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 (TDnaK.J 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 TDnaK.J 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 TDnaK.J complex, TGrpE, TClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that TGroEL/ES could not substitute TClpB.

Figure 1

Effect of the TDnaK⋅J-GrpE set and TClpB on aggregation of LDH. LDH, biotinylated for detection, was incubated in the presence of indicated components at 73°C for 30 min. The solutions were centrifuged, and supernatant (sup) and precipitate (ppt) were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. Electrophoresed proteins were blotted to the membrane, and biotinylated LDH was detected by alkaline phosphatase-conjugated streptavidin. KJ, E, and B represent TDnaK⋅J complex, TGrpE and TClpB, respectively.

Figure 2

Effect of chaperones on reactivation of heat-inactivated LDH. Indicated components were added to the solutions containing LDH (final concentration, 0.2 μM), and the solutions were incubated at 73°C for 30 min. Then the temperature was shifted down to 55°C, and other indicated components were immediately supplemented to the solutions (0 min in abscissa). Incubation at 55°C continued, and LDH activities were measured at the indicated times. Final concentrations of added components were 0.4 μM (TDnaK⋅J complex), 0.8 μM (TGrpE), 0.4 μM (TClpB), 0.4 μM (TGroEL/ES), and 3 mM (nucleotides). Note that components added at 73°C remained present after the temperature shift. The KJ, E, and B represent the TDnaK⋅J complex, TGrpE, and TClpB, respectively. The orders of addition are shown. (A) Effect of the TDnaK⋅J complex and TGrpE. (B) Effect of TClpB. (C) Effect of TDnaK⋅J complex, TGrpE, TClpB, and TGroEL/ES. (D) Effect of nucleotides.

Figure 3

Effect of order of additions of the TDnaK⋅J-GrpE set and TClpB on the recovery of LDH activity. The order of addition of chaperones are indicated. For all experiments, ATP (3 mM) was added from the beginning of the 73°C incubation. The LDH activities at 0 min (blank bars) and 60 min (black bars) after the temperature shift to 55°C were measured.

Figure 4

Reactivation of heat-inactivated α-glucosidase and G6PDH mediated by the TDnaK⋅J-GrpE set and TClpB. α-glucosidase (0.2 μM) and G6PDH (0.2 μM) were incubated at 73°C for 20 min (α-glucosidase) or 10 min (G6PDH) and then subsequently incubated at 55°C. The activities of these enzymes were measured at 0 min and 60 min after the temperature shift to 55°C. Activities of α-glucosidase and G6PDH are expressed as percent of those before heat inactivation.