅐J has been studied with a general interest because the current model for the mechanism of DnaK function assumes only transient but not stable interaction between DnaK and DnaJ (4,11,16,19,23). Related to this contention, the chaperone activity of uncomplexed TDnaK and TDnaJ (termed KϩJ) has been reported (14,23,24), but the difference from that of K⅐J is not clear.…”

“…The central cellular defense against protein aggregation consists of molecular chaperones that bind unfolded proteins and prevent aggregation (1,2). Once the aggregates are formed, however, the usual chaperones cannot handle them, and the cooperative function of HSP104 and HSP70 can dissolve aggregates and help the proteins to restore their native structures (3)(4)(5)(6)(7). In eukaryotes, this cooperation helps yeast cells to recover from severe heat stress (8 -10), and in vitro experiments have shown that HSP104-HSP70 cooperation facilitates reactivation of the proteins that had been chemically denatured and aggregated (3).…”

Walker‐A threonine couples nucleotide occupancy with the chaperone activity of the AAA+ ATPase ClpB

“…5 ClpB reactivates aggregated proteins in cooperation with the DnaK chaperone system. [6][7][8] The ClpB activity is linked to extraction of single polypeptides from aggregated particles and their forced unfolding by translocation through a narrow channel located at the center of the hexameric ring. 9 The substrate translocation by ClpB is driven by ATP hydrolysis and is analogous to the degradationpreceding unfolding/translocation of substrates by the proteasome and other ATP-dependent proteases.…”