©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transient Interaction of Hsp90 with Early Unfolding Intermediates of Citrate Synthase
IMPLICATIONS FOR HEAT SHOCK IN VIVO(*)

(Received for publication, October 24, 1994; and in revised form, December 13, 1994)

Ursula Jakob Hauke Lilie Ines Meyer Johannes Buchner (§)

From the Institut für Biophysik & Physikalische Biochemie, Universität Regensburg, 93040 Regensburg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells. Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for helping cells to survive under these conditions. However, studies so far have been almost exclusively concerned with the function of Hsp90 under non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock. As a model for heat shock in vitro, we have monitored the inactivation and subsequent aggregation of dimeric citrate synthase (CS) at elevated temperatures. Hsp90 effectively ``stabilized'' CS under conditions where the enzyme is normally inactivated and finally aggregates very rapidly. A kinetic dissection of the unfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions. Hsp90 apparently interacts transiently with these highly structured early unfolding intermediates. Binding and subsequent release of the intermediates favorably influences the kinetic partitioning between two competing processes, the further unfolding of CS and the productive refolding to the native state. As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is especially evident in the suppression of aggregation, the major end result of thermal unfolding events in vivo and in vitro. These effects, which are ATP-independent, seem to be a general function of members of the Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homologue from Escherichia coli gave similar results. It seems likely that this function also reflects the role of Hsp90 under heat shock conditions in vivo. We therefore propose that members of the Hsp90 family convey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggregation and stabilizing the active species.


INTRODUCTION

Members of the 90-kDa heat shock family are extremely abundant heat shock proteins under physiological conditions (Lindquist and Craig, 1988). Despite this fact, only specific interactions with a limited number of relatively non-abundant substrate proteins have been described, the best analyzed being the association of Hsp90 with unliganded steroid receptors and newly synthesized tyrosine kinases (for review, see Pratt, 1993; Jakob and Buchner, 1994). In addition, interactions with various other kinases and tubulin have also been detected (Sanchez et al., 1988; Miyata and Yahara, 1992; Xu and Lindquist, 1993). Recently, we presented evidence that Hsp90 may act as a general chaperone in protein folding at normal temperatures (Wiech et al., 1992). In vitro folding experiments showed that Hsp90 interacts with folding polypeptide chains. Hsp90 promoted functional refolding to the native state by suppressing unspecific side reactions. Further evidence for the chaperone activity of Hsp90 has been obtained for the folding of the muscle specific DNA-binding protein MyoD (Shaknovich et al., 1992; Shue and Kohtz, 1994). Given these in vitro results it is likely that Hsp90 is involved in folding events in vivo under non-stress conditions.

At elevated temperatures the concentration of Hsp90 increases severalfold, suggesting a protective or restoring role of Hsp90 under heat shock conditions (Lindquist, 1980; Nover, 1991; Parsell and Lindquist, 1994). In agreement with this notion, reactivation of thermally inactivated firefly luciferase is effectively stimulated by reticulocyte lysate and also by a purified system which contained Hsp90, Hsp70, and ATP (Schumacher et al., 1994).

In addition to its role in assisting refolding, we wondered if Hsp90 may also influence the thermal unfolding process of proteins in vitro. Mitochondrial citrate synthase (CS) (^1)was chosen as a model substrate because it is inactivated and aggregates rapidly upon incubation at 43 °C. CS is a dimeric protein composed of two identical 48 kDa subunits, which catalyzes the reaction of oxaloacetic acid (OAA) and acetyl-CoA to form citric acid and CoA. In the absence of the substrates, the midpoint of the thermal unfolding transition of CS has been shown to be at 48.0 °C (Zhi et al., 1991). The change in structure is accompanied by a loss in enzymatic activity. Binding of OAA and acetyl-CoA produces large conformational changes in CS (Remington et al., 1982; Wiegand et al., 1984; Karpusas et al., 1990) and significant stabilization under thermal denaturing conditions (Wieland et al., 1964; Srere, 1966). A shift of T from 48.0 to 66.5 °C in the presence of both substrates has been observed (Zhi et al., 1991).

We have kinetically analyzed the thermal unfolding pathway of CS using ligand binding as a tool to identify unfolding intermediates. We demonstrated that Hsp90 binds transiently to unfolding intermediates of thermal unfolding CS. Upon release from Hsp90, the intermediates are able to refold rapidly to the native state. Hsp90 apparently stabilizes the native CS and slows dramatically the subsequent aggregation process.


EXPERIMENTAL PROCEDURES

Proteins and Materials

Hsp90 was purified from both bovine pancreas (Wiech et al., 1993) and yeast. (^2)Escherichia coli Hsp90 was the kind gift of Dr. James Bardwell, University of Regensburg. Mitochondrial CS from porcine heart (E.C.4.1.3.7.), MAK33 IgG, acetyl-CoA, and proteinase K were obtained from Boehringer Mannheim GmbH. BSA was obtained from Serva. Oxaloacetic acid (OAA) and trypsin were from Sigma.

The concentrations of yeast Hsp90 and E. coli Hsp90 were determined using the extinction coefficient of 0.72 and 1.25, respectively, for a 1 mg/ml solution in a 1-cm cuvette (Wetlaufer, 1962). The concentration of bovine Hsp90 was determined according to Bradford(1976) using BSA as a standard. For CS the published extinction coefficient of 1.78 for a 1 mg/ml solution in a 1-cm cuvette was used (West et al., 1990). CS was stored in 50 mM Tris, 2 mM EDTA, pH 8.0. Concentrations of CS and Hsp90 (Minami et al., 1994) are given for the dimeric form.

Determination of CS Activity

Enzymatic assays for CS were performed according to Srere et al.(1963) with the modification that 40 mM HEPES, pH 7.5, was used instead of Tris buffer. The activity assays were carried out at 25 °C.

Thermal Inactivation and Reactivation of CS

Thermal inactivation of CS was achieved by incubating the native enzyme in 40 mM HEPES, pH 7.5, in 1.5-ml Eppendorf plastic tubes at the temperatures given in the figure legends. Unless otherwise indicated, the CS concentration was 0.075 µM. The inactivation kinetics of CS were slightly dependent on the buffer conditions. Therefore, independently of the additives, identical buffer compositions were used within every experiment. To initiate CS reactivation, 100 mM OAA (in 50 mM Tris, 2 mM EDTA, pH 8.0) was diluted 1:100 into the inactivation reactions, and incubation was continued at the given temperature. To determine the inactivation or reactivation kinetics, aliquots were taken at the time points indicated, and activity measurements were performed as described. While inactivation and reactivation kinetics were highly reproducible within every set of experiments, day to day errors of about 10% occurred. This could be due to small changes in the temperature. Furthermore, slight differences between the rate constants of the reactivation kinetics in the absence of any additional proteins (Fig. 4B, CS) and in the presence of IgG (Fig. 2) were observed. To test whether reactivation occurs in the test assay, aliquots of the inactivation reaction were withdrawn and incubated for 1 min in the presence of 0.3 mg/ml trypsin and 0.6 mg/ml proteinase K, respectively, before being added to the test assay. No difference in the time course of CS inactivation was observed, indicating that no significant reactivation occurred during activity assays.


Figure 4: Influence of Hsp90 on the reactivation kinetics of citrate synthase at 43 °C. A, time course of OAA-induced reactivation of CS in the presence of a 10-fold molar excess of yeast Hsp90. The inactivation of CS (0.075 µM) was performed at 43 °C in the presence of 0.75 µM yeast Hsp90. After 5 min of incubation, reactivation was initiated by the addition of 1 mM OAA to the inactivation assay. At the time points indicated, aliquots were taken and the enzymatic activity was determined (bullet). The reactivation reaction followed an apparent first-order reaction (-) with a rate constant of k = 6.1 times 10 sInset, after 5 min of incubation of CS at 43 °C in the presence of Hsp90 (as indicated by the arrow), either inactivation was continued (bullet) or reactivation of CS was initiated by adding 1 mM OAA to the inactivation reaction (circle). The inactivation reaction followed a first-order kinetic with k = 1.7 times 10 s. B, influence of the Hsp90/CS ratio on the rate constants of the fast and slow refolding reaction of CS. CS (0.075 µM) was incubated at 43 °C in the presence of increasing amounts of yeast Hsp90. At the time point where about 60% active citrate synthase was left, reactivation of CS was initiated by adding OAA (final concentration, 1 mM). The rate constants for the fast (bullet) and slow (circle) refolding reaction were obtained by analyzing the reactivation kinetics of CS depending on the Hsp90-concentration used. Inset, influence of the Hsp90/CS ratio on the relative amount of intermediates I(1) and I(2) represented by the amplitudes A(1) and A(2) in percent. The amplitudes were calculated according to the rate constants shown in the main part of the figure.




Figure 2: Inactivation and subsequent OAA-induced reactivation of CS at 43 °C. A, influence of OAA on the thermal inactivation of CS at 43 °C. The inactivation of CS (0.075 µM) was performed at 43 °C in the presence of 1.7 µM IgG. After 5 min of incubation at 43 °C (as indicated by the arrow), either inactivation was continued (bullet) or reactivation was initiated by adding 1 mM OAA to the inactivation reaction (circle). At the time points indicated, aliquots were taken and the enzymatic activity was determined. The inactivation reaction followed a first-order kinetics with k = 7.4 times 10 s. Inset, inactivation of CS (0.075 µM) in the absence (bullet) or presence (circle) of 1 mM OAA at 43 °C. B, time course of OAA-induced reactivation of CS. Correlation of the experimental data (bullet) with single(- - -) and two exponential (-) fits. After 2 min of CS incubation in the presence of 1.7 µM IgG at 43 °C, OAA-induced reactivation was initiated and monitored as described under A. The reactivation reaction followed the two exponential fit better with the two rate constants being k = 50 times 10 s, k = 4.2 times 10 s.



Spectroscopy

Light scattering and fluorescence was measured in stirred quartz cells in a Perkin Elmer MPF44A luminescence spectrometer with thermostated cell holder. To monitor thermal unfolding/aggregation, 15 µM native CS was diluted 1:200 into HEPES buffer (40 mM, pH 7.5) equilibrated at the temperatures given in the figure legends. To determine the aggregation kinetics, both excitation and emission wavelengths were set to 500 nm, with a spectral bandwidth of 2 nm. Changes in fluorescence during the incubation of CS at elevated temperatures were monitored at an excitation wavelength of 285 nm and an emission wavelength of 336 nm. The spectral bandwidth was 5 and 10 nm for excitation and emission, respectively.


RESULTS

Thermal Inactivation and Subsequent Aggregation of CS Is Decelerated by Hsp90

CS is very sensitive to thermal inactivation; activity is almost completely lost following incubation at 43 °C for 10 min (Fig. 1A). The inactivation of CS followed apparent first-order kinetics (Table 1). The slight acceleration of inactivation with increasing protein concentration (data not shown) might be due to subsequent aggregation processes which follow second or higher order kinetics (Zettlmeissl et al., 1979). Within 2 min of incubation at 43 °C, measurable aggregates of CS could be detected by light scattering. Within 12 min the light scattering signal reached a plateau (Fig. 1B).


Figure 1: Inactivation and subsequent aggregation kinetics of citrate synthase (CS) at elevated temperatures. A, influence of Hsp90 on the thermal inactivation of CS. CS (0.075 µM) was incubated at 43 °C in the presence of 0.075 µM Hsp90 (circle), 0.15 µM Hsp90 (), 0.3 µM Hsp90 (Delta), 0.6 µM Hsp90 (), 1.7 µM IgG (box), and in the absence of additional protein (bullet). At the times indicated, aliquots were withdrawn and the activity was determined as described. The solid lines represent single exponential functions with apparent rate constants given in the inset and in Table 1. Inset, influence of the Hsp90/CS ratio on the apparent rate constant of the thermal inactivation of CS at 43 °C. The rate constants were obtained from the inactivation kinetics shown in the main part of the figure. B, influence of the temperature on the thermal aggregation of CS. CS was diluted to a final concentration of 0.075 µM at (box) 37 °C, (bullet) 40 °C, or (circle) 43 °C. No additional protein was present in the incubation reaction. Aggregation was determined by light scattering. C, influence of Hsp90 from different sources on the thermal aggregation process of CS at 43 °C. CS was diluted to a final concentration of 0.075 µM in the presence of 0.15 µM yeast Hsp90 ± 1 mM MgATP (box), 0.15 µM bovine Hsp90 (), 0.3 µME. coli Hsp90 (circle), 0.4 µM BSA (bullet), or 1 mM OAA (). The kinetics of aggregation were determined by light scattering. Inset, thermal unfolding of CS (0.075 µM) in the presence of 0.15 µM bovine Hsp90 or 0.4 µM BSA; the kinetics represent the change in fluorescence at 336 nm.





The presence of stoichiometric amounts of yeast Hsp90 during the incubation of CS at 43 °C changed the inactivation kinetics significantly (Fig. 1A). An equimolar ratio of yeast Hsp90 to CS slowed down the inactivation process by 50%. In the presence of an 8-fold molar excess of Hsp90, the obtained apparent rate constant of inactivation decreased 5-fold (Table 1). Thus, this effect was strongly dependent on the Hsp90/CS ratio (Fig. 1A, inset). Similar results were obtained with bovine and E. coli Hsp90 (Table 1). In control experiments IgG and BSA were added to a 23- and 16-fold molar excess, respectively. Only minor changes in the inactivation kinetics were observed (Fig. 1A, Table 1), showing that the stabilizing effect of Hsp90 is not simply due to additional protein being present during the time course of inactivation.

The presence of Hsp90 also affected the aggregation behavior of CS at 43 °C (Fig. 1C). A 2-fold molar excess of either yeast or bovine Hsp90 or slightly higher amounts of E. coli Hsp90 were sufficient to suppress the thermal aggregation of CS. BSA, again used as a control for unspecific protein effects, showed no significant influence. In contrast to Hsp70 and GroEL whose actions are ATP dependent, addition of ATP shows no detectable effect on the function of eu- and procaryotic Hsp90s in slowing the aggregation process of CS (Fig. 1C and data not shown).

The influence of Hsp90 on the overall unfolding/aggregation reaction of CS could also be monitored by following changes in intrinsic fluorescence of CS (Fig. 1C, inset). In the absence of Hsp90 a strong decrease in the fluorescence of CS was observed during the first 2 min of incubation at 43 °C. This was followed by an increase in fluorescence, indicating CS aggregation. The addition of Hsp90 again resulted in significant differences in the kinetics of CS unfolding. The initial decrease in fluorescence was slower, and no subsequent increase in fluorescence could be detected. This indicated that insignificant aggregation of CS occurred in the presence of the heat shock protein during the time course of the experiment.

Detection of Unfolding Intermediates of CS during Thermal Inactivation

Since little is known about the thermal unfolding behavior of CS, we decided that a description of the thermal unfolding pathway was a necessary prerequisite for understanding the function of Hsp90 in protecting CS from thermal inactivation. We exploited the stabilizing effect of OAA, a substrate of CS (Wieland et al., 1964), to characterize the thermal unfolding pathway of CS. In the presence of this substrate, no significant decrease in enzymatic activity of CS was observed over a 30-min incubation period at 43 °C (Fig. 2A, inset) in contrast to the rapid inactivation normally seen at these temperatures. The dissociation constant of the CSbulletOAA complex is about 6 times 10M (Srere, 1966), and, therefore, the OAA concentrations used may be considered to be saturating. Surprisingly, the addition of OAA during the whole time course of inactivation led not only to the stabilization of still native CS molecules but to a significant renaturation of already inactive molecules (Fig. 2, A and B). After 5 min of incubation at 43 °C in the presence of 1.7 µM IgG (to exclude unspecific protein effects), only about 20% of CS activity remained. Addition of OAA at this time more than doubled the number of CS molecules in the native functional conformation (Fig. 2A).

Analysis of the Formation of Intermediates on the Thermal Unfolding Pathway of CS

To analyze the formation of intermediates during the thermal unfolding process of CS at 43 °C, we initiated reactivation by addition of OAA at different time points during the inactivation. Sixty min after the addition of OAA and further incubation at 43 °C, reactivation was completed and the activity of CS was determined. The activity obtained corresponded to the sum of native CS at the time point of OAA addition plus reactivated CS molecules. The percentage of CS present as unfolding intermediates which are able to reactivate, could thus be calculated as a function of the time of OAA addition (Fig. 3). This experiment allowed us to monitor the time range of the appearance of unfolding intermediates. It should be noted that the values obtained represent minimum concentrations of the intermediates, since irreversible aggregation of the intermediates could decrease the amount observed. Within the first minutes of thermal inactivation at 43 °C, the proportion of intermediates increased, reaching a maximum value of about 30% after 6 min. The concentration of intermediates declined with further incubation at 43 °C, probably due to subsequent irreversible unfolding-aggregation processes, and after 30 min no intermediates could be detected. Further evidence that aggregation limits the accumulation of intermediates came from experiments performed at 37 °C, where CS is rapidly inactivated but only slight aggregation occurred (Fig. 1B, data not shown). After 10 min of incubation at 37 °C, only about 25% native CS was left (t = 4.8 min). Most of the inactive CS molecules were probably unfolding intermediates, since about 70% active species could be regained after the addition of OAA (data not shown). This is presumably due to the smaller influence of aggregation.


Figure 3: Analysis of intermediates on the CS thermal unfolding pathway. CS was incubated at 43 °C in the presence of 1.7 µM IgG. At the time points indicated, aliquots were withdrawn and the activity was measured (bullet). The apparent rate constant obtained was k = 7.4 times 10 s. At the same time reactivation of inactive CS molecules was initiated by adding OAA (final concentration, 1 mM). After further incubation for 60 min at 43 °C, the activity of the reactivated samples was determined. The amounts of intermediates were obtained by subtracting the percentage of native species at the time points, at which the reactivation was initiated, from the total amount of active species determined after the end of the reactivation process (circle). Open squares (box) represent the calculated amount of irreversibly denatured species.



The reactivation reaction of CS could be described by two first-order reactions with a fast phase (k = 50 times 10 s) and a slower refolding reaction with a rate constant of k = 4.2 times 10 s (Fig. 2B). In addition, Fig. 2B shows the correlation of a single and a two-exponential fit with the experimental data. A single exponential fit could not represent the experimental data adequately. A possible explanation for this biphasic reactivation kinetic is the formation of at least two unfolding intermediates I(1) and I(2). Intermediate I(1), which is in a rapid equilibrium with the native state and I(2) which is in a slower equilibrium with I(1) (see ``Discussion''). Due to the presence of both intermediates and the distinct rate constants of the individual refolding reactions, we could apply parallel reactivation mechanisms to distinguish between intermediate I(1) and I(2). We analyzed the amplitudes of the individual reactivation reactions initiated by addition of OAA at different time points during the thermal inactivation of CS. Within the first 2 min of inactivation, reactivation kinetics were dominated by the renaturation of the fast refolding species intermediate I(1) (Table 2). After longer inactivation periods, the fraction of slow refolding CS (intermediate I(2)) increased. This accumulation of I(1) within the first 2 min of thermal inactivation implied that the formation of I(1) is slightly faster than the conversion from I(1) to I(2). That no significant reactivation could be detected within the first 75 s might be due to adsorption of intermediate I(1) to the surface of the reaction vessel or denatured CS which does not aggregate or reactivate.



Intermediates I(1) and I(2) Are Probably Still in Their Dimeric Conformation

The next question we addressed was whether dissociation of the CS molecules takes place during the first steps of thermal inactivation. We first monitored the kinetics of inactivation of CS with varying protein concentrations at lower temperatures, at which the subsequent aggregation of CS should only marginally influence the inactivation kinetics (Fig. 1B). Assuming a dissociation-association reaction during the unfolding of native CS an increase in half-time of the thermal inactivation process with growing protein concentrations would be expected. However, no increase in half-time could be observed, although the CS concentration used varied by a factor of five (0.075-0.375 µM). The inactivation process was understandably slower at 37 than at 43 °C, but the protein concentration still accelerated the time course of inactivation indicating that slight aggregation occurred (data not shown). As a second approach, we followed the concentration dependence of CS reactivation. While the fast refolding reaction could not be adequately resolved to strictly exclude a second-order reaction, the slow refolding reaction of CS did not involve a bimolecular folding step. This data, together with thermal transition measurements performed by Srere and co-workers(1991), allows one to conclude that both intermediates are likely to be still in their dimeric conformation.

Hsp90 Interacts Transiently with Early Unfolding Intermediates of CS

Hsp90 apparently stabilized thermally unfolding CS when present in stoichiometric amounts (Fig. 1A, Table 1). The dissection of the thermal unfolding pathway of CS allowed us to address the question at which step Hsp90 intervenes. We therefore performed reactivation experiments of thermally unfolding CS with OAA (Fig. 2) in the presence of Hsp90 (Fig. 4A, inset). After 5 min of incubation of CS at 43 °C in the presence of a 10-fold molar excess of Hsp90, when about 60% active species was left, OAA was added. The reactivation kinetics could be described by a single first-order reaction with an apparent rate constant of 6.1 times 10 s (Fig. 4A). This rate was slower than the rate constants of the fast refolding reaction (I(1) to N) and comparable to the reactivation reaction from I(2) in the absence of Hsp90. With decreasing amounts of Hsp90 in the reaction, the reactivation kinetics again could be described by two first-order reactions: a fast folding reaction, which was slower than the fast refolding reaction in the absence of Hsp90 and a slow refolding rate, which approached the rates obtained in the absence of the heat shock protein (Fig. 4B). Analysis of the amplitudes revealed that the relative amount of intermediate I(1) increased with increasing concentrations of Hsp90, present during inactivation and subsequent reactivation (Fig. 4B, inset). These results could be explained by Hsp90 binding to the early unfolding intermediate I(1). The lack of the fast refolding reaction of I(1) in the presence of a 10-fold molar excess of Hsp90 implies that the concentrations of free intermediate I(1) are kept low during the inactivation process because Hsp90 binds to this intermediate. As a consequence, the dissociation from Hsp90 becomes rate-limiting for reactivation, consistent with the apparent first-order kinetics. In addition, interaction of Hsp90 with the unfolding intermediate I(2) seems to be likely, since the reactivation kinetic of I(2) (slow phase) also depends on the Hsp90/CS ratio (Fig. 4B). Our experiments also showed that the interaction of substrate with Hsp90 is transient, since if a stable complex were formed, no reactivation would be expected.


DISCUSSION

The data presented here provide evidence that Hsp90 ``chaperones'' protein unfolding under heat shock conditions in vitro. Irreversible aggregation of proteins is the major reaction occurring upon heat shock in the cell (Pelham, 1985; Bensaude et al., 1990; Gragerov et al., 1991). Cells respond to the accumulation of unfolding and aggregating proteins by increasing the synthesis of several Hsps. In most eucaryotic cells, three classes of heat shock proteins, Hsp90, Hsp70, and small Hsps, are predominantly synthesized (Lindquist, 1980; Nover, 1991). Co-localization of Hsps with aggregates formed upon heat shock in vivo suggests that these proteins are involved in the prevention of intracellular aggregation (Bensaude et al., 1990). While the role of Hsp70 as a chaperone in this context was established early (Pelham, 1985), the function of Hsp90 under heat shock has remained enigmatic. Here we demonstrate that the presence of Hsp90 during the incubation of CS at elevated temperatures has significant effects both on inactivation kinetics as well as on the subsequent aggregation process. These effects are comparable to the effects of GroEL (data not shown) and small heat shock proteins on CS inactivation (Jakob et al., 1993). While CS is almost completely inactivated after 10 min of incubation at 43 °C in the absence of Hsp90, almost 40% of active molecules are still present if CS is incubated the same period in the presence of an 8-fold molar excess of yeast Hsp90. Similar results could be obtained using Hsp90 from bovine pancreas and E. coli. Control experiments using similar or even higher concentrations of BSA had no influence on the inactivation kinetics.

Early manifestations of structural changes in CS are accompanied by the loss of enzymatic activity. These minor changes are followed by irreversible aggregation processes due to the increasing influence of intra- and intermolecular hydrophobic interactions at higher temperatures (Jaenicke, 1987). We examined if Hsp90 exerts an influence not just on the initial inactivation of CS but also on the subsequent aggregation process by performing light scattering measurements either in the absence or presence of this heat shock protein. We found stoichiometric amounts of Hsp90 were able to slow down the aggregation process of CS significantly. About the same amount of Hsp90 was required to suppress aggregation as was required for the ``stabilization'' during inactivation. Hsp90 from different sources exerted the same suppression of aggregation, whereas BSA did not change the aggregation kinetics. These effects seem therefore to reflect general properties of members of the Hsp90 family. Unlike GroEL and Hsp70, whose actions require ATP, the function of Hsp90 is ATP-independent.

To elucidate the mechanism of Hsp90 action, an analysis of the thermal unfolding pathway of CS seemed necessary. The stabilizing effect of the substrate OAA was used as a tool to elucidate a kinetic description for the first steps in this thermal unfolding pathway. In the absence of OAA at 43 °C, the inactivation of CS proceeded with a half-time of 1.7 min. The inactivation, which apparently followed first-order kinetics, was only slightly dependent on the CS concentration used. The concentration dependence observed might be due to a subsequent aggregation process, observable by light scattering. After incubation of CS at 43 °C for 2 min, measurable aggregates could be detected. However, when OAA was added, neither inactivation nor aggregation of CS was observed within the time range of the experiment (Fig. 2A, inset, and 1C). These results were in good agreement with experiments by Srere and co-workers, who showed that the formation of the CSOAA complex results in a significantly higher thermostability of CS (Zhi et al., 1991). Next, we investigated how addition of OAA after start of the thermal inactivation affects CS. We could show that the substrate not only stabilized the still native molecules but also led to the reactivation of about 30% of already inactive molecules. Since inactivation of CS might be due to early manifestations of structural changes at the active site (Zhi et al., 1992), it is unlikely that direct binding of OAA to the inactive intermediates triggers their functional refolding. We therefore suggest an equilibrium between native CS and early unfolding intermediates, where the interaction of active CS with OAA will shift the equilibrium toward the native state. As a consequence the refolding of already inactive molecules can occur (). The reactivation kinetics could be explained by two consecutive first-order reactions with a fast and a slow refolding reaction. Thus the existence of at least two unfolding intermediates is proposed. Intermediate I(1) refolds rapidly to the native state (k = 50 times 10 s) and intermediate I(2), which refolds with significantly slower rate constant of k = 4.2 times 10 s. Our experimental data and the thermal transition measurements performed by Srere and co-workers(1991) suggest that both intermediates undergo only unimolecular rearrangements, while remaining in a dimeric conformation. We determined the concentrations of the intermediates I(1) and I(2) during the inactivation process by analyzing the amplitudes of the individual refolding reactions, initiated at different time points after start of the incubation at 43 °C. The accumulation of intermediate I(1) within the first 2 min of inactivation indicated that the unfolding to I(1) is faster than the following unimolecular rearrangement to I(2) and is also faster than the refolding to the native state (k(1) > k(2) and k(1) > k). The thermal unfolding pathway of CS can be described by the following model:

where N-OAA is the native enzyme complexed with oxaloacetic acid, N is the native state, I(1) and I(2) are the dimeric intermediates, and A are aggregates (k(1) > k, k(1) > k(2)).

Upon incubation at elevated temperatures in the absence of OAA, the native enzyme unfolds rapidly to intermediate I(1), which is in a fast equilibrium with the native state. Intermediate I(1) is in slow equilibrium with I(2) which can undergo an irreversible unfolding and/or aggregation reaction. This irreversibility shifts the overall equilibrium toward the aggregates A (k(3) > k). In the presence of OAA, most of the native enzyme is complexed with the substrate. This complex provides higher thermostability, and thus CS is not inactivated at these temperatures. When OAA is added during the inactivation process, native CS molecules are complexed. As a consequence, intermediate I(1) refolds rapidly to the native state, with k apparently >k(2). Since the concentration of free N becomes very small, the refolding of I(1) to N is also essentially irreversible. The refolding of intermediate I(2) is limited by the subsequent unfolding/aggregation reaction with the rate constant k(3).

We could now address the question of the underlying mechanism by which Hsp90 slows the thermal inactivation and subsequent aggregation process of CS. We concluded that transient interactions of Hsp90 with the substrate protein play a major part in regulating the inactivation kinetics of CS. If Hsp90 binds irreversibly to folding intermediates, no positive kinetic effect on slowing the thermal inactivation would be expected. Interestingly, this result was obtained in studies on the effect of GroEL on thermally denaturing alpha-glucosidase (Höll-Neugebauer et al., 1992). GroEL formed stable complexes with the thermal unfolding intermediates, thereby keeping the molecules inactive. As a consequence, no GroEL-dependent deceleration in the rate of thermal inactivation could be observed. On the other hand, transient interactions of GroEL and thermal unfolding rhodanese resulted in a significant increase in the half-time of inactivation (Mendoza et al., 1992), comparable to the results presented here with Hsp90 and CS. This ``thermoprotective'' effect is due to transient interactions of GroEL with rhodanese conformers, which are native-like or are able to renature in the test assay (Mendoza et al., 1992). That Hsp90 interacts transiently with CS unfolding intermediates was further confirmed by performing reactivation studies of thermally unfolding CS in the presence of Hsp90. If OAA was added after the start of the inactivation, part of the inactive molecules could refold to the native state. If partially unfolded CS molecules were irreversibly bound to Hsp90, no reactivation would be expected. Additional evidence for the transient character of the Hsp90 interaction came from refolding studies on chemically denatured proteins in the presence of Hsp90 (Wiech et al., 1992) as well as studies on the helix-loop-helix protein MyoD (Shaknovich et al., 1992). Both reports describe a transient interaction of Hsp90 with substrate proteins. Also in the case of progesterone receptor-Hsp90 interactions steady state assembly and disassembly cycles have been demonstrated (Smith, 1993). Taking these results together we propose that isolated Hsp90 is mainly engaged in transient association-dissociation reactions with non-native substrate proteins. Whenever stable complexes between Hsp90 and substrate proteins (steroid receptors, kinases) could be detected, partner proteins like Hsp70, Hsp56, p60, or p23 were also part of the complex (Pratt, 1993; Stancato et al., 1993; for review see Jakob and Buchner, 1994), and ATP was required for dissociation (Schumacher et al., 1994). The complexes of isolated Hsp90 with substrate proteins, however, are intrinsically unstable, and do not require ATP to trigger dissociation.

The next question we asked was how Hsp90 exerts its function in ``stabilizing'' CS. Therefore, we examined the OAA-induced refolding kinetics of CS in the presence of Hsp90 at high temperature. In the presence of a 10-fold molar excess of Hsp90, only one apparent rate constant could be detected which was comparable to the rate constant of the slow refolding reaction of CS in the absence of additional protein. Reactivation experiments in the presence of lower amounts of Hsp90 led to reactivation kinetics which followed two first-order reactions. The fast rate constant was still smaller than the rate constant of the fast refolding reaction of I(1) in the absence of Hsp90 while the slow refolding reaction approached the refolding rate of intermediate I(2). We suggest that Hsp90 interacts transiently with intermediate I(1) and I(2), since both refolding reactions were slower in the presence of Hsp90. Furthermore, we conclude that after initiating the refolding reaction by adding OAA in the presence of Hsp90, the rate of refolding of intermediate I(1) is slowed by the association and dissociation of Hsp90 with I(1). Upon refolding of intermediate I(2) to I(1) again, the kinetic competition between binding to Hsp90 and refolding to the native state determines the rate constants. Therefore, the rate constants observed mirror the overall refolding reaction of intermediate I(1) and I(2) influenced by the binding to and release from Hsp90. Although the interaction of Hsp90 with I(1) and I(2) and the subsequent suppression of aggregation has been clearly demonstrated, these effects alone cannot be the entire reason for the observed deceleration of the inactivation kinetics. Kinetic simulations (using the program KinSim) suggest that a further, still enzymatically active unfolding intermediate may exist, which is stabilized by interaction with Hsp90. The existence of this intermediate cannot be demonstrated using the techniques employed in this study. However, such ``native-like'' structures which possess enzymatic activity but differ significantly from the crystal structure are not without precedence (Cioni et al., 1994). Our proposal that Hsp90 interacts with highly structured unfolding intermediates seems to be reminiscent of its function in the ``activation'' of steroid receptors and certain kinases (Pratt, 1993; Smith, 1993). Hsp90 seems to bind to these molecules when they are native-like but not in a fully active conformation since they either lack interactions with ligands (in the case of the receptors) or with membranes (in the case of kinases).

Despite the interaction of unfolding intermediates with Hsp90, part of I(2) will unfold irreversibly, mainly dependent on the rate constant k(3). This explains why in the presence of Hsp90 a slow increase in light scattering could still be detected. Based on these findings we propose the following model:

We draw the conclusion that Hsp90 binds transiently to very early, dimeric unfolding intermediates, therefore allowing the stabilizing of still active species and functional refolding of I(1) and I(2) to the native state by keeping the amount of aggregation-sensitive intermediates low.

Taking all these results together and taking into account what is known about the function of Hsp90 in vivo, we propose that structured folding intermediates are the preferential substrates for Hsp90. In many studies reported so far, Hsp90 seems to interact with structured, inactive proteins, missing only the final conversion to the native state (Shue and Kohtz, 1994; Smith, 1993). Recently, this could also be shown for Grp94, the Hsp90 homologue from the endoplasmic reticulum. On the folding pathway of immunoglobulins, Grp94 interacts with late folding intermediates, which are already oxidized, fully folded but not yet assembled (Melnick et al., 1994). The action of Hsp90 in binding to highly structured folding intermediates appears to be rather different from the action of two other chaperones Hsp70 and GroEL which bind to peptide sequences and more disordered folding intermediates, respectively (cf. Hendrick and Hartl, 1993).

In the case of Hsp90 it seems possible, that the folding-unfolding rate of polypeptides determines the interaction with Hsp90. This would give a rational explanation for the ability of Hsp90 to interact with unrelated substrates (Jakob and Buchner, 1994).


FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie (to J. B.) and a scholarship from the Studienstiftung des Deutschen Volkes (to U. J.). 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: Institut für Biophysik & Physikalische Biochemie, Universität Regensburg, 93040 Regensburg, Germany. Tel.: +49-941-943-3039; Fax: +49-941-943-2813.

(^1)
The abbreviations used are: CS, citrate synthase (E.C. 4.1.3.7.); BSA, bovine serum albumin; CoA, coenzyme A; GroEL, 60-kDa heat shock protein from E. coli; OAA, oxaloacetic acid; sHsp, small heat shock protein.

(^2)
U. Jakob, I. Meyer, H. Bügl, S. André, J. C. A. Bardwell, and J. Buchner, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank S. Lindquist for providing the Hsp90 overexpressing yeast strain. We thank Robert Seckler, Rainer Rudolph, Jim Bardwell, Rudi Glockshuber, and Rainer Jaenicke for stimulating discussions and Kari Nadeau for experimental help and continuous interest in the project.


REFERENCES

  1. Bensaude O., Pinto, M., Nguyen, V. T. & Morange, M. (1990) in Stress Proteins: Induction and Function (Schlesinger, M. J., Santoro, G. & Garaci, E. eds) pp. 89-99, Springer Verlag, Berlin
  2. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  3. Cioni, P., Gabellieri, E., Gonelli, M. & Strambini, G. B. (1994) Biophys. Chem. 52, 25-34 [CrossRef]
  4. Gragerov, A. J., Martin, E. S., Krupenko, M. A., Kashlev, M. V. & Nikiforov, V. G. (1991) FEBS Lett. 291, 222-224 [CrossRef][Medline] [Order article via Infotrieve]
  5. Hendrick, J. P. & Hartl, F. U. (1993) Annu. Rev. Biochem. 62, 349-384 [CrossRef][Medline] [Order article via Infotrieve]
  6. Höll-Neugebauer B., Schmidt, M., Rudolph, R. & Buchner, J. (1992) Biochemistry 30, 1609-1614
  7. Jaenicke, R. (1987) Prog. Biophys. Mol. Biol. 49, 117-237 [CrossRef][Medline] [Order article via Infotrieve]
  8. Jakob, U. & Buchner, J. (1994) Trends Biochem. Sci. 19, 205-211 [CrossRef][Medline] [Order article via Infotrieve]
  9. Jakob, U., Gaestel, M., Engel, K. & Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520 [Abstract/Free Full Text]
  10. Karpusas, M., Branchaud, B. & Remington, S. (1990) Biochemistry 29, 2213-2219 [Medline] [Order article via Infotrieve]
  11. Lindquist, S. (1980) Dev. Biol. 77, 463-479 [Medline] [Order article via Infotrieve]
  12. Lindquist S. & Craig E. A. (1988) Ann. Rev. Genet. 22, 631-677 [CrossRef][Medline] [Order article via Infotrieve]
  13. Melnick, J., Dul, J. & Argon, Y. (1994) Nature 370, 373-375 [CrossRef][Medline] [Order article via Infotrieve]
  14. Mendoza, J. A., Lorimer, G. H. & Horowitz, P. M., (1992) J. Biol. Chem. 267, 17631-17634 [Abstract/Free Full Text]
  15. Minami, Y., Kimura, Y., Kawasaki, H., Suzuki, K. & Yahara, I. (1994) Mol. Cell. Biol. 14, 1459-1464 [Abstract]
  16. Miyata, Y. & Yahara, I. (1992) J. Biol. Chem. 267, 7042-7047 [Abstract/Free Full Text]
  17. Nover, L. (1991) in The Heat Shock Response (Nover, L., ed), CRC Press, Boca Raton, FL
  18. Parsell, D. A. & Lindquist, S. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R. I., Tissi è res, A. & Georgopoulos, C., eds) pp. 457-494, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  19. Pelham, H. R. B. (1985) EMBO J. 3, 3095-3100 [Abstract]
  20. Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458 [Free Full Text]
  21. Remington, S., Wiegand, G. & Huber, R. (1982) J. Mol. Biol. 158, 111-152 [Medline] [Order article via Infotrieve]
  22. Sanchez, E. R., Redmond, T., Scherrer, L. C., Bresnick, E. H., Welsh, M. J. & Pratt, W. B. (1988) Mol. Endocrinol. 2, 756-760 [Abstract]
  23. Schumacher, R. J., Hurst, R., Sullivan, W. P., McMahon, N. J., Toft, D. O. & Matts, R. L. (1994) J. Biol. Chem. 269, 9493-9499 [Abstract/Free Full Text]
  24. Shaknovich, R., Shue, G. & Kohtz, S. (1992) Mol. Cell Biol. 12, 5059-5068 [Abstract]
  25. Shue, G. & Kohtz, D. S. (1994) J. Biol. Chem. 269, 2707-2711 [Abstract/Free Full Text]
  26. Smith, D. F. (1993) Mol. Endocrinol. 7, 1418-1429 [Abstract]
  27. Srere, P. A. (1966) J. Biol. Chem. 241, 2157-2165 [Abstract/Free Full Text]
  28. Srere, P. A., Brazil, H. & Gonen, L. (1963) Acta Chem. Scand. 17, S.129-S.134
  29. Stancato, L. F., Chow, Y. H., Hutchison, K. A., Perdew, G. H., Jove, R. & Pratt, W. B. (1993) J. Biol. Chem. 268, 21711-21716 [Abstract/Free Full Text]
  30. West, M. W., Kelly, S. M. & Price, N. C. (1990) Biochim. Biophys. Acta 1037, 332-336 [Medline] [Order article via Infotrieve]
  31. Wetlaufer, D. B. (1962) Adv. Protein Chem. 17, 303-390
  32. Wiech, H., Buchner, J., Zimmermann, R. & Jakob, U. (1992) Nature 358, 169-170 [CrossRef][Medline] [Order article via Infotrieve]
  33. Wiech, H., Buchner, J., Zimmermann, M., Zimmermann, R. & Jakob, U. (1993) J. Biol. Chem. 268, 7414-7421 [Abstract/Free Full Text]
  34. Wiegand, G., Remington, S., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 174, 205-219 [Medline] [Order article via Infotrieve]
  35. Wieland, O., Weiss, L. & Eger-Neufeldt, I. (1964) Biochem. Z. 339, 501-513 [Medline] [Order article via Infotrieve]
  36. Xu, Y. & Lindquist, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7074-7078 [Abstract]
  37. Zettlmeissl, G., Rudolph, R. & Jaenicke, R. (1979) Biochemistry 18, 5567-5571 [Medline] [Order article via Infotrieve]
  38. Zhi, W., Srere, P. A. & Evans, C. T. (1991) Biochemistry 30, 9281-9286 [Medline] [Order article via Infotrieve]
  39. Zhi, W., Landry, S. J., Gierasch, L. M. & Srere, P. A. (1992) Protein Sci. 1, 552-529

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