T H E JOURNAL OF BIOLOGICAL CHEMISTRY 8 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 2, Issue of January 15. pp. 1479-1487,1993 Printed in U.S.A.

Hsp90 Chaperonins Possess ATPase Activity and Bind Heat Shock Transcription Factors and Peptidyl Prolyl Isomerases*

(Received for publication, August 28, 1992)

Kari Nadeau, Ananya Das, and Christopher T. Walsh From the Department of Biological Chemistry and Molecu.lar Pharmacology, Harvard Medical School, Boston, Massachusetts 021 15

Heat shock proteins of the 82-90 kDa class (hsp82 and hsp90) are abundant, conserved, and ubiquitous from prokaryotes to eukaryotes. Although proposed to be chaperones, they had not been reported to possess enzymatic activity until our recent observation that pure trypanosomatid hsp83 had potent ATPase activ- ity (Nadeau, K., Sullivan, M., Engman, D., and Walsh, C. T. (1992) Protein Sci. 1, 970-979). We have now purified the hsp90 homolog from Escherichia coli (HtpG) and from Saccharomyces cereuisiae (hsp82) to homogeneity and observe ATPase activity with kc,, values of 3 min” and 140 rnin”. In addition, exami- nations of purified rat hsp9O and human hsp90 detect ATPase activity with a kcat of 0.6 min” and 10 rnin”. Each of these hsp9Os undergoes autophosphorylation on serine or threonine residues. In prokaryotes and eukaryotes, the induction of hsps during heat shock is controlled, respectively, by the binding of an alternate a32 or a transcriptional activator (heat shock factor or HSF) at heat shock promoter elements. Here we show that E. coli HtpG immobilized to Affi-Gel beads selec- tively retains a32 while the yeast hsp90 and rat hsp90 retain HSF. The peptidyl prolyl isomerase hsp59 of the FK506 binding class is known to bind to hsp90. We also detect binding of the other family of PPIases, the cyclophilins, to immobilized hsp90, consistent with a functional convergence of protein foldases.

Heat shock proteins (hsps)’ are implicated as cofactors or chaperones in major cell growth-related processes, transcrip- tion, translation, DNA synthesis, protein folding and trans- port, cell division, and membrane function (Morimoto et al., 1990; Gething, and Sambrook, 1992). Of the three major families of heat shock proteins, the 60 kDa class (i.e. prokar- yotic GroEL and eukaryotic hsp60), the 70 kDa class (i.e. prokaryotic DnaK and eukaryotic hsp70s and BiP), and the 90 kDa class (i.e. prokaryotic HtpG and eukaryotic hsp82, hsp90, and grp94), the 60 and 70 kDa classes have been the most extensively studied. Hsp6O or chaperonin 60, a weak ATPase (1 rnin”), plays an important role in preventing the aggregation of misfolded proteins (Buchner et al., 1991; Clarke

* This work was supported in part by National Institutes of Health GM 20011 and by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: hsps, heat shock proteins; hsp90, heat shock protein of 90,000 daltons; HSF, heat shock factor; FKBP, FKBO6-binding protein); CypA, cyclophilin A; hsc, heat shock protein cognate; PAGE, polyacrylamide gel electrophoresis; ATP+, adeno- sine 5’-0-(thiotriphosphate).

et al., 1988) and has been shown to aid in the folding pathways of mitochondrial proteins in eukaryotes (Manning-Krieg et al., 1991). Hsp70, also a weak ATPase (0.1 rnin”), has been deemed the “work horse” of protein folding in prokaryotic and eukaryotic systems because of its tendency to bind and escort a large number of unfolded proteins (Morimoto et al., 1990). The amino-terminal domain of hsp70 contains an ATP-binding site in a kinase-like architecture while the car- boxyl-terminal domain is responsible for its ability to bind peptides and proteins: ATP hydrolysis is necessary for the release of “substrate proteins” from hsp7O (Flaherty et al., 1990).

In prokaryotes, the hsp90 family is represented by a single gene (HtpG) encoding a 624-amino-acid, 71.4-kDa protein which has 40% identity with various eukaryotic hsp90s. The smaller size of HtpG (71 kDa) compared with its eukaryotic counterparts (82-94 kDa) is due to an internal deletion of 50 hydrophilic amino acids in the amino domain and 35 amino acids at the carboxyl terminus (Bardwell and Craig, 1987). HtpG becomes phosphorylated in vivo and is present as a dimer (Spence and Georgopoulos, C., 1989). Deletion of the HtpG gene only slightly reduces the rate of growth a t high temperatures (Bardwell and Craig, 1988).

In eukaryotic cells, the hsp90s are highly abundant proteins under normal conditions (up to 1-2% of cytoplasmic protein in L cells and in yeast), and their levels increase under stress conditions likely to cause unfolding of proteins (i.e. heat, contact with heavy metal ions, oxygen-free radicals, and an- ticancer agents). Cytosolic (hsp90) and endoplasmic reticulum (grp 94) forms of the hsp90 class range in molecular mass in different organisms from 82 to 94 kDa. Hsp9Os are reported to associate with several proteins including kinases (the re- troviral transforming viral protein pp60src (Brugge et al., 1981), as well as casein kinase I1 (Miyata and Yahara, 1992), eIF2a kinase (Matts and Hurst, 1989)), and cytoskeletal proteins (actin and tubulin (Lindquist and Craig, 1988)). Under normal conditions, cytosolic dimers of hsp90 associate in higher eukaryotes with steroid receptors for estrogen, glu- cocorticoid, androgen, and progesterone so that the steroid receptors are stabilized in a partially unfolded, non-DNA binding conformation until the steroid hormone binds to the receptor and hsp90 dissociates (Catelli et al., 1985).

In these different interactions, hsp90 seems to stabilize target proteins in an inactive, partially unfolded, or unassem- bled state. Recently, Wiech et al. (1992) have demonstrated that bovine pancreas hsp90 prevents non-native proteins from unproductive aggregative interactions and have suggested that hsp90 acts as a general chaperone. Genetic experiments in Saccharomyces cereuisiae reveal two versions of the essential hsp9O gene, one heat shock inducible (hsp90) and one ex- pressed constitutively (hsc90); double mutants are lethal at any temperature (Borkovich et al., 1989). This defect can be

1479

compensated by the expression of mammalian hsp90, reflect- ing the functional conservation of the hsp90 among eukar- yotes (Picard et al., 1990).

Because the chaperone functions of hsp60 and hsp70 are associated with their ATPase activity, we asked if hsp90 also might possess an ATPase activity that could drive folding or unfolding of affiliated proteins. While several reports have suggested hsp90 has no ATPase activity, Csermely and Kahn (1991) noted a slow, substoichiometric autophosphorylation of rat hsp90 by ATP and a potential nucleotide-binding site that might explain its binding to ATP-agarose columns. We have recently reported potent, peptide-stimulated ATPase activity in pure hsp90 (hsp83) proteins from the trypanoso- matid parasites Crithidia fasciculata and Trypanosoma cruzi (Nadeau et al., 1992). In the work reported here, we have purified two additional hsp90s to homogeneity, HtpG from Escherichia coli and the S. cereuisiae hsp82 and reexamined rat and human hsp90s. All show ATPase activities and undergo slow, stoichiometric autophosphorylations. In a search for partner proteins with pure, immobilized hsp90s, we confirm the binding of hsp59 and hsp7O and also detect binding of the cyclophilin family of peptidyl prolyl isomerases. Furthermore, the transcriptional activator for hsp gene regu- lation, heat shock factor (HSF) binds to eukaryotic hsp90s while the RNA polymerase alternate a32 binds to E. coli HtpG.

EXPERIMENTAL PROCEDURES

Materials

Chromatography resins used during hsp purification were Q Fast Flow Sepharose and Mono P from Pharmacia LKB Biotechnology Inc. Protein concentrations were determined using Bio-Rad dye re- agent following the manufacturer's directions with bovine serum albumin as standard. All Western blot materials were obtained from Bio-Rad. Immobilon was purchased from Millipore. Bio-Rad hydrox- ysuccinimide-activated Affi-Gel beads were used for affinity chro- matography experiments. Dialysis tubing was from Spectrum. Human hsp9O was purchased from StressGen (Vancouver, Canada). All chem- icals for polyacrylamide gel electrophoresis were from Bio-Rad. Glu- tathione agarose (S-linked) and ATP-agarose (A4793) were from Sigma. [y3'P]ATP was bought from Amersham Corp. a t 6000 cpm/ pmol. The peptide VRLYEA was provided by Dr. Athan Kuliopulos from these laboratories.

All other reagents and chemicals were of the highest grade com- mercially available. Samples of pure rat hsp90 were obtained from Dr. Peter Csermely and pure E. coli, yeast, and human cyclophilin A were provided by Dr. Lesley Stolz, Dr. Stephen Ferguson, Zhi Yu Chang, and Dr. Felicia Etzkorn of these laboratories.

Strains, Plasmids, Extracts

The S. cereuisiae strains W303ECUa and W303ECUa containing the plasmid pKAT6 which carries the yeast hsc82 gene was con- structed by Dr. K. Borkvovich and Dr. L. Arwood. E. coli DH5a cells were grown harboring the GST-human HSFl fusion construct in a pGEX2T vector which was donated by Tom Scheutz and Dr. Robert Kingston. The HtpG gene on pl6bp from Dr. James Bardwell was grown in E. coli DHB4 strain. Rat tissue was obtained from adult rat liver. Yeast HSF (pure and partially pure) were a gift of Dr. Peter Sorger (University of California, San Francisco).

Enzyme Assay for ATPase Activity

To assay the hydrolysis of ATP by the heat shock proteins, release of inorganic phosphate was determined using a malachite green assay (Geladopoulos et al., 1991). Enzyme was diluted with buffer (50 mM HEPES, 2 mM M F , pH 7.2) and ATP was added to 2 mM concen- tration. Typically the total reaction volume was 100 pl after addition of 10 pl (500-1000 ng) of enzyme, and the reaction was incubated at 25 "C for 10-20 min. The assay was quenched with 900 p1 of malachite green reagent (34), and after 10 min at 25 "C the absorbance at 630 nm was determined. The pyruvate kinase/lactate dehydrogenase as-

1480 Biochemical Characterization of Hsp9Os

say for coupled assay of ADP produced by ATP hydrolysis was performed according to (Duncan and Walsh, 1988).

Purification of Proteins S. cereuisiae Hsp82 and HtpG Purification-The buffer used for all

steps was 20 mM bis-tris propane at pH 7.4 with 1 mM dithiothreitol and 1 mM EDTA. All operations were carried out at 4 "C. In the case of yeast hsp82, the procedure was started with -18 g of packed S. cereuisiae which had been grown to late log phase and were lysed in glass bead buffer for 5 min in a glass bead beater (Ausebel et al., 1990) in the presence of antiproteases (0.2 mg of phenanthroline, 0.2 mg of benzamidine, 0.001 mg of phenylmethylsulfonyl fluoride, 1.2 pg of trypsin inhibitor, and 1.2 pg of aprotinin). For E. coli HtpG 1 g of packed E. coli cells which had been grown to late log phase were French pressed in phosphate-buffered saline at pH 7.4 with 0.1% Triton with the same antiprotease solution. Crude cell lysates for both S. cereuisiae and E. coli were obtained after spinning at 10,000 rpm for 20 min and collecting the supernatant.

Ammonium Sulfate Fractionation-A 20430% ammonium sulfate cut was dissolved in -15 (v/v) ml of buffer and dialyzed against 4 liters of the same buffer. The dialysate was centrifuged at 15,000 rpm, and the supernatant was saved for the next step.

Q Sepharose Fast Flow Chromatography-This mixture was then applied to a Q Sepharose column (0.9 X 15 cm, 38 ml) which had been equilibrated with bis-tris propane buffer. Over a 200-ml linear gradient of 0.0-1.0 M KCl, fractions were tested for ATPase activity and correct molecular weight, pooled, and dialyzed. Yeast hsp90 and E. coli HtpG elute at approximately 0.3 M KC1.

Mono P Chromatography-Dialysate was concentrated to 5 ml and loaded on to a Mono P column (HR 5/5) at a rate of 0.3 ml/min over 72 ml in a linear gradient from 0.0 to 1.0 M KC1 of buffer. Yeast hsp90 and E. coli HtpG elute at about 0.5 M KCl. At this point 2 mg of 90% pure S. cereuisiae hsp9O and 1 mg of 95% pure HtpG were obtained. Fractions were pooled and dialyzed against bis-tris propane buffer for the next step.

ATP-Agarose Purification-Each heat shock protein was then in- cubated with ATP-agarose beads (200 pl) in the presence of 5 mM M%+ in 50 mM HEPES at pH 7.2. The column was washed 4 X 0.3 ml of the same buffer, and the bound hsp 83 was eluted with 4 X 0.3 ml of 50 mM HEPES at pH 7.2,lO mM EDTA solution. From 18 g of yeast cells, 1 mg of hsp82 was purified; from 1 g of E. coli cells, 2 mg of HtpG was obtained.

Purification of Human HSFl from E. coli From an overnight culture of DH5a cells harboring the pGEX

fusion human HSFl protein gene, 1 liter of LB medium was inocu- lated. At log phase, isopropyl-1-thio-P-D-galactopyranoside was added to 0.1 mM, and cells were grown for another 5 h. Cells were harvested (-1 g) and resuspended in 1/50 volume of MTPBS. Cells were lysed by passing through a French press twice at 1,200 lb/in'. The bacterial lysate was centrifuged at 10,000 rpm for 15 min at 4 "C to remove cellular debris. Incubation at 4 ' C with GSH-agarose (5 ml) occurred for 15 h, and elution was performed with 150 mM GSH in 2 ml of 50 mM Tris, pH 7.4 buffer.

Molecular Weight Determination and Protein Concentration The molecular mass for all proteins analyzed were determined by

SDS-PAGE with standards of myosin heavy chain (200 kDa), phos- phorylase b (97 kDa), bovine serum albumin (68 kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa).

All protein concentrations were determined according to manufac- turer's instruction (Bio-Rad).

Kinetic Analysis Michaelis constants (Km) and maximal initial velocities (Vmax)

for ATP, and peptides were determined by the malachite green assay. Initial velocity measurements were made at 5 substrate concentra- tions which ranged from 5 p~ to 1 mM for ATP. Kinetic constants were calculated from Lineweaver Burk plots.

Phosphoamino Acid Analysis Phosphoamino acid analysis was carried out by the method of

Kamps and Sefton (1989). 7 pg of hsp9O was incubated in the presence of 50 mM HEPES, pH 7.2, 200 p~ of [y3'P]ATP (6000 cpm/pmol) with 5 mM M e for 30 min at 37 "C. The reaction was stopped by boiling for 5 min in the presence of Laemmli buffer. The hsp90 band

Biochemical Characterization of Hsp9Os 1481

was excised from Immobilon (Millipore) after being transferred from a n SDS-9.5% PAGE and hydrolyzed with constant boiling 5 N HCI for 1 h a t 110 "C. The hydrolysates were lyophilized and resuspended in pH 1.9 buffer with phosphorylated standards (threonine, serine, and tyrosine). Two-dimensional electrophoresis was carried out a t pH 1.9 buffer and then pH 3.5 buffer. Plates were dried, stained with ninhydrin, and analyzed by autoradiography.

Phosphorylation of HspSOs For time course studies, the hsp9Os from ATP-agarose steps were

incubated with [[gamma]-32P]ATP as described above with M e , a t 37 "C, and for various time points. The radioactivity of the hsp83 bands was quantitated by densitometry of autoradiograms of SDS- 9.5% PAGE. Each of these studies was performed twice.

Immunoblotting and Antibodies Western blot analysis was performed according to (Ausebel et al.,

19891, and antibodies were used in 1:500 or 1:lOOO dilutions. Poly- clonal antisera against hsp90 was donated by Dr. David Toft (AC88) and Dr. David Engman (IC1 anti hsp90), anti-human HSF was a gift of Tom Scheutz (Massachusetts General Hospital, Harvard Medical School), a n t i 3 cereuisiae HSF was a gift from Dr. Peter Sorger, and anti-human FKBP59 was obtained from Mark Albers (Harvard Uni- versity Chemistry Dept.). a32 antibodies were a gift from Dr. Carol Gross (University of Wisconsin). Antibodies to casein kinase I1 B subunit were a gift of Dr. Yoshihiko Miyata. Antisera to E. coli, yeast, and human CypA were a gift of Dr. Kim Mcintyre from Hofmann La Roche.

Preparation of Tissue Extracts and Affinity Chromatography Adult rat liver extract was prepared according to Liu et al. (1991)

and adsorption of hsp9O-Affi-Gel beads was performed with hydrox- ysuccinimide activated Affi-Gel-15 beads in NaHC03, pH 8.5 (4 ml) with 2 ml of 2 mg of hsp90 sample (beads were prepared on smaller scales depending on amount of pure hsp90 available). Incubation with proteins from rat liver extracts, E. coli extracts, and S. cereuisiae extracts was carried out as follows: 0.1 ml of Affi-Gel beads which had been protected with P-mercaptoethanol were incubated with 5- 10 pl of tissue extract (5 mg/ml) with 100 mM HEPES, pH 7.5, to a total volume of 0.2 ml for 2 h at 4 "C. The supernatant from this incubation (0.1 ml) was added to 0.1 ml of hsp9O-Affi-Gel beads (1 mg/ml hsp90 attached/packed column volume) with 0.1 ml of buffer A (50 mM Tris, pH 7.5, 150 mM NaCI, 5 mM M e , and 5 mM Ca2+) and then incubated for 2 h a t 4 "C. The beads were washed with buffer A with 0.1% Triton X-100 three times, removing the super- natant each time. Finally, the 0.1 ml of spun beads were mixed with 2 x SDS dye buffer, boiled for 5 min, and loaded on an SDS-PAGE gel for subsequent silver staining or for immunoblotting.

Proteolysis of HspSOs-Experiments were carried out according to the procedure in Liberek et al. (1991).

RESULTS

Purification of E. coli HtpG and Yeast Hsp90 and Charac- terization as ATPases-E. coli HtpG, a 71.4-kDa protein, was discovered by homology to Drosophila hsp83 in hybridization analysis. It has 40% identity to eukaryotic hsp82-90s and no significant homology to the hsp7O class of heat shock proteins represented by E. coli dnaK (69.5 kDa). To assess potential enzymatic activity of HtpG, we have purified it to homoge- neity as shown in Fig. 1. HtpG separates from DnaK by selective elution on ATP-agarose and pure HtpG has no contaminating dnaK by SDS-PAGE analysis. Similarly, we have purified the S. cereuisiae hsp90 to apparent homogeneity (Fig. l), and Western analysis with polyclonal antibodies against hsp70s fails to reveal any hsp7O contaminants (de- tectable to 3 ng of hsp7O protein) compared to a 90% pure mouse hsp70 control (data not shown). As reported for hsp90s from higher eukaryotes, HtpG and yeast hsp9Os migrate pri- marily as dimers on native gels (data not shown).

E. coli HtpG is an active ATPase as measured both by Pi formation using a malachite green assay and by ADP forma- tion by coupled pyruvate kinase/lactate dehydrogenase assay. The kcat value is 3 min" and the ATP K,,, is 500 PM (Table

1 2

FIG. 1. Silver-stained 12% SDS-PAGE gel with yeast hsp90 (lane I ) and E. coli HtpG (lane 2).

TABLE I ATPase oarameters of Durified hspgos

ATPase kinetic Autophosphorylation

parameters

K, L, k., Stoichiometry" PAAb

Hsp9Os E. coli C. fasciculata Yeast Rat Human

Hsp7Os DnaK BiP

500 60 70

160 80

200 55

rnin"

3 150' 140

10 0.6

51 0.2

x104

6.4 1.5 S, T

4.2 2.0 S, Twu 2.5 1.0 S, T

NDd ND ND

4.3 1.0 S, T

ND 0.01' S, T ND 0.03' S, T

Stoichiometry is defined as the moles of Pi incorporated/mole of

PAA, phosphoamino acid analysis. E Nadeau et al., 1992.

ND, not determined. Liberek et al., 1991.

monomer hsp90.

'Rothman, 1989.

I). For comparison, the ATPase activity of the E. coli hsp70 representative, DnaK, is reported as 1.0 min" (Liberek et al., 1992), 3-fold lower. These kat differences rule out contami- nation by DnaK (69.5 kDa) as a possible source of the more potent ATPase activity in HtpG preparations.

Yeast hsp90 is an even more potent ATPase with a kcat of 140 min" and an ATP K,,, of 70 PM (Table I). This high turnover number for ATP hydrolysis matches that which we have recently described for trypanosoma1 hsp90 proteins of 150 min" (Nadeau et al., 1992). Again the higher ATPase hat of yeast hsp90 argues strongly against the possibility that the observed ATPase activity is due to contaminating hsp7O none of which is detectable by silver staining or by Western blot. Although yeast hsp7O ATPase activities have not been re- ported, kcat values for other hsp70s (Table I) are three orders of magnitude lower.

At this juncture, we reexamined the hsp90s of higher eu- karyotes, using purified rat and human hsp90. Both proteins were also free of hsp70 by Western blot analysis (data not shown). The rat hsp90 has a low but reproducible, detectable ATPase activity, with a kcat of 0.6 min", 3-fold higher than the kat of 0.2 min" reported for bovine hsp70. Pure human hsp90 shows ATPase activity (Fig. 2) with a kcat of lO/min with a K, for ATP of 80 PM. This ATPase level is 30-60-fold higher than kcat values reported for hsp70. In aggregate these data validate that the proteins of the hsp90 family from bacteria to human do have ATPase activity.

1482 Biochemical Characterization of HspSOs

/ *,'

I / I

0- 0 10 20

time In mlnuter FIG. 2. Time course of the catalysis of ATP hydrolysis by

1.2 pg (0.014 nmol) (-) and 2.4 pg (0.028 nmol) (- - -) of human hsp9O compared to control of 0.0 nmol of human hsp9O ( filled squares) using Pi release assay (Geladopoulos et al., 1991) at indicated incubation times with 1 mM ATP/Mg.

We have previously noted that trypanosoma1 hsp90 ATPase activity is stimulated up to 5-fold by the addition of peptides (Nadeau et al., 1992). When the same peptides were used with HtpG or rat hsp90, no stimulation of ATPase activity was detected. The hexamer VRLYEA did produce a 1.5-2.0-fold stimulation of the yeast hsp9O ATPase activity. As an initial effort to assess the role of partner proteins on ATPase activ- ity, pure cyclophilins from E. coli, yeast, and human, which bind hsp9Os (below), had no effect on the ATPase activities of their homologous hsp90s. Heat shock factor isolated from HeLa cells (Schuetz et al., 1991) enhanced the ATPase activ- ity of human Hsp9O from 10 min-' to 33 min" at a molar stoichiometric ratio of 1:l with an apparent K,,, of 500 nM. The physiological implications and quantitative measure- ments of HSF-hsp9O interactions are now under investigation.

Autophosphorylation of HspSOs-In view of reports of in vivo phosphorylation of hsp90s that arise from the action of such kinases as casein kinase I1 (Miyata and Yahara, 1992), the newly detected ATPase activity of hsp9Os raises the prospect that some of the hsp90 phosphate groups may be due to autophosphorylation during ATP hydrolysis. In the hsp70 family, slow autophosphorylation to low fractional stoi- chiometries have been reported for BiP (Rothman, 1989) and DnaK (McCarty and Walker, 1991). Csermely and Kahn (1991) observed autophosphorylation of rat hsp90 to 0.01 mol/ mol in a 30-min incubation. Three hsp90s, yeast, E. coli, and rat, at 90-95% purity, were incubated with [y3'P]ATP for periods up to 7 h and the time course of autophosphorylation followed by SDS gel analysis and autoradiography as sum- marized in Fig. 3. Yeast hsp90 can acquire up to two phos- phoryl groups/709-amino-acid polypeptide chain; HtpG ac- quires 1.5 phosphate groups/molecule within 4 h while rat hsp9O acquires one stoichiometric phosphoryl group by 400 min. Phosphoamino acid analysis (Table I) indicates phos- phorylation predominantly on serine with a lower amount on threonine. At values of 2.5 X min" to 6.4 X min" which is 1,000-10,000-fold slower than an ATPase catalytic cycle (Table I), these phosphorylations are too slow to repre- sent competent phosphoenzyme intermediates. However, they could possibly reflect regulatory events. We have determined that stoichiometric phosphorylation of yeast, rat, human hsp90, and E. coli htpG has no effect on ATPase activity (as assessed by the pyruvate kinase/lactate dehydrogenase assay) but phosphorylation state could affect partner protein recog-

time in minutes

FIG. 3. Time course and stoichiometry of autophosphory- lations of pure E. coli. HtpG (-) with a molar equivalent of 1.5 mol of 32P to 1.0 mol of HtpG, yeast hsp90 (- --) with a molar equivalent of 2.0 mol of 32P to 1.0 mol of yeast hsp90 and rat hsp90

hsp907 pg of each hsp90 was incubated with [Y-~'P]ATP and with M e , at 37 "C, for various time points. The radioactivity of the hsp90 bands was quantitated by densitometry of autoradiograms of SDS-

nition. Subsequent identification of which specific hydroxy- amino acid side chains are phosphorylated may help to iden- tify the catalytic site. Further such autophosphorylation sites will serve as markers to distinguish these from other sites, such as serine 231 and 263, on hsp9Os that may be phos- phorylated in vivo by kinases such as casein kinase I1 (Lees- Miller and Anderson, 1989).

Given the reports that casein kinase I1 and eIF2a kinase copurify tenaciously with hsp90s (Miyata and Yahara, 1992; Matts and Hurst, 1989) and that hsp90 stimulates casein kinase I1 activity up to 15-20-fold, casein kinase I1 had to be eliminated as a possible source of the ATP hydrolysis activity associated with the purified hsp90s. First, hydrolysis by hsp90s yields equal amounts of ADP and Pi from ATP whereas a kinase would yield ADP and a phosphorylated product such as phospho-hsp90. This argues against contam- inating kinase activity being responsible for ATP consump- tion. Second, casein kinase I1 is separable from hsp90 by heparin-Sepharose chromatography (Miyata and Yahara, 1992). When HtpG and yeast hsp90 were further purified on such columns, there was no dimunution in ATPase-specific activities, and no change in the kinetics of autophosphoryla- tion was seen after incubation of the yeast hsp9O protein with heparin-Sepharose. Third, while casein kinase I1 is reported to phosphorylate 2 serines on human hsp90 (Ser-231 and Ser- 263) (Lees-Miller and Anderson, 1989) that process would yield a limit of two ADP/hsp90 substrate molecule. In our ATPase kinetic assays, yeast hsp90 assays were followed for 1500 turnovers of ATP to ADP and Pi, HtpG for 30 turnovers, and human hsp90 for at least 30 turnovers, numbers far in excess of any stoichiometric phosphorylation by a kinase.

Proteins That Associate with Purified HspSO-Most of the evidence for formation of complexes between hsp90 and other proteins comes from immunoprecipitation studies. As a com- plementary method, we have employed affinity columns of purified hsp90 coupled to Affi-Gel to probe for protein inter- actions under physiological, non-stress conditions (150 mM NaC1, and pH 7.4). Background levels were much lower when pure hsp90s were immobilized on this column rather than GST-hsp9O fusion proteins on glutathione-agarose columns (data not shown). In each case controls were performed using P-mercaptoethanol-blocked beads. Post-nuclear supernatants of cell extracts a t 5 mg/ml were passed through the columns, and bound proteins were eluted by boiling in 2 x SDS buffer.

Many proteins were selectively retained (Fig. 4A) on the

(-.-.- ) with a molar equivalent of 1.0 mol of 32P to 1.0 molof rat

Biochemical Characterization of HspSOs A B C

- 97 kd 4

- 71 kd

- 4 3 k d

- - 29 kd

- 18 kd

FIG. 4. Associated proteins detected by immobilized hsp90 chromatography of E. coli HtpG)A) yeast hsp90 ( B ) and rat hsp90 (C). Affi-Gel-preabsorbed tissue extracts (5 mg/ml) were added to 0.1 ml of hsp9O-Affi-Gel beads (1 mg/ml hsp90 attached/ packed column volume) with 0.1 ml of buffer A and then incubated for 2 h a t 4 "C. The beads were washed with buffer A with 0.1% Triton X-100. 0.1 ml of spun beads were mixed with 2 X SDS dye buffer, boiled for 5 min, and loaded on a 12% SDS-PAGE gel for silver staining or immunoblotting.

HtpG column, two of which were identified by specific anti- body detection on Western blots (Fig. 5a) corresponding to bands on a silver-stained gel (Fig. 4.4) migrating a t 18 and 32 kDa. Higher molecular weight bands, which have not been identified yet, were found to be associated with an HtpG column after washing extensively with detergent and isotonic buffer conditions. One of the lower molecular weight bands was the RNA polymerase subunit alternate a32 (Gross and Craig, 1991). u32 is required for transcription of heat shock genes in E. coli and previously has been shown to bind DnaK, DnaJ, and GrpE (Gamer, J. et al., 1992; Liberek et al., 1992). The other bound protein was E. coli cyclophilin, 18 kDa. We have previously purified a periplasmic E. coli Cyp, and there is a highly homologous cytoplasmic E. coli cyclophilin (Liu et al., 1990). Each has peptidyl prolyl isomerase activity and both are thought to be involved in protein folding processes. When purified E. coli Cyp was tested directly for retention by the HtpG column, it was retained. Notably retention was dependent upon both ATP and Mg". The ATP requirement for Cyp-HtpG interaction could be met also by ATP-yS, which is not detectably hydrolyzed by hsp90. u32 binds to HtpG independently of ATP/Mg (Fig. 5a).

In proteins retained by immobilized yeast hsp90, four major species could be detected by silver staining, ranging in size from 27-29,52-54, and 62 kDa (Fig. 4B). Through the use of specific antibodies and Western blotting, three retained pro- teins were identified (Fig. 5b). One, the yeast transcriptional activation factor HSF, at 130 kDa was present at low abun- dance and detectable by Western blot analysis but not by the less sensitive silver staining. Second, the yeast homolog of hsp59 (a heat shock protein also known as FKBP59) (Renoir et al., 1990; Tai et al., 1992) migrated as a doublet between 53 and 54 kDa (Fig. 4B) detectable by silver staining and by Western blot with goat polyclonal antibodies against human FKBP59 (Fig. 56). These 53- and 54-kDa bands most likely correspond to the yeast homolog of hsp59 although this ten- tative identification will require further studies with purified yeast proteins. Third, a band not detected by silver staining (Fig. 4B) but detected by Western blot was identified as a the 18-kDa yeast cyclophilin (Fig. 5b). Yeast CypA has been purified previously in these laboratories (Zydowsky et al., 1992) and indeed when pure does bind selectively to yeast hsp90 columns (data not shown). As with the HtpG-E. coli Cyp pair, the yeast hsp9O-yeast CypA interaction requires Mg and ATP for retention on the hsp90 column, but the yeast

a:

1

b: 1

C 1

2

2

1483

3

3 4

FIG. 5. Identification of associated proteins released from columns of each hsp90 described in Fig. 4 by Western analy- sis. Note: since the primary antibodies were prepared from different

blots. a, E. coli 032 a t 32 kDa (lane 1 ) with 1 mM ATP/Mg and mammalian sources, single Western blots were needed for certain

without ATP/Mg (lane 2) and E. coli cyclophilin a t 18 kDa with 1mM ATP/Mg (lane 3). b, yeast hsp90-associated proteins with lane 1, yeast HSF (band at 130 kDa); lane 2, yeast FKBP59 homologs (bands a t 53-54 kDa); lane 3, yeast Cyp (band a t 18 kDa) with 1 mM ATP/ Mg; and lane 4, yeast Cyp without ATP/Mg. c, rat hsp90-associated proteins with lane 1, rat HSF (83 kDa band) and FKBP59; lane 2, rat CypA (18 kDa) with, and lane 3 without 1 mM ATP/Mg.

HSF-yeast hsp90 complex does not (Fig. 5b, lanes 3 and 4). When rat tissue post-nuclear supernatant extracts were

applied to a rat hsp9O column, five proteins (Fig. 4C) of 18, 27, 42, 59, and 96 kDa were retained selectively as assessed by silver stain. Of these five, three have been identified by immunoblotting (Fig. 5c). The first protein identified is the cognate heat shock factor, an 83-kDa species in mammals (Larson et al., 1990; Schuetz et al., 1991; Rabindran et al., 1991). Again this protein is of iow abundance such that it is seen with anti-HSF antibody but not in silver staining. The 59-kDa protein is the hsp59 (FKBP59) known to be affiliated with hsp90 in steroid receptor complexes. The 18-kDa protein is rat cyclophilin A in accord with the HtpG and yeast hsp9O results above. Although we did not have samples available of

1484 Biochemical Characterization of HspSOs

pure rat cyclophilins or HSF, we did prepare pure human CypA and GST-human HSF (Rabindran et al., 1991), and they are selectively bound to the rat hsp90 columns (not shown). Both the HSF and FKBP59 binding to hsp90 are independent of ATP, but the rat hsp90-cyclophilin A inter- action required ATP and Mg (Fig. 5c). A column was run under similar conditions with pure immobilized mouse hsp70, and human cyclophilin was found to bind in the presence of ATP/Mg. Again, ATPyS could be substituted for ATP, sug- gesting that ATP binding but not ATP hydrolysis is involved in hsp90-cyclophilin recognition. The 27-kDa band was iden- tified as casein kinase I1 /3 subunit (27 kDa) by immunoblots (data not shown). We presume the 42-kDa band is the casein kinase I1 a subunit (42 kDa). Casein kinase I1 has already been reported to be a partner protein for higher eukaryotic hsp90s (Miyata and Yahara, 1992). Finally, when we probed the SDS gel from the retentate on the rat hsp9O column with antibody to hsp70, a positive response was detected on West- ern blots even though the level of hsp7O was below the limit of silver stain detection. Clearly, the profiles of proteins associated with hsp90s yield a combination of proteins with detectable affinity that vary in abundance.

Human cyclophilin A was immobilized and extracts of Jurkat T cells, rat liver, rat heart, and rat brain tissue were passed through the column (Fig. 6) in the presence of ATP/ Mg. Among the several (eight to nine) tissue-specific proteins which bound to the CypA column, immunoblotting with anti- hsp90 and anti hsp70 antibodies indicated that hsp90 was present in each tissue, whereas hsp70 was present only in liver tissue. While these may reflect as yet undetermined relative abundances of hsp70 and hsp90 in specific tissues, it may also signify that the cyclophilin class of protein foldases have higher affinity for hsp90 than hsp70, a point to be further investigated quantitatively.

Proteolytic Susceptibility ofHsp90s"Given previous studies on the altered sensitivity of DnaK to proteolytic digestion in the presence and absence of ATP (Liberek et al., 1991) and the fact that a 44-kDa fragment of hsc70 accumulates in vitro and has yielded a crystal structure (Flaherty et al., 1990), we analyzed the sensitivity of purified hsp90s to protease action. Yeast and trypanosoma1 hsp90s (data not shown) were resist- ant to trypsin digestion in the presence or absence of ATP over a 40-min period under conditions used to digest hsp70s (Liberek et al., 1991). The HtpG was differentially sensitive to trypsin in the presence of ATP, with ATP accelerating cleavage without altering the pattern of proteolytic fragments (Fig. 7, a and b). After 10 min, a 62-kDa band which represents the whole HtpG protein was still visible with small quantities of 52-, 44-, and 28-kDa band fragments present. At 15 min, there was clear evidence that without 1 mM ATP, the 62-kDa HtpG band remained; yet, with 1 mM ATP, the band at 62

1 2 3 4

FIG. 6. Tissue-specific rat hsp90 and rat hsp70 binding to an immobilized human CypA Affi-Gel column as detected by Western blotting. Lane 1, hsp90 from Jurkat T cells; lane 2, hsp90 and hsp70 from rat liver; lane 3 hsp90 from rat heart, and lane 4, hsp90 from rat brain.

a b

Tune In rnlnutes after trypsm dlgestlon

"

I

C: Time: 0 5 10 15 5 10 1 5 (min) r"

d:

v 4 - 8 6 kd

!.r* &b -1 4 - 4 7 kd -" :-A 4-36 kd

FIG. 7. Time course of proteolysis with trypsin according to Liberek et al. (1991) with 0.12 pg trypsin, 2.0 pg of Hsp90, 40 mM HEPES at pH 7.6, 8 mM MgClz, 0.3 mM EDTA, 2.0 mM dithiothreitol, 20 mM NaCl, 20 mM KC1 at 25 'C. Reactions were stopped by adding SDS-gel sample buffer and boiling for 10 min. Trypsin digestion of HtpG (a, without ATP and b with 1 mM ATP) at 10, 15,30 and 45 min. Trypsin digestion of Human hsp90 ( c with 1 mM ATP and d with 1 mM ATP, 10 p ~ C y p A ) a t 0,5,10, and 15 min. Breakdown products are indicated as 36,47, and 86 kDa.

kDa was not present indicating that ATP increased the sus- ceptibility of HtpG to trypsin digestion. At 30 and 45 min, the 52-kDa fragment was still detected when HtpG had been digested without ATP; this band did not exist a t 30 and 45 min in the presence of ATP. Once digested to 40 min, full HtpG ATPase activity was present even though only a 43- and 28-kDa fragment remained. The 43-kDa fragment could correspond to the ATPase functionality of the HtpG protein (Fig. 7, a and b).

Human hsp90 sensitivity to trypsin increased detectably on addition of ATP (Fig. 7c). On the other hand human hsp90 digestion by trypsin could be protected kinetically by addition of human cyclophilin in the presence of ATP (Fig. 7 4 . When Hsp9O was incubated with ATP and trypsin for 5 min, the 90-kDa band representing Human Hsp9O was present with additional 47- and 36-kDa fragments and a t 10 min, the 90- kDa band was almost completely degraded. In Fig. 7d, incu- bation with ATP and CypA rendered the human Hsp9O band less susceptible to digestion with trypsin since the 900-kDa band was still detected a t 15 min by silver staining. The 47- and 36-kDa fragments were still present a t 15 min after CypA, ATP, trypsin, and Hsp9O had been incubated. Human hsp90 ATPase activity no longer persisted after trypsin digestion at 40 min. These data suggest ATP and cyclophilin may be altering the conformation of some hsp90s. They also confirm the ATP-dependent cyclophilin interactions detected with immobilized hsp90 (Figs. 4-6) columns. Subsequent sequence analysis of fragments may help define regions of hsp90 that interact with these substrates.

DISCUSSION

The results reported here for pure E. coli HtpG, yeast hsp90, rat and human hsp90, and from our recent work on purified trypanosomatid hsp83s from C. fusciculuta and T. cruzi estab- lish that hsp90 proteins are all ATPases. This knowledge may lead to a redefinition of hsp90 in various cellular functions as active rather than passive chaperones. The range of hsp9O kcat values as ATPases is almost 250-fold from 0.6 min" (rat hsp90) to 3-6 min" (HtpG and human), to 150 min" (tryp- anosomes and yeast). For comparison the ATPase activity of hsp7O members are 0.2 min" to 1.0 min" for DnaK, hsc70, and BiP (grp78) as noted in Table I. The analysis of catalytic efficiency ratios (kccat/Km) of hsp90s and hsp7Os as ATPases

Biochemical Characterization of HspSOs 1485

thus reveals that the hsp90s are from 10-1000-fold more effective catalysts than the hsp70s.

Since the ATPase activity of hsp7Os has been suggested as a major thermodynamic force in release and/or folding of hsp70-associated proteins, the more potent intrinsic ATPase action of hsp90s may suggest they also may be active protein foldases in addition to more passive chaperoning roles. The particularly potent ATPase action of yeast and parasite hsp90s may require regulation and suppression in vivo to avoid running down ATP stores. In this connection, GroES, a 10-kDa monomer, binds to, regulates, and decreases the intrinsic ATPase activity of GroEL, the E. coli hsp60. Further evidence for regulation of hsp ATPase activity is revealed by the observation that the 44-kDa NH2-terminal fragment that contains ATPase activity of hsc70 has 2-fold higher ATPase than intact protein (Chappell et al., 1987). It may be that some partner proteins/subunits of the hsp90 chaperones will exert negative regulation of the hsp90 ATPase activity.

Peptide stimulation of ATPase activity of hsp70s by up to 3-5-fold has previously been reported (Rothman, 1989), and i t is well established for hsp7Os such as DnaK that ATP induces release of partner proteins from immobilized DnaK (Rothman, 1989). We have previously noted up to a 5-fold stimulation of C. fasciculatu hsp90 ATPase activity (to about 1000 min") by peptides and note a 1.5-%fold stimulation of yeast hsp90, human hsp90, and HtpG ATPase here, support- ing a functional similarity between hsp7O and hsp90 chaper- ones as peptide/protein recognizing ATPases.

An ATP-dependent interaction of three hsp90s from E. coli, yeast, and human, with their cognate cyclophilins was de- tected in this work giving indication that ATP hydrolysis by hsp90 is likely to alter conformation and partner protein recognition. The ATP requirement could be fulfilled also by ATPyS which is not detectably hydrolyzed by the purified hsp90s. ATP also alters the kinetics of protease digestion of HtpG and rat hsp90 (increasing rates of proteolysis) as did the partner protein cyclophilin for rat hsp90 (decreasing rates of proteolysis). It is likely that there are two (or more) discrete domains in hsp90, an ATP hydrolysis domain and a protein- binding domain, as in the hsp7Os, but this remains to be established.

Heat shock proteins are known to be phosphorylated in vivo and while exogenous kinases are clearly implicated, part of the covalent phosphorylation may derive from slow auto- phosphorylation on threonines and serines. In the case of DnaK, threonine 199 is the major autophosphorylation site as established by phosphopeptide isolation and subsequent mutagenesis (McCarty and Walker, 1991). The T199A mutant of DnaK was inactive as an ATPase, kcat dropping from 0.06 min" to below the detection limit of 0.01 min". To assess the propensity of hsp90 ATPases to produce autophosphoryl- ated species, we have conducted time course and stoichiometry determinations with the pure hsp9Os and demonstrate phos- phorylations that achieve or surpass stoichiometric modifi- cation on serines or threonines for HtpG, yeast hsp90, try- panosomatid hsp90s (Nadeau et al., 1992) and rat hsp90. These results set the stage for subsequent phosphopeptide analysis to identify residues phosphorylated. While these autophosphorylations are clearly too slow by orders of mag- nitude to be relevant to catalysis, it is likely these serine and threonine side chains are in or near to the hsp90 ATPase active sites and will help establish and define those sites. There is a crystal structure of the NH,-terminal 44-kDa fragment of hsc70 (Flaherty et al., 1990), but there is essen- tially no primary sequence homology between hsp7O and hsp9O to aid in topology assignments in hsp90s. It may also

be of interest to see how mutants of hsp90 defective for autophosphorylation act not only as ATPases but also as chaperones for specific partner proteins. Finally, identifica- tion of specific residues that comprise the autophosphoryla- tion sites will provide a base line to analyze any additional in vivo phosphorylation sites which arise from association and intermolecular phosphorylation by such kinases as casein kinase 11.

The major role attributed to the highly abundant cytosolic hsp90s in eukaryotes has been the recognition of unfolded proteins, the prevention of aggregation (Wiech et al., 1992), and more interestingly the formation of specific complexes with partner proteins (Picard et al., 1990; Dalman et ul., 1991). While hsp90s are found in association with cytoskeletal pro- teins, protein kinases, viral proteins, and other proteins, the relevance of any particular interaction physiologically has been difficult to document although it is known that the protein is essential (Borkovich et al., 1989) and required in yeast for active transcription factor-glucocorticoid function (Picard et al., 1990). Much attention has been focused on complexes of hsp90 in higher eukaryotes with steroid recep- tors, for glucocorticoids, estrogen, and progesterone. The glu- cocorticoid receptor hsp90 complexes are largely cytosolic, and heat shock leads to dissociation of steroid receptors, translocation to the nucleus, and transcriptional activation of steroid-responsive genes. Thus, hsp90s act as functional cy- toplasmic anchors to keep these transcription factors out of the nucleus. The mechanism by which steroid binds the receptor and releases dimeric hsp9O from these complexes is obscure, although ATP is required (Hutchinson et al., 1992). The colocalization of hsp70 was thought to account for the ATPase activity in complexes of hsp90, hsp70, hsp59, and steroid receptors. While that premise may still hold, the potent catalytic efficiency ratio of hsp9O as ATPase now merits examination of its catalytic function in steroid recep- tor-hsp90 dissociation.

To address the rules for molecular recognition of partner proteins by hsp9Os, we immobilized purified hsp90s and ex- amined proteins selectively retained from post-nuclear super- natants in E. coli, yeast, and rat liver. This approach is limited in that it gives only qualitative information, is biased toward high abundance partner proteins, and could be clouded by nonspecific interactions. However, it offers a starting point to identify associating proteins for further characterization. We have been able to identify several proteins involved in both protein folding and transcriptional activation which may be relevant to hsp90 function.

The E. coli HtpG protein is potently induced by heat shock, with some 26-fold elevation of protein level (Spence and Georgopoulos, 1989). This and other E. coli heat shock genes are transcribed by RNA polymerase containing the alternate a32. The availability of u32, a low abundance protein esti- mated at 20-30 molecules/cell (Gamer et al., 1992), for inter- action with RNA polymerase core may be controlled by whether 032 is free or sequestered in complex with other proteins. Recently, it has been shown that DnaK, DnaJ, and GrpE bind a32 in a complex (Gamer et al., 1992; Liberek et al., 1992). Thus, a proposed feedback loop has been validated for an hsp70 family member. Our findings that the hsp90 type chaperone HtpG also binds a32 indicate that the hsp90 family may also help regulate hsp gene transcription by controlling availability of u32. Presumably as E. coli proteins unfold upon heat shock, they could compete with a32 for HtpG, raising the pool of free a32 and increasing hsp gene transcription by RNA polymerase-a32.

TWO partner proteins for hsp90 in both yeast and rat liver

1486 Biochemical Characterization of HspSOs

extracts are from the two separate families of peptidyl prolyl isomerases implicated in protein folding, the FK506-binding proteins (FKBPs) and the cyclosporin-binding cyclophilins. Recently, Callebaut et al. (1992) have named the heat shock protein-binding immunophilins as HBIs. The particular FKBP detected, FKBP59, has recently been identified by two groups (Renoir et al., 1990; Tai et al., 1992) as the previously known hsp59, a heat shock protein detected in hsp90-steroid receptor complexes by immunoprecipitation analysis (Catelli et al., 1985). It is clear from Western blots that hsp59 (FKBP59) is a major component retained by rat, and it is suggestive from immunoblots in the case of immobilized yeast hsp9O. If the hsp59 yeast homolog associated with yeast hsp9O is further validated (i.e. by amino-terminal sequencing), this would suggest a direct interaction of hsp90 and hsp59 since yeast does not contain steroid receptors. The observation of the 18-kDa cyclophilin family retained by hsp90 is also in- triguing given cyclophilin's established role in protein folding in vitro (Walsh et al., 1992), and while one must always worry about nonspecific affinity of one abundant cytosolic protein to another, the ATP dependence of the interaction argues for specificity. The interaction of human hsp90 and human CypA is not antagonized by CsA (up to 10 p ~ ) suggesting that hsp90 is not binding to the peptidylprolyl isomerase active site of cyclophilin. It will take mutant studies to establish which chaperone may be playing an active foldase role in the Cyp. hsp9O and in the FKBP59. hsp90 complexes.

Hsp9Os show selective binding of several proteins, have established function as chaperones (Picard et al., 1990; Wiech et al., 1992), and have an ATPase activity which can be stimulated up to 5-fold by peptides (such as hsp7Os). Addi- tional association of hsp90s with both structural variants of PPIases may indicate that higher order complexes exist in the cell for the return of unfolded and misfolded proteins to native states. It is quite possible then that hsp70, hsp90, FKBP59, and Cyp may all interact to refold proteins in a heat shock response. A presumably analogous cascade in prokar- yotes of successive action of hsp70 and hsp60 species has recently been suggested for conversion of extended nascent polypeptides into compact, folded native structures (Langer et al., 1992).

An equally interesting association with hsp90 from yeast and higher eukaryotes is that of the cognate heat shock factors, a protein of apparently 110-130 kDa in yeast and 83 kDa in humans. Each HSF were selectively bound from crude cell extracts to immobilized hsp90s, and in addition, the purified yeast and human HSFs were specifically liganded. This is the first demonstration of an hsp9O-HSF interaction and parallels a recent observation of hsp70-HSF interaction (Abravaya et al., 1992). The hsp7O. HSF complex was detected by distinct methods; in murine extracts, HSF binding to an oligonucleotide containing the heat shock element sequences was supershifted by hsp70 detected by hsp7O antibodies. These in vitro hsp-HSF interactions need now to be quantified and assessed for in vivo relevance.

The transcriptional activation of HSPs (hsp70, hsp90, and hsp59) in eukaryotes is logically parallel but mechanistically distinct from the prokaryotic paradigm noted above for ~ 3 2 . Hsp gene transcription is effected by HSF binding to heat shock element sequences in the hsp gene promoter regions but it had been known that new protein synthesis of HSF after heat shock was not required. Preexisting HSF was converted from an inactive to active state on heat shock, and one hypothesis had been that perhaps preexisting HSF was complexed with hsp70 and/or hsp90 (Hightower, 1991; Abra- vaya et al., 1992; Sorger, 1991). On thermal shock, as unfolded

proteins accumulated and became ligands for hsps, the com- petition would release free HSF to act as transcriptional activator to turn on synthesis of more hsps to increase the chaperone/refolding capacity of the heat-shocked cell. The data here provide the first evidence in support of this proposal for hsp9O-HSF interaction and corroborate those being re- ported for hsp7O-HSF (Abravaya et al., 1992). The on and off rates, the role of ATP hydrolysis, and the basis of hsp9O and HSF recognition sites remain to be determined. Of particular interest is to study what effects the multiple phosphorylation and oligomerization states of HSF, known to affect transcrip- tional activation, have on hsp90 association. If hsp9O-HSF interactions play a role in maintaining HSF in the cytoplasmic compartment and keeping it transcriptionally silent much in the same way hsp90 keeps the steroid receptor/transcription factors tethered and inactive, then it will be of interest to see if common domains of hsp9O are involved.

Acknowledgments-We thank Dr. Susan Lindquist, Jane Chang, and Laura Arwood for the yeast hsc82 plasmid and for reading the manuscript. We also thank Jim Bardwell for the HtpG plasmid, Peter Csermely for pure rat hsp90, and Tom Scheutz of the Kingston Laboratories (MGH, HMS) for human H S F l and 2 genes. We appre- ciate antibodies from Stuart Calderwood (anti-hsp70), and David Engman (anti-hsp90). We would like to thank Paul Jackson for donating the rat liver tissue. We would also thank S. Ferguson, L. Stolz, Z. Chang, and F. Etzkhorn for the CypA from E. coli, yeast, and human. We appreciate the peptides provided by the laboratories of Dr. Mary Jane Gething. We appreciate the helpful conversations from Dr. Susan Lindquist, Dr. Mary Jane Gething, Dr. Peter Sorger, Dr. Johannes Buchner, Dr. Alfred Goldberg, Dr. Linda Hendershot, Dr. Robert Kingston, and Dr. Miles Brown.

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