©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Heat-induced Chaperone Activity of HSP90 (*)

(Received for publication, May 3, 1995; and in revised form, November 2, 1995)

Minako Yonehara Yasufumi Minami Yasushi Kawata (1) Jun Nagai (1) Ichiro Yahara (§)

From the Department of Cell Biology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Tokyo 113, Japan and the Department of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 90-kDa stress protein, HSP90, is a major cytosolic protein ubiquitously distributed in all species. Using two substrate proteins, dihydrofolate reductase (DHFR) and firefly luciferase, we demonstrate here that HSP90 newly acquires a chaperone activity when incubated at temperatures higher than 46 °C, which is coupled with self-oligomerization of HSP90. While chemically denatured DHFR refolds spontaneously upon dilution from denaturant, oligomerized HSP90 bound DHFR during the process of refolding and prevented it from renaturation. DHFR was released from the complex with HSP90 by incubating with GroEL/ES complexes in an ATP-dependent manner and refolded into the native form. alpha-Casein inhibited the binding of DHFR to HSP90 and also chased DHFR from the complex with HSP90. These results suggest that HSP90 binds substrates to maintain them in a folding-competent structure. Furthermore, we found that HSP90 prevents luciferase from irreversible thermal denaturation and enables it to refold when postincubated with reticulocyte lysates. This heat-induced chaperone activity of HSP90 associated with its oligomerization may have a pivotal role in protection of cells from thermal damages.


INTRODUCTION

Stress response and acquired stress tolerance are commonly seen in all organisms from bacteria to higher vertebrates and are considered to be essential defense mechanisms of the cell against various environmental stresses(1) . When cultured mammalian cells are exposed to 45 °C or higher temperatures, they are killed rapidly. However, if they are preheated at nonlethally high temperatures, they can survive the subsequent exposure to 45 °C(2, 3) . These results were interpreted as indications that the acquisition of thermotolerance is attributed to the accumulation of stress proteins. This has been supported by the fact that overexpression of stress proteins such as HSP90(3) , HSP70(4) , and HSP27 (5, 6, 7, 8) rendered cells stress-tolerant. In the case of budding yeast, HSP104 plays a crucial role in stress tolerance, and mutations in this gene drastically reduced survival at extreme temperatures(9) .

It is now accepted that stress proteins prevent catastrophic protein aggregation induced by heat and other stresses and also assist refolding of damaged proteins during recovery from stress(10, 11, 12, 13) . For instance, Escherichia coli molecular chaperones, DnaK/DnaJ/GrpE(14, 15, 16) , GroEL/ES(16, 17) , and ClpA(18) , mitochondrial HSP60(19) , and HSP90 (20) protect substrate proteins from thermally induced aggregation. Furthermore it was revealed that HSP104 does not prevent protein aggregation but rather disaggregates and reactivates stress-damaged proteins(21, 22) . DnaK/DnaJ/GrpE and GroEL/ES are also proposed to have reactivation activities(14, 16) .

HSP90 interacts with a specialized class of proteins such as steroid hormone receptors, protein kinases, and cytoskeletal proteins, and regulates their functions(11, 23) . Since the mode of action of HSP90 has not been fully investigated, its function as a molecular chaperone remains elusive. It has been shown recently, however, that HSP90 functions as a molecular chaperone to prevent protein aggregation (24, 25) and assist refolding of denatured proteins(26) . The endoplasmic reticulum homologue of HSP90, Grp94, was shown to mediate folding and assembly of immunoglobulin chains(27) .

We report here that, distinct from these chaperone activities expressed under normal conditions, HSP90 acquires another chaperone activity, which is latent under normal conditions but is induced by heating, to bind substrates and prevent their irreversible aggregation.


MATERIALS AND METHODS

Refolding of Dihydrofolate Reductase in the Presence of HSP90

HSP90 was purified from porcine brains as described previously(28) . Chicken dihydrofolate reductase (DHFR) (^1)(80 µg/ml; Sigma) was denatured in buffer A (6 M guanidinium HCl (GdmCl), 20 mM Tris-HCl, pH 7.4, 2 mM dithiothreitol (DTT)) by incubation for 30 min at 25 °C and diluted 160-fold (0.5 µg/ml, 22 nM) into buffer B (30 mM Tris-HCl, pH 7.4, 50 mM KCl, 5 mM Mg(CH(3)COO)(2), 2.5 mM DTT) containing varying concentrations of HSP90 as indicated. The mixtures were immediately transferred to a water bath at 25, 43, 46, or 49 °C, respectively, and incubated for 2 min, after which DHFR activities were determined with NADPH (50 µM) and dihydrofolate (50 µM) by monitoring the decrease in absorbance at 340 nm (29) .

S-Labeled DHFR

To introduce an NcoI site at the initiation methionine of mouse DHFR, the DNA fragment was constructed via polymerase chain reaction by using NcoI primer (5`-CCCCATGGTTCGACCATTGAACTGC-3`) and EcoRV primer (5`-GGGATATCTTCCTGTTAGTCTTTCTTCTC-3`), which was used to introduce an EcoRV site 10-base pairs downstream from the termination codon, and the mouse DHFR cDNA clone, pGEM-DHFR provided by T. Endo, as a template. The polymerase chain reaction-amplified DNA was digested with NcoI and EcoRV, and the obtained DNA fragment was ligated to the pET11d (Novagen, Inc.), which had been digested with BamHI followed by blunting and additional digestion with NcoI, to create the plasmid pET11d-DHFR expressing mouse DHFR. The plasmid pET11d-DHFR was transformed into BL21(DE3) and grown in LB medium at 37 °C. Then, growing cells in 10-ml of culture medium were transferred into 2 ml of M9 medium containing 0.2% glycerol and 200 µCi (7.4 MBq) of [S]methionine (Amersham Corp.; 370 MBq/ml), and expression of DHFR was induced with 0.4 mM isopropyl-1-thio-beta-D-galactopyranoside for 2.5 h. Cells were harvested and lysed by freezing and thawing in 200 µl of buffer C (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 2 µg/ml leupeptin) after extensive washing with buffer C. S-Labeled DHFR (4.8 times 10^7 cpm/mg) was purified according to the method described previously(30) .

Binding of S-Labeled DHFR to HSP90

S-Labeled DHFR (8.4 µg) denatured in buffer D (4 M GdmCl, 20 mM Tris-HCl, 0.2 mM DTT) was diluted 60-fold into buffer B containing 1, 2, or 4 µM HSP90 as indicated. The mixtures were heated at 49 °C or kept on ice for 2 min and then resolved by nondenaturing polyacrylamide gel electrophoresis (native PAGE)(31) . The gel was stained with Coomassie Brilliant Blue followed by autoradiography by an image analyzer BAS2000 (Fuji Photo Film Co., Ltd.).

Refolding of DHFR Released from HSP90 by GroEL/ES Complexes

GroEL and GroES were purified from an overexpressing strain, DH1/pKY206 as described previously(32) . GdmCl-denatured DHFR (22 nM at a final concentration) was diluted into buffer B containing 2.2 µM HSP90 and incubated at 49 °C for 2 min. After cooled to 25 °C, 1 µM GroEL/ES complexes composed of GroEL and GroES at a molar ratio of 14:7 were added to the reaction mixture and incubated for further 2 min at 25 °C, followed by addition of NADPH and dihydrofolate in the presence or absence of 1 mM ATP. DHFR activity was measured after a 3-min incubation at 25 °C.

Substrate Binding Activity of HSP90 Induced by Heating in the Presence of Calmodulin-binding Peptide

The calmodulin-binding peptide from Lys to Ile of mouse HSP90alpha (alpha-peptide) was synthesized as described previously(33) . HSP90 (3.6 mg/ml) was incubated in PN buffer (30 mM PIPES, pH 6.7, 100 mM NaCl) at 43 °C for 40 min in the presence or absence of alpha-peptide (0.6 mg/ml) and then cooled to 25 °C. GdmCl-denatured DHFR (22 nM at a final concentration) was diluted at 25 °C into buffer B containing the indicated concentrations of HSP90 preheated with or without alpha-peptide, and DHFR activity was measured.

Effects of alpha-Casein and R-CMLA on Substrate Binding Activity of HSP90

For the experiments of competition (see Fig. 5A), bovine alpha-casein (0.05, 0.1, 0.5, and 1 µM; Sigma) or reduced carboxymethyl bovine alpha-lactalbumin (R-CMLA) (1 µM; Sigma) was mixed with buffer B with or without 1 µM HSP90 preheated with alpha-peptide as described above and incubated for 2 min at 25 °C. To the reaction mixtures, GdmCl-denatured DHFR was added to 22 nM, and DHFR activity was measured as described above. On the other hand, for the experiments of replacement (see Fig. 5B), GdmCl-denatured DHFR (22 nM) was diluted into buffer B with or without 1 µM HSP90 preheated with alpha-peptide and incubated for 2 min at 25 °C, followed by addition of alpha-casein (0.25, 0.5, 1, or 2 µM) or R-CMLA (2 µM). After a further 3-min incubation, DHFR activity was measured.


Figure 5: Effects of alpha-casein or R-CMLA on inhibition of DHFR refolding by HSP90 preheated with alpha-peptide. A, alpha-casein or R-CMLA at the indicated concentrations was mixed with buffer solutions with or without HSP90 preheated with alpha-peptide and incubated at 25 °C for 2 min. After adding denatured DHFR, the activity of DHFR was measured. In each pair of experiments with and without HSP90 at the same concentrations of alpha-casein or R-CMLA, inhibition of DHFR refolding is expressed as percent of the activity obtained without HSP90. The activity of DHFR in the absence of HSP90 was not altered by addition of alpha-casein or R-CMLA (data not shown). B, denatured DHFR was diluted into buffer solutions with or without HSP90 preheated with alpha-peptide and incubated at 25 °C for 2 min. After adding alpha-casein or R-CMLA at the indicated concentrations, DHFR activity was measured. Inhibition of DHFR refolding is expressed as described above.



Effects of HSP90 on Thermal Inactivation of Luciferase

Firefly luciferase (250 nM; Sigma) was heated at 45 or 50 °C for 5 min in buffer E (30 mM Tris-HCl, pH 7.4, 2 mM DTT) in the presence or absence of HSP90 (20 µM). 1-µl aliquots of the reaction mixture were diluted into 10-µl of buffer F (30 mM HEPES, pH 7.4, 50 mM KCl, 2 mM DTT, 5 mM MgCl(2)) with or without 50% (v/v) rabbit reticulocyte lysates (Promega) and incubated at 25 °C for 1 h, followed by measurement of luciferase activity using Promega luciferase assay system and TD4000SP lumiphotometer (NDS, Japan).


RESULTS

Heat-induced HSP90 Oligomers Bind Denatured DHFR and Prevent Its Spontaneous Refolding

We have previously shown that HSP90 exists mainly as a dimeric form in both purified preparations and crude cell extracts(31) . When purified HSP90 was incubated at temperatures higher than 43 °C, oligomeric forms of HSP90, instead of dimers, appeared as ladder-like discrete bands on native PAGE ((33) ; cf. Fig. 2). Since HSP90 is known to function as a molecular chaperone, the oligomerization may be a consequence of recognizing substrate HSP90 by chaperone HSP90. On the other hand, when crude cell extracts of L5178Y murine lymphoma cells were incubated at elevated temperatures, HSP90 dimers were significantly reduced and instead smear rather than discrete staining of HSP90 appeared above HSP90 dimers on native PAGE, which were visualized by immunostaining (data not shown). Taken together, we assume that HSP90 may be induced by incubating at high temperatures to acquire high binding affinity for substrates; that is, if HSP90 is present alone, it undergoes self-oligomerization, and if there are additionally other proteins, HSP90 binds them to form complexes.


Figure 2: Binding of S-labeled DHFR to oligomerized HSP90. GdmCl-denatured S-labeled DHFR was diluted into buffer solutions containing HSP90 at varying concentrations (lanes 1 and 5, 1 µM; lanes 2 and 6, 2 µM; lanes 3, 4, 7, and 8, 4 µM) and heated at 49 °C (lanes 1-3 and 5-7) or left on ice (lanes 4 and 8) for 2 min. The mixtures were resolved by native PAGE, and the gel was stained with Coomassie Brilliant Blue (lanes 1-4), followed by autoradiography (lanes 5-8). Dimeric (2-mer) to octameric (8-mer) forms of HSP90 are indicated on the left.



In order to test this hypothesis, we have first analyzed the interaction between HSP90 and denatured DHFR. It is well known that GdmCl-denatured DHFR spontaneously refolds upon dilution from denaturant(34, 35) . Recovery of the activity was significantly reduced when denatured DHFR was diluted into buffer solutions containing HSP90 at temperatures higher than 46 °C (Fig. 1A, lanes 4 and 6). When HSP90 was omitted, DHFR activity was fully recovered even when incubated at 49 °C (Fig. 1A, lanes 1, 3, and 5). In addition, bovine serum albumin did not affect DHFR refolding at 49 °C (Fig. 1A, lane 7). The inhibitory effect of HSP90 on the renaturation of DHFR observed at 49 °C was concentration-dependent, while no inhibition was observed at 25 °C (Fig. 1B). These results suggest that unfolded DHFR could be bound to HSP90, which is probably converted to oligomeric forms, thereby leading to prevention of DHFR refolding. To examine this possibility, DHFR was labeled with [S]methionine, and its binding to HSP90 was analyzed by native PAGE using GmdCl-denatured S-labeled DHFR. As demonstrated before(33) , incubation of HSP90 at 49 °C induced self-oligomerization (Fig. 2, lanes 1-3). The proportion of oligomers was evidently dependent upon the concentration of HSP90. We found that S-labeled DHFR was bound to oligomerized HSP90 but not to HSP90 dimers (Fig. 2, lanes 5-7). It is detectable for the bands of HSP90 tetramers (4-mer in Fig. 2) that the radioactive bands slightly more slowly migrated compared with the Coomassie Brilliant Blue-stained bands, which resulted from DHFR binding to HSP90 tetramers (Fig. 2, compare lanes 1-3 with lanes 5-7, respectively). When the mixtures were kept on ice, neither HSP90 oligomerization nor DHFR binding to HSP90 occurred (Fig. 2, lanes 4 and 8). Once HSP90 oligomerized by heating, the oligomeric forms stably remained even after the temperature was lowered. ATP did not affect the oligomerization of HSP90 (data not shown). It should be mentioned that HSP90, which had been incubated at 49 °C for 2 min and then cooled to 25 °C, was still oligomeric but unable to prevent DHFR refolding (data not shown; cf. Fig. 4).


Figure 1: Heat-induced activity of HSP90 to prevent DHFR refolding. A, GdmCl-denatured DHFR (22 nM) was diluted into buffer solutions in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 2.2 µM HSP90 and incubated at 43, 46, and 49 °C for 2 min followed by measurement of DHFR activity that is expressed as percent of the recovered activity when incubated at 25 °C for 2 min without HSP90. GdmCl-denatured DHFR was diluted into a buffer solution containing 2 µM bovine serum albumin and incubated at 49 °C for 2 min (lane 7). B, GdmCl-denatured DHFR (22 nM) was diluted into buffer solutions containing HSP90 at indicated concentrations and incubated at 25 or 49 °C for 2 min, followed by measurement of DHFR activity. The activity of DHFR spontaneously refolded at 25 °C for 2 min was set as 100%.




Figure 4: Prevention of DHFR refolding by HSP90 preheated with alpha-peptide. GdmCl-denatured DHFR was diluted at 25 °C into buffer solutions containing the indicated concentrations of HSP90, which had been preheated at 43 °C for 40 min in the presence or absence of alpha-peptide and then cooled to 25 °C, after which DHFR activity was measured. The activity of DHFR refolded in the absence of HSP90 was set as 100%.



Release from Complexes with HSP90 and Refolding of DHFR

The heat-induced binding of DHFR to HSP90 shown above does not appear to be interpreted by the trivial possibility that heated HSP90 was co-aggregated with GdmCl-denatured DHFR, because bovine serum albumin could not substitute for HSP90 (Fig. 1A). Furthermore, it is more important that DHFR complexed with HSP90 was released and refolded to an active form by postincubation with E. coli GroEL/ES complexes without dissociation of oligomerized HSP90. The activity of DHFR, whose spontaneous refolding had been suppressed by approximately 60% by incubating with HSP90 at 49 °C (Fig. 3, lanes 1 and 2), was almost fully recovered by the addition of GroEL/ES in the presence of ATP (Fig. 3, lane 4). When ATP was omitted, GroEL/ES complexes could not show such an activity (Fig. 3, lane 3). Incubation with GroEL/ES and ATP did not alter the ladder-like profile of oligomerized HSP90 on native PAGE, however (data not shown). It is suggested that DHFR is not irreversibly captured by HSP90 but bound to HSP90 in a folding-competent form that can be released by GroEL/ES. Thus HSP90 oligomerization is functionally coupled with this DHFR binding.


Figure 3: Refolding of DHFR complexed with HSP90 by GroEL/ES. GdmCl-denatured DHFR (22 nM) was diluted into buffer solutions without (lane 1) or with (lanes 2-4) HSP90 (2.2 µM) and incubated at 49 °C for 2 min, followed by incubation at 25 °C for 2 min in the presence or absence of 1 µM GroEL/ES complexes. After addition of 1 mM ATP when indicated, DHFR activity was measured. The activity obtained for lane 1 is set as 100%.



We have previously shown that the synthetic 21-residues peptide corresponding to the calmodulin-binding domain of mouse HSP90alpha, alpha-peptide, is able to bind to HSP90 itself and to stimulate the self-oligomerization(33) . When GdmCl-denatured DHFR was diluted at 25 °C into buffer solutions containing HSP90 that had been preincubated at 43 °C for 40 min in the presence of alpha-peptide and cooled to 25 °C, DHFR refolding was significantly prevented (Fig. 4). Furthermore the inhibitory activity of HSP90 preheated with alpha-peptide lasted at least for 2 h even after cooled to 25 °C (data not shown), indicating that HSP90 may be fixed in a binding-competent state for substrates by preheating with alpha-peptide (see ``Discussion''). HSP90 (Fig. 4) or alpha-peptide (data not shown) preheated alone did not exhibit such an inhibitory activity on DHFR refolding.

When alpha-casein, which has properties of partially denatured proteins in its native state (35, 36) and is bound to GroEL(37, 38) , was added to the solutions of HSP90 preheated with alpha-peptide before mixing with denatured DHFR, the inhibitory effect of HSP90 was remarkably reduced in a manner dependent on concentrations of alpha-casein (Fig. 5A). This suggests that the binding of alpha-casein to HSP90 competitively prevented access of DHFR to HSP90. Moreover, postaddition of alpha-casein to the mixtures containing complexes of DHFR and HSP90 caused significant reduction of the inhibitory effect of HSP90, which is due to release and renaturation of DHFR (Fig. 5B). Thus alpha-casein, which does not have a chaperone activity on its own, seems to release DHFR bound to HSP90, probably by replacing DHFR on the HSP90 molecule because it has affinity to HSP90 as shown in Fig. 5A. In contrast to alpha-casein, R-CMLA, which exists in an extended form in aqueous solutions without detergent(38, 39, 40) , did not affect the inhibition of DHFR refolding by HSP90, irrespective of whether its addition was before or after addition of denatured DHFR (Fig. 5, A and B, lanes 6). Therefore HSP90 seems to recognize a folding intermediate structure that is represented by alpha-casein, and DHFR bound to HSP90 is probably in such a structure.

Protection of Luciferase from Irreversible Denaturation by HSP90

We have next examined whether the heat-induced activity of HSP90 to bind denatured DHFR is effective to other proteins that are not able to spontaneously refold differently from DHFR. Firefly luciferase was chosen because it requires the aid of molecular chaperones to refold from a denatured form(15, 41, 42, 43) .

Luciferase was completely inactivated upon incubation for 5 min at temperatures higher than 45 °C (Fig. 6, lanes 1 and 2). Incubation with reticulocyte lysate after thermal inactivation could not reactivate thermally damaged luciferase (Fig. 6, lanes 3 and 4). When HSP90 was present during thermal inactivation, luciferase could not be protected from denaturation (Fig. 6, lanes 5 and 6), but reactivation of luciferase was induced by postincubation with reticulocyte lysates after thermal inactivation (Fig. 6, lanes 7 and 8). Thus it is likely that HSP90 prevented irreversible denaturation of luciferase and maintained it in a folding-competent state during thermal inactivation, from which luciferase could be reactivated by the unidentified component(s) in reticulocyte lysates. Interestingly, the activity of luciferase was reproducibly higher at 50 than at 45 °C (Fig. 6, lanes 7 and 8). This may be explained by the following possibility that at higher temperatures, HSP90 is more rapidly converted to a form that is able to capture denatured proteins, thereby leading to more effective reactivation of luciferase.


Figure 6: Protection of luciferase from irreversible denaturation by HSP90. Luciferase (250 nM) was incubated at 45 or 50 °C for 5 min in buffer solutions with (+) or without(-) HSP90 (20 µM). After cooled to 25 °C, aliquots were incubated at 25 °C for 1 h with (+) or without(-) reticulocyte lysates and then luciferase activity was determined. The activity before thermal denaturation is set as 100%.




DISCUSSION

In this study, we demonstrated using two substrates, chemically denatured DHFR and thermally denatured luciferase, that HSP90 newly acquires a chaperone activity upon incubation at high temperatures (Fig. 1A and Fig. 6). This heat-induced chaperone activity is not detectable before heating at all (Fig. 1B) and is coupled with self-oligomerization (Fig. 2). DHFR bound to oligomerized HSP90 was released and refolded by GroEL/ES in the presence of ATP (Fig. 3). Furthermore alpha-casein chased bound DHFR from the HSP90 molecule to cause its spontaneous refolding (Fig. 5B). On the other hand, the HSP90 oligomers were not dissociated in either case. (^2)alpha-Casein, which possesses a folding intermediate-like structure in its native state(35, 36) , inhibited the binding of DHFR to oligomerized HSP90 (Fig. 5A), probably because HSP90 recognized a structure of folding intermediates and bound alpha-casein. Thus these results exclude the trivial possibility that DHFR binding to oligomerized HSP90 was a consequence of co-aggregation of the two proteins and indicate that oligomerized HSP90 keeps DHFR in a folding-competent state.

HSP90 rapidly lost the newly acquired chaperone activity to bind denatured DHFR while HSP90 oligomers stably remained when it was cooled to 25 °C after heating.^2 Thus it should be emphasized that oligomerized HSP90 per se is not responsible for the novel chaperone activity but that conformational changes of HSP90 leading to its oligomerization are required for induction of the chaperone activity. Both self-oligomerization and the novel chaperone activity are suggested to be attributable to the same conformational changes induced on HSP90 by heat. In fact, the conformation of HSP90 alters upon heating with an increase of hydrophobicity(44, 45, 46) . Although HSP70 is also reported to undergo oligomerization at high temperatures(39) , it is unclear whether the chaperone activity of HSP70 is facilitated. Analysis by native PAGE revealed that DHFR is bound exclusively to oligomerized HSP90 larger than tetramers but not to dimeric forms (Fig. 2). It would be possible that these dimers might not efficiently undergo conformational changes. Therefore they remained dimers and could not bind denatured DHFR.

Although the activated state of HSP90 is labile unless the temperature is left high, when HSP90 is incubated in the presence of alpha-peptide, which corresponds to the calmodulin-binding domain of mouse HSP90alpha (33) , the novel chaperone activity of HSP90 to capture denatured DHFR is prolonged at least for 2 h even after cooled to 25 °C.^2 Furthermore, this chaperone activity was induced at 43 °C (Fig. 4). As discussed previously, HSP90 probably has an intrinsic site interacting with its own calmodulin-binding domain and therefore alpha-peptide can bind to HSP90(33) . In addition, this peptide enhances self-oligomerization of HSP90(33) . Taken together, the hypothetical intramolecular interaction between the calmodulin-binding domain and its counterpart may be involved in a heat-induced process of HSP90 oligomerization. Dissociation of this interaction is likely to be caused by heating, thereby leading to conformational changes of HSP90 responsible for self-oligomerization. If it is the case, it is conceivable that alpha-peptide can stimulate HSP90 oligomerization and stabilize conformational changes of HSP90, which are labile otherwise, even after cooling. Therefore HSP90 preheated with alpha-peptide can long retain the activity to bind denatured DHFR.

When cells are exposed to high temperatures, a significant portion of proteins is denatured and tends to form aggregates, which are extremely harmful to cells. Following are two ways to protect cells from such a catastrophe: one is prevention of off-pathway reactions(14, 15, 16, 17, 18, 19, 20) , and the other is solubilization and reactivation of aggregated proteins (14, 16, 21, 22) . The former seems primarily important in protection of cells, since no matter how effective the latter system is, it cannot work if cells do not survive stress. Assuming that the duration of heat shock is prolonged to exhaust all the preexisting chaperones, more chaperones are necessary. To meet this demand, cells are equipped with a specific system of inducing the synthesis of chaperones, which were therefore called heat-shock (or stress) proteins at first(1, 47) . Furthermore, we propose here another system of inducing a novel chaperone activity without increase in the amount of chaperones. As seen above, HSP90 acquires the chaperone activity to bind partially unfolded proteins, which is latent under normal conditions but appears upon incubation at elevated temperatures. Although HSP90 can bind and protect proteins from irreversible aggregation, it cannot release and reactivate the bound proteins without assistance of other chaperones. Since what is required in an emergency is such an action, the heat-induced activity of HSP90 appears quite important for a defense system of the cell. E. coli chaperone ClpA was reported to have a similar activity toward thermally denatured luciferase in which DnaK/DnaJ/GrpE can reactivate luciferase bound to ClpA(18) . Although we have found in this study that reticulocyte lysates can reactivate luciferase captured by HSP90, the factor(s) in lysates has not been identified yet. Since GroEL/ES complexes can release DHFR bound to HSP90 as shown above, the functionally equivalent eukaryotic chaperonin TRiC (11, 13) might be the most likely candidate for the factor, which remains to be tested.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture of Japan and by a grant from the Human Frontier Science Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-5685-2264; Fax: 81-3-5685-2932; :yahara{at}rinshoken.or.jp.

(^1)
The abbreviations used are: DHFR, dihydrofolate reductase; alpha-peptide, the calmodulin-binding peptide from Lys-Ile of mouse HSP90alpha; DTT, dithiothreitol; GdmCl, guanidinium HCl; native PAGE, nondenaturing polyacrylamide gel electrophoresis; R-CMLA, reduced carboxymethyl bovine alpha-lactalbumin.

(^2)
M. Yonehara, Y. Minami, Y. Kawata, J. Nagai, and I. Yahara, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. T. Endo (Nagoya University) for pGEM-DHFR.


REFERENCES

  1. Parsell, D. A., and Lindquist, S. (1993) Annu. Rev. Genet. 27, 437-496 [CrossRef][Medline] [Order article via Infotrieve]
  2. Li, G. C., and Werb, Z. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3218-3222 [Abstract]
  3. Yahara, I., Iida, H., and Koyasu, S. (1986) Cell Struct. Funct. 11, 65-73 [Medline] [Order article via Infotrieve]
  4. Li, G. C., Li, L., Liu, Y.-K., Mak, J. Y., Chen, L., and Lee, W. M. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1681-1685 [Abstract]
  5. Landry, J., Chretien, P., Lambert, H., Hickey, E., and Weber, L. A. (1989) J. Cell Biol. 109, 7-15 [Abstract]
  6. Rollet, E., Lavoie, J. N., Landry, J., and Tanguay, R. M. (1992) Biochem. Biophys. Res. Commun. 185, 116-120 [Medline] [Order article via Infotrieve]
  7. Mehlen, P., Briolay, J., Smith, L., Diaz-Latoud, C., Fabre, N., Pauli, D., and Arrigo, A.-P. (1993) Eur. J. Biochem. 215, 277-284 [Abstract]
  8. Aoyama, A., Frohli, E., Schafer, R., and Klemenz, R. (1993) Mol. Cell. Biol. 13, 1842-1835
  9. Sanchez, Y., and Lindquist, S. L. (1990) Science 248, 1112-1115 [Medline] [Order article via Infotrieve]
  10. Gething, M.-J., and Sambrook, J. F. (1992) Nature 355, 33-45 [CrossRef][Medline] [Order article via Infotrieve]
  11. Hendrick, J. P., and Hartl, F.-U. (1993) Annu. Rev. Biochem. 62, 349-384 [CrossRef][Medline] [Order article via Infotrieve]
  12. Georgopoulos, C., and Welch, W. J. (1993) Annu. Rev. Cell Biol. 9, 601-634 [CrossRef]
  13. Hartl, F.-U., Hlodan, R., and Langer, T. (1994) Trends Biochem. Sci. 19, 20-25 [CrossRef][Medline] [Order article via Infotrieve]
  14. Skowyra, D., Georgopoulos, C., and Zylicz, M. (1990) Cell 62, 939-944 [Medline] [Order article via Infotrieve]
  15. Schröder, H., Langer, T., Hartl, F.-U., and Bukau, B. (1993) EMBO J. 12, 4137-4144 [Abstract]
  16. Ziemienowicz, A., Skowyra, D., Zeilstra-Ryalls, J., Fayet, O., Georgopoulos, C., and Zylicz, M. (1993) J. Biol. Chem. 268, 25425-25431 [Abstract/Free Full Text]
  17. Hartman, D. J., Surin, B. P., Dixon, N. E., Hoogenraad, N. J., and Hø, J. P. B. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2276-2280 [Abstract]
  18. Wickner, S., Gottesman, S., Skowyra, D., Hoskins, J., McKenney, K., and Murizi, M. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12218-12222 [Abstract/Free Full Text]
  19. Martin, J., Horwich, A. L., and Hartl, F.-U. (1992) Science 258, 995-998 [Medline] [Order article via Infotrieve]
  20. Jakob, U., Lilie, H., Meyer, I., and Buchner, J. (1995) J. Biol. Chem. 270, 7288-7294 [Abstract/Free Full Text]
  21. Parsell, D. A., Kowal, A. S., Singer, M. A., and Lindquist, S. (1994) Nature 372, 475-478 [CrossRef][Medline] [Order article via Infotrieve]
  22. Vogel, J. L., Parsell, D. A., and Lindquist, S. (1995) Curr. Biol. 5, 306-317 [Medline] [Order article via Infotrieve]
  23. Jacob, U., and Buchner, J. (1994) Trends Biochem. Sci. 19, 205-211 [CrossRef][Medline] [Order article via Infotrieve]
  24. Miyata, Y., and Yahara, I. (1992) J. Biol. Chem. 267, 7042-7047 [Abstract/Free Full Text]
  25. Miyata, Y., and Yahara, I. (1995) Biochemistry 34, 8123-8129 [Medline] [Order article via Infotrieve]
  26. Wiech, H., Buchner, J., Zimmermann, R., and Jakob, U. (1992) Nature 358, 169-170 [CrossRef][Medline] [Order article via Infotrieve]
  27. Melnick, J., Dul, J. L., and Argon, Y. (1994) Nature 370, 373-375 [CrossRef][Medline] [Order article via Infotrieve]
  28. Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8054-8058 [Abstract]
  29. Huang, S., Delcamp, T. J., Tan, X., Smith, P. L., Prendergast, N. J., and Freisheim, J. H. (1989) Biochemistry 28, 471-478 [Medline] [Order article via Infotrieve]
  30. Viitanen, P. V., Donaldson, G. K., Lorimer, G. H., Lubben, G. H., and Getenbly, A. A. (1991) Biochemistry 30, 9716-9723 [Medline] [Order article via Infotrieve]
  31. Minami, Y., Kawasaki, H., Miyata, Y., Suzuki, K., and Yahara, I. (1991) J. Biol. Chem. 266, 10099-10103 [Abstract/Free Full Text]
  32. Mizobata, T., Akiyama, Y., Ito, K., Yumoto, N., and Kawata, Y. (1992) J. Biol. Chem. 267, 17773-17779 [Abstract/Free Full Text]
  33. Minami, Y., Kawasaki, H., Suzuki, K., and Yahara, I. (1993) J. Biol. Chem. 268, 9604-9610 [Abstract/Free Full Text]
  34. Touchette, N. A., Perry, K. M., and Matthews, C. R. (1986) Biochemistry 25, 5445-5452 [Medline] [Order article via Infotrieve]
  35. Waxman, L., and Gorldberg, A. L. (1986) Science 232, 500-503 [Medline] [Order article via Infotrieve]
  36. Ostoa-Saloma, P., Ramirez, J., and Perez-Montfort, R. (1990) Biochim. Biophys. Acta 1041, 146-152 [Medline] [Order article via Infotrieve]
  37. Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, F.-U. (1991) Nature 352, 36-42 [CrossRef][Medline] [Order article via Infotrieve]
  38. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer-Hartl, M.-K., and Hartl, F.-U. (1992) Nature 356, 683-689 [CrossRef][Medline] [Order article via Infotrieve]
  39. Palleros, D. R., Welch, W. J., and Fink, A. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5719-5723 [Abstract]
  40. Ewbank, J. J., and Creighton, T. E. (1993) Biochemistry 32, 3694-3707 [Medline] [Order article via Infotrieve]
  41. Frydman, J., Nimmesgern, E., Erdjument-Bromage, H., Wall, J. S., Tempst, P., and Hartl, F.-U. (1992) EMBO J. 11, 4767-4778 [Abstract]
  42. Nimmesgern, E., and Hartl, F.-U. (1993) FEBS Lett. 331, 25-30 [CrossRef][Medline] [Order article via Infotrieve]
  43. Szabo, A., Langer, T., Schr der, H., Flanagan, J., Bukau, B., and Hartl, F.-U. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10345-10349 [Abstract/Free Full Text]
  44. Yamamoto, M., Takahashi, Y., Inano, K., Horigome, T., and Sugano, H. (1991) J. Biochem. (Tokyo) 110, 141-145
  45. Lanks, K. W., London, E., and Dong, D. L.-Y. (1992) Biochem. Biophys. Res. Commun. 184, 394-399 [Medline] [Order article via Infotrieve]
  46. Csermely, P., Kajtár, J., Hollósi, M., Jalsovszky, G., Holly, S., Kahn, C. R., Gergely, P., Jr., Söti, C., Mihály, K., and Somogyi, J. (1993) J. Biol. Chem. 268, 1901-1907 [Abstract/Free Full Text]
  47. Ashburner, M., and Bonner, J. J. (1979) Cell 17, 241-254 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.