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Originally published In Press as doi:10.1074/jbc.M308782200 on January 16, 2004

J. Biol. Chem., Vol. 279, Issue 16, 15723-15727, April 16, 2004
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Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

HEAT STRESS CONVERTS THE FORMER TO THE LATTER, AND ONLY THE LATTER CAN DO DISAGGREGATION IN COOPERATION WITH ClpB*

Yo-hei Watanabe{ddagger} and Masasuke Yoshida§

From the Chemical Resources Laboratory, R-1, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226-8503, Japan

Received for publication, August 8, 2003 , and in revised form, January 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
DnaK from Thermus thermophilus (TDnaK) is unique because significant fractions of cellular TDnaK exist as a trigonal K·J complex that consists of three copies each of TDnaK, TDnaJ, and an assembly factor TDafA. Here, chaperone functions of the K·J complex and free TDnaK plus free TDnaJ (K+J) were compared. Substrate proteins were completely denatured at 72–73 °C or 89 °C in the absence or the presence of K·J complex or K+J and were subsequently incubated at a moderate temperature of 55 °C. TGrpE and ATP were always included in the K·J complex and K+J, and TClpB was supplemented at 55 °C. At 72–73 °C, both the K·J complex and K+J suppressed heat aggregation of substrate proteins. During the next incubation at 55 °C, K+J, assisted by TClpB, was able to disaggregate the heat aggregates and efficiently reactivate activities of the proteins, whereas the K·J complex was not; it reactivated only the soluble inactivated proteins. When substrate proteins were heated to 89 °C, both the K·J complex and K+J were no longer able to prevent heat aggregation, and because of selective, irreversible denaturation of TDafA the K·J complex dissociated into K+J, which then exhibited disaggregation activity during the next incubation at 55 °C. Thus, TClpB-assisted disaggregation activity belongs only to K+J, and TDafA is a potential thermosensor for converting the K·J complex to K+J in response to heat stress.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein aggregation is one of the major damaging consequences of a stress situation such as heat shock. 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 (3–7). 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). In prokaryotes, as we have demonstrated for chaperones from a thermophilic eubacteria, Thermus thermophilus, heat-damaged 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 (12–17). DnaK also has ATPase activity and works with DnaJ and GrpE (termed the DnaK set) (2). Cooperation between the DnaK set and ClpB in disaggregation and reactivation of the aggregated proteins was also reported for Escherichia coli and mitochondrial homologues (5–7, 18).

Unlike other bacteria, however, approximately half the population of cellular DnaK of T. thermophilus is purified as a stable trigonal ring complex (19). This complex (330 kDa), termed K·J1 hereafter, is composed of three copies each of TDnaK (69 kDa), TDnaJ (33 kDa), and TDafA (the prefix T designates proteins of T. thermophilus). TDafA is a small protein necessary to assemble TDnaK and TDnaJ into K·J (20); TGrpE (22 kDa) is isolated as a homodimer (21–23). The stable K·J has been reported only for DnaK and DnaJ from T. thermophilus, but the chaperone function of K·J 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 K+J) has been reported (14, 23, 24), but the difference from that of K·J is not clear. Here, we carefully compared chaperone activities of the K·J set (K·J plus TGrpE) and those of the K+J set (K+J plus TGrpE). The results indicate that the ability of disaggregation of the previously formed aggregate in cooperation with TClpB belongs only to K+J and that K·J must be dissociated into K+J to elicit this function, which indeed occurs under heat stress, in which TDafA is heat-inactivated.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—A plasmid containing genes for TDafA(L2V) named pET-DafA(L2V) was constructed as follows. A DNA fragment containing the sequence of the tdafA gene with an L2V mutation was prepared with PCR by using Ex-Taq DNA polymerase (Takara). The pMKJ8 plasmid (20) was used as a PCR template. The forward primer was an oligonucleotide having an NdeI site, and a 5'-end sequence of the tdafA gene with an L2V mutation was used. As the reverse primer, an oligonucleotide having a 3'-end sequence of the gene and a BamHI site was used. The amplified DNA fragment was cloned into the NdeI and BamHI sites of pET23c (Novagen), and the base sequence was confirmed. To make a pET-TDnaK(N-His10) for expression of His-tagged TDnaK, a TDnaK-expression plasmid, pMDK6 (20), was digested with NdeI-BamHI, and the fragment was ligated into NdeI-BamHI sites of the pET16b (Novagen).

Proteins—Glucose-6-phosphate dehydrogenase (G6PDH) and {alpha}-glucosidase from Bacillus stearothermophilus were purchased from Unitika and Sigma, respectively. 3-D-threo-isopropylmalate dehydrogenase from T. thermophilus (TIPMDH) was expressed in E. coli and purified as described (25). K·J 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 K·J to remove trace amounts of free TDnaK and TDnaJ. TDnaK was expressed in E. coli BL21(DE3) carrying pMDK6. The cells were suspended in Buffer A (25 mM Tris-HCl, pH 7.5, and 5 mM MgCl2) and disrupted by sonication. The cell extracts were heat-treated at 80 °C for 30 min and centrifuged at 100,000 x g for 40 min. The supernatant was applied to a Toyopearl DEAE-column (Tosoh) equilibrated with Buffer A. The column was eluted with Buffer A containing 300 mM NaCl. Fractions containing TDnaK were pooled, and solid ammonium sulfate was added, to a concentration of 800 mM. The solution was applied to a Toyopearl butyl column (Tosoh) and eluted with a linear reverse gradient of ammonium sulfate (800–0 mM). Fractions containing TDnaK were pooled, concentrated by Ultra-free (Millipore), and applied to a PD10 gel filtration column (Amersham Biosciences) to exchange the buffer to 50 mM MOPS-NaOH buffer, pH 7.5, 150 mM KCl, and 5 mM MgCl2. The TDnaK fractions were frozen by liquid nitrogen and stored at –80 °C until used. His-tagged TDnaK was expressed in E. coli carrying pET-TDnaK(N-His10) and purified by the same procedures as TDnaK. TDnaJ was expressed in E. coli carrying pMDJ10 (20). One-half of the amount of expressed TDnaJ was in the soluble fraction and was heat-stable. TDnaJ was purified with the same procedures used for TDnaK purification, except that the pass-through fraction of the Toyopearl DEAE-column (Tosoh) contained TDnaJ and was used for further purification. To isolate the TDafA expressed in E. coli, we used the mutation L2V, which prevents rapid degradation in the cell (23). For simplicity, hereafter we call this mutant TDafA. TDafA was expressed as inclusion bodies in E. coli carrying pET-DafA(L2V). The cells were suspended in Buffer B (25 mM Tris-HCl, pH 7.5, and 1 mM EDTA), disrupted by a French press, and centrifuged at 12,000 x g for 20 min. The pellet was washed twice with Buffer B. From the pellet, TDafA was extracted using Buffer B containing 4 M urea. The extract was concentrated by Ultra-free and loaded to a G3000SWXL gel filtration column equilibrated with the buffer (50 mM MOPS-NaOH, pH 7.5, 150 mM KCl, 5 mM MgCl2, and 4 M urea). The peak fraction was frozen by liquid nitrogen and stored at –80 °C until use. Throughout this article concentrations of proteins are expressed as monomers except for K·J (expressed as a trigonal complex), TGrpE (expressed as a dimer), and TClpB (expressed as a hexamer).

Aggregation and Disaggregation of Proteins—G6PDH (53 kDa, tetramer), {alpha}-glucosidase (65 kDa, monomer), and TIPMDH (37 kDa, dimer) were used as substrate proteins. These substrate proteins (0.2 µM, expressed as a monomer) were dissolved in 250 µl of the reaction mixture (50 mM MOPS-NaOH, pH 7.5, 5 mM MgCl2, 150 mM KCl, 1 mM dithiothreitol, and 3 mM ATP) containing the indicated chaperones. When the temperature was reduced to 55 °C after heat treatment and the reactivation incubation at 55 °C for 90 min was finished, an aliquot of the solution was centrifuged at 20,000 x g for 10 min at 4 °C. Precipitated proteins were washed by the buffer, and proteins in the supernatant were precipitated by 10% trichloroacetic acid. Both samples were analyzed with polyacrylamide gel (13%) electrophoresis in 0.1% sodium dodecyl sulfate.

Reactivation of Heat-inactivated Proteins—Substrate proteins (0.2 µM, expressed as monomers) were dissolved in 250 µl of the reaction mixture (50 mM MOPS-NaOH, pH 7.5, 5 mM MgCl2, 150 mM KCl, 1 mM dithiothreitol, and 3 mM ATP) containing chaperones as indicated. The reaction mixture was incubated at 72 °C for 8 min for G6PDH, 73 °C for 10 min for {alpha}-glucosidase, or at 89 °C for 30 min for TIPMDH, and then the temperatures were brought down to 55 °C with the supplementation of the indicated components. After a 90-min incubation at 55 °C, recovered enzymatic activities were assayed as follows: G6PDH activity was assayed at 55 °C by the absorbance at 340 nm in the assay solution (100 mM Tris-HCl, pH 8.8, 40 mM MgCl2, 1 mM NADP+, 3 mM glucose-6-phosphate) (26); {alpha}-glucosidase activity was assayed at 55 °C by the absorbance at 405 nm in the assay solution (50 mM sodium phosphate, pH 6.8, 2 mM p-nitrophenyl-{alpha}-D-glucopyranoside); and TIPMDH activity was assayed at 55 °C by the absorbance at 340 nm in the assay solution (100 mM potassium phosphate, pH 7.8, 1.0 M KCl, 1 mM MgCl2, 0.8 mM NAD+, 0.4 mM isopropylmalate) (25).

K·J Complex Formation—Indicated chaperone components were dissolved in 250 µl of the reaction mixture (50 mM MOPS-NaOH, pH 7.5, 5 mM MgCl2, 150 mM KCl, and 1 mM dithiothreitol) and incubated as indicated. After incubation, a 200-µl aliquot was loaded to a Ni-NTA super flow column (Qiagen), equilibrated with the buffer (50 mM MOPS-NaOH, pH 7.5, 5 mM MgCl2, and 150 mM KCl), and washed with the same buffer. If needed, the column was also washed by the same buffer containing 50 mM imidazole. The proteins bound to the column through the His tag were eluted with the same buffer containing 200 mM imidazole. The proteins in each fraction were precipitated by 10% trichloroacetic acid and loaded to SDS-PAGE (15%).

TDafA Aggregation—The K·J complex was dissolved at 2 µM in 25 µlof the reaction mixture (50 mM MOPS-NaOH, pH 7.5, 5 mM MgCl2, 150 mM KCl, 1 mM dithiothreitol, and 3 mM ATP) containing 0.4 mg/ml bovine serum albumin as a carrier protein to recover small amounts of aggregates. The mixture was incubated at 55 or 89 °C for 30 min, and an aliquot of the solution was centrifuged at 20,000 x g for 10 min at 4 °C. Precipitated and supernatant fractions were analyzed with SDS-PAGE (15%).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Preventing Aggregation by K+J and K·J—The substrate proteins were heat-treated for the indicated period and subjected to centrifugation to separate soluble and aggregate fractions that were visualized by SDS-PAGE. When G6PDH and {alpha}-glucosidase, both from B. stearothermophilus, were heat-treated at 72 and 73 °C, all the proteins were recovered in the precipitation after 8 and 10 min, respectively (Fig. 1, A and B). However, if the TDnaK set, in the form of either K+JorK·J, was included in the solution during the heat treatment, a significant fraction of the proteins remained soluble after 8 and 10 min, respectively, with the K+J set being slightly more efficient (Fig. 1, A and B, right panels). Thus, both the K+J and K·J set have a protective effect on the substrate proteins from heat aggregation. It should be added, however, that the proteins that remained soluble were not native but denatured because the enzyme activities of G6PDH and {alpha}-glucosidase were completely abolished, irrespective of the presence or absence of the K+J and K·J sets. Next, TIPMDH was examined as a representative substrate protein derived from the same bacterium from which the chaperones were isolated. TIPMDH was more heat-stable than the enzymes from B. stearothermophilus, and complete aggregation required a 30-min incubation at 89 °C, an upper limit temperature of T. thermophilus growth (Fig. 1C). In contrast to G6PDH and {alpha}-glucosidase, both the K+J and K·J sets were apparently unable to prevent aggregation of TIPMDH at 89 °C (Fig. 1C, right panel). Hereafter, conditions of heat treatment were fixed to 72 °C for 8 min for G6PDH, 73 °C for 10 min for {alpha}-glucosidase, and 89 °C for 30 min for TIPMDH.



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  		FIG. 1.
Progress of heat aggregation and effect of the K·J and K+J sets. G6PDH (A), {alpha}-glucosidase (B), and TIPMDH (C) were heat-treated at 72, 73, and 89 °C, respectively. After incubation for the indicated times, the solutions were centrifuged, and supernatant (sup) and precipitates (ppt) were analyzed by SDS-PAGE. Right, the heat treatment was performed in the presence of the K·J and K+J sets, respectively. The concentrations of chaperones were 0.2 µM K·J (trigonal) complex, 0.6 µM TDnaK (monomer), 0.6 µM TDnaJ (monomer), and 0.1 µM TGrpE (dimer). Experimental details are described under "Experimental Procedures."

 
TClpB-assisted Disaggregation by K+J and K·J—The ability of TClpB-assisted disaggregation was compared between the K+J and K·J sets. The ability was assessed from the comparison of the amount of aggregates before and after a 90-min incubation at 55 °C. In the experiments shown in Fig. 2, columns 1 and 2, all the proteins (G6PDH, {alpha}-glucosidase, and TIPMDH) were aggregated during heat treatment in the absence of chaperones. These aggregates did not spontaneously become soluble during subsequent incubation at 55 °C (not shown). When TClpB plus the K+J set was added at the time of the temperature shift to 55 °C, a large fraction of the aggregates (>50%) was disaggregated during subsequent incubation at 55 °C (Fig. 2, column 1). By contrast, if TClpB plus the K·J set, instead of the K+J set, was added at the time of the temperature shift, only a small amount of aggregates (<15%) was disaggregated and transferred into the soluble fraction (Fig. 2, column 2). These results show the clear difference between the K+J and K·J sets; the former has TClpB-assisted disaggregation activity of the previously formed aggregates, but the latter has only a trivial amount of such activity.



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  		FIG. 2.
TClpB-assisted disaggregation by the K+J and K·J sets. As illustrated, after substrate proteins were subjected to the heat treatment (G6PDH, 72 °C, 8 min; {alpha}-glucosidase, 73 °C, 10 min; and TIPMDH, 89 °C, 30 min), the temperatures were shifted to 55 °C, and incubations were continued for another 90 min. At the time of temperature shift (0') and the end of the 90-min incubation at 55 °C (90'), aliquots of the solutions were centrifuged, and whole proteins in the supernatant (sup) and precipitated (ppt) fractions were analyzed by SDS-PAGE. Only the bands of substrate proteins are shown. In the case of the supernatant fraction of {alpha}-glucosidase, the nearby thick band of TDnaK was artificially removed for clarity. In all experiments, 3 mM ATP was included from the beginning of heat treatment, and TClpB was added at the time of the temperature shift. The K+J set (column 1) or the K·J set (column 2) was added at the time of the temperature shift. The K+J set (column 3) or the K·J set (column 4) was included from the beginning of heat treatment. The concentrations of chaperones were 0.6 µM K+J (0.6 µM TDnaK and 0.6 µM TDnaJ), 0.2 µM K·J (as trigonal complex), 0.1 µM TGrpE (as dimer), and 0.05 µM TClpB (as hexamer). Other experimental details are described under "Experimental Procedures."

 
As described, when G6PDH and {alpha}-glucosidase were heat-treated in the presence of the K+J or K·J set, a significant amount of the proteins remained soluble. The subsequent incubation at 55 °C with supplemented TClpB resulted in marginal (K+J set) or no further (K·J set) decrease in the amount of aggregates (see the band densities of precipitates, Fig. 2, columns 3 and 4). For TIPMDH, all the proteins were aggregated by heat treatment in the presence of the K+J or the K·J set, but, in either case, a significant fraction was disaggregated during the subsequent incubation at 55 °C (Fig. 2, columns 3 and 4). This result for the K+J set is consistent because the K+J set has activity of TClpB-assisted disaggregation, but the result for the K·J set is enigmatic because the K·J set does not have such activity, as described above; this is because of the dissociation of K·J into K+J at 89 °C, as described later.

Reactivation of Heat-treated Proteins by K+J and K·J—Before the 55 °C incubation, the proteins showed no enzyme activities (<5%) even when a significant amount of proteins existed in the soluble fractions. However, after an incubation at 55 °C, a significant amount of enzyme activities of G6PDH, {alpha}-glucosidase, and TIPMDH was reactivated (Fig. 3, A–C, columns 1, 3, and 4), except when K·J was added after heat treatment (Fig. 3, A–C, column 2). A comparison with the results in Fig. 2 shows that the yields of reactivation are roughly parallel to the amount of protein in the soluble fractions after a 90-min incubation at 55 °C. It appears that during the 55 °C incubation, the K+J and K·J sets reactivated the pre-existing but inactivated soluble proteins with the aid of TClpB. In addition, the K+J set reactivated newly solubilized proteins by the action of disaggregation, giving a slightly better yield of reactivation than the K·J set. The good recovery of TIPMDH by the K·J set shown in Fig. 3C, column 4, is the result of the dissociation of K·J into K+J at 89 °C as described later.



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  		FIG. 3.
TClpB-assisted reactivation of heat-inactivated proteins by the K+J and K·J sets. Recovered activities are shown of heat-inactivated G6PDH, {alpha}-glucosidase, and TIPMDH after incubation at 55 °C for 90 min. The experimental procedures were the same as those in Fig. 2 (columns 1–4). After the heat treatment, enzyme activities of three proteins were nearly completely lost (<5%), and the recovered activities after subsequent incubation at 55 °C for 90 min are shown as percentages of the activities before the heat treatment. Other experimental details are described under "Experimental Procedures."

 
TDafA Inhibits TClpB-assisted Disaggregation Activity of K+J Set—The effect of isolated TDafA on the TClpB-assisted disaggregation/reactivation of G6PDH by the K+J set was tested. G6PDH was heat-treated at 72 °C for 8 min in the absence of a chaperone and subsequently incubated at 55 °C with TClpB, K+J set, and various concentrations of TDafA. The results showed that the reactivation yield of the heat-aggregated G6PDH decreased as concentration of TDafA in the solution increased (Fig. 4). When the amount of TDafA reached 1:1 molar stoichiometry to TDnaK, the reactivation was nearly completely inhibited. In addition, as G6PDH was heat-treated with the K+J set and TDafA (equimolar to TDnaK and TDnaJ), 50–60% of G6PDH activities were recovered by subsequent incubation with TClpB (not shown). This reactivation yield was almost the same as when G6PDH was heat-treated with the K·J set, and less than that was obtained for K+J (~80%). These results suggest that TDafA acts on TDnaK and TDnaJ to form the K·J complex, resulting in a loss of disaggregation activity of previously formed aggregates.



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  		FIG. 4.
Effect of TDafA on TClpB-assisted reactivation of G6PDH by the K+J set. The experimental procedures were essentially the same as Fig. 3A, column 1. Various concentrations of TDafA were added to the reaction mixture at the time of the temperature shift to 55 °C. Recovered activities of G6PDH were plotted against the concentrations of TDafA.

 
Dissociation of K·J into K+J at 89 °C—To test whether TDafA really induced the assembly of TDnaK and TDnaJ, we prepared TDnaK that had a His10 tag at the N terminus. The chaperone activity of the TDnaKhis set was confirmed to be intact (data not shown). The formation of Khis·J was detected by a Ni-NTA column. In the absence of TDafA, only TDnaKhis was adsorbed to the column, and all of the TDnaJ passed alone through the column (Fig. 5A, lane 1). With the addition of TDafA, by contrast, most TDnaJ was retained on the column and was eluted together with TDnaKhis and TDafA by the wash with imidazole (Fig. 5A, lane 2). It was confirmed that Khis·J was stable during the procedures of experiments in Fig. 2, column 4 (Fig. 5A, lane 3), and therefore interaction with denatured protein did not induce decay of Khis·J. By using the reconstituted Khis·J, the heat stability of this complex was examined. Khis·J was exposed to the indicated temperatures for 30 min and applied to a Ni-NTA column (Fig. 5B). From 25 to 85 °C, a majority of TDnaJ was retained on the column and was eluted together with TDnaKhis and TDafA by the wash with imidazole. At 89 °C, however, ~70% of Khis·J decayed in 30 min, producing uncomplexed, individual components. It appears that activity of TDafA as an assembly factor was destroyed at 89 °C. Indeed, when K·J complex was heated to 89 °C for 30 min and centrifuged, TDafA was recovered in the precipitated fraction, whereas most TDnaK and TDnaJ remained in the supernatant fraction (Fig. 5C). To know if the products of decayed K·J have chaperone, especially disaggregation, activity, we substituted K·J in the experiment shown in Fig. 3, column 2, for the K·J that had been already heat-treated at 89 °C for 30 min and measured the reactivated enzyme activities of the substrate proteins after a 90-min incubation at 55 °C (Fig. 6). The results were very different from those of unheated K·J in that heated K·J could disaggregate and reactivate the aggregated proteins as efficiently as K+J. Moreover, the observed reactivation of the aggregated proteins was nearly completely blocked by the addition of native TDafA (Fig. 6). These results supported the conclusion that K·J decayed into K+J and denatured TDafA during incubation at 89 °C. Returning to the enigmatic result of TIPMDH disaggregation by K·Jin Fig. 2, column 4, we can now give a consistent explanation. During heat treatment at 89 °C, K·J was dissociated and exhibited disaggregation activity during subsequent incubation at 55 °C. The same explanation is applicable for the result shown in Fig. 3C, column 4, in which significant activity was recovered by K·J.



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  		FIG. 5.
Temperature-dependent dissociation of K·J into K+J. A, the solution containing TDnaKhis and TDnaJ was incubated at 72 °C for 8 min (lane 1). The solution containing TDnaKhis, TDnaJ, and TDafA was incubated at 72 °C for 8 min (lane 2). The solution containing TDnaKhis, TDnaJ, TDafA, TGrpE (0.1 µM dimer), G6PDH (0.2 µM monomer), and 3 mM ATP was incubated at 72 °C for 8 min and then at 55 °C for 90 min. TClpB (0.05 µM hexamer) was added at the time of the temperature shift (lane 3). B, the same solutions of lane 2 (A), treated at 72 °C for 8 min, were subsequently incubated at 25, 75, 80, 85, and 89 °C for 30 min. These solutions were loaded on the Ni-NTA column and eluted with the buffer containing 0, 50, and 200 mM imidazole. The amounts of TDnaKhis and TDnaJ in the fractions eluted with the buffer containing 200 mM imidazole were analyzed by SDS-PAGE. The step in which the column was washed with 50 mM imidazole buffer, which is less effective, was omitted in B, and only the bands of TDnaJ eluted with the buffer containing 0 and 200 mM imidazole are shown. The concentrations of TDnaKhis, TDnaJ, and TDafA were 0.6 µM (as monomer) throughout experiments A and B. C, 2 µM K·J complex was heated at 55 or 89°C for 30 min in the presence of 0.4 mg/ml bovine serum albumin. After centrifugation, the amount of TDafA in the fractions of supernatant and precipitates were analyzed by SDS-PAGE. Experimental details are described under "Experimental Procedures."

 



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  		FIG. 6.
TClpB-assisted disaggregation activities of heated K·J complex. The experimental procedures were essentially the same as Fig. 3, column 2, except for the use of the K·J complex. Open bar, intact K·J (control); black bar, K·J heated at 89 °C for 30 min; gray bar, TDafA (0.6 µM monomer) added to K·J and heated to 89 °C for 30 min.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
As reported previously (4), the K·J set from T. thermophilus can suppress heat aggregation of substrate proteins and help reactivate heat-inactivated proteins in cooperation with TClpB. However, it is now clear that only soluble fractions of the denatured proteins can be reactivated by the K·J set and that efficient disaggregation of the previously formed aggregates in cooperation with TClpB is possible only by the K+J set (Fig. 7A). The mechanism underlying this difference is yet unknown, but it is likely that TClpB-assisted disaggregation activities are mediated through the transient and sequential action of TDnaK and TDnaJ that cannot be carried out by K·J. Another possibility is that K·J cannot bind to the aggregate because K·J and isolated TDnaK have different peptide-binding repertoires (19, 23).



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  		FIG. 7.
Models for functions of the DnaK chaperone of T. thermophilus. A, chaperone functions of K·J and K+J. Both the K·J and K+J sets can prevent formation of aggregation and reactivate soluble denatured proteins in the presence of TClpB. TClpB-assisted disaggregation activity, however, belongs only to the K+J set. GrpE and ATP are required for all functions but are not shown in the illustration. B, TDafA is a potential thermosensor. In response to heat shock, TDafA is irreversibly denatured, and K·J dissociates into K+J, which starts disaggregation of the heat aggregates.

 
Because the K+J set can carry out all aspects of chaperone function, one might wonder why T. thermophilus cells possess one-half the cellular fraction of TDnaK as K·J. In the case of E. coli, a counterpart of neither TDafA nor the K·J complex has been found; DnaK and DnaJ exist individually in the cell. We speculate that K·J in T. thermophilus cells, with an optimum growth temperature of approximately 75 °C, participates in reactivation of the soluble denatured proteins and suppression of occasional aggregation in cooperation with other chaperones under normal growth conditions. However, when exposed to a stress temperature above 85 °C, TDafA is denatured, and K·J dissociates to K+J to counteract increasing aggregates (Fig. 7B). If this really is the case, TDafA is a kind of thermosensor that strengthens the cellular ability to defend against heat shock. For this purpose, TDafA may be made as a heat-labile sticky protein. In fact, isolated TDafA tend to stick to each other even at room temperature, and 4 M urea is needed to prevent aggregation.


   FOOTNOTES
 
* This work was supported by Grant-in-aid for Scientific Research on Priority Area No. 14037217 (to M. Y.) from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported in part by a research fellowship of the Japan Society for the Promotion of Science for Young Scientists. Back

§ To whom correspondence should be addressed. Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.

1 The abbreviations used are: K·J, trigonal ring complex consisting of three copies each of TDnaK, TDnaJ, and TDafA; T, proteins of T. thermophilus; K+J, uncomplexed TDnaK and TDnaJ; G6PDH, glucose-6-phosphate dehydrogenase; IPMDH, 3-D-threo-isopropylmalate dehydrogenase; MOPS, 3-morpholinopropanesulfonic acid; Ni-NTA, nickel-nitrilotriacetic acid. Back


   ACKNOWLEDGMENTS
 
We thank T. Murakami and J. Suzuki for technical assistance of DNA sequencing, Dr. F. Motojima for providing us with purified TIPMDH, and Drs. K. Motohashi and Y. Kato-Yamada for the helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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