Reconstitution of the steroid receptor.hsp90 heterocomplex assembly system of rabbit reticulocyte lysate.

Rabbit reticulocyte lysate contains a multiprotein system that assembles steroid receptors into a heterocomplex with hsp90. In the case of the glucocorticoid receptor (GR), the receptor must be bound to hsp90 to bind steroid, and assembly of the GR.hsp90 complex restores the hormone binding domain of the receptor to the steroid binding conformation. Using both direct assay of heterocomplex assembly by Western blotting and indirect assay of assembly by steroid binding, it has previously been determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) are required to form a functional GR.hsp90 complex. It is also thought that a 60-kDa protein (p60) may be required for progesterone receptor.hsp90 heterocomplex assembly, but a complete heterocomplex assembly system has never been reconstituted from individual components. In this work, we separate the proteins of rabbit reticulocyte lysate into three fractions by DEAE chromatography and then reconstitute the GR.hsp90 heterocomplex assembly system in a manner that requires the presence of each fraction. Fraction A contains most of the hsp70 and all of the p60 in lysate, and elimination of p60 by immunoadsorption inactivates this fraction, with bioactivity being restored by the addition of bacterially expressed human p60. The activity of fraction A is replaced by a combination of highly purified rabbit hsp70 and lysate from p60-expressing bacteria. Fraction B contains hsp90, and its activity is replaced by purified rabbit hsp90. Fraction C contains p23, and its activity is replaced in the recombined system by highly purified bacterially expressed human p23. A minimal GR.hsp90 heterocomplex assembly system was reconstituted with purified rabbit hsp70 and hsp90 and bacterially expressed human p23 and p60. This reports the first reconstitution of this apparently ubiquitous protein folding/heterocomplex assembly system.

In cytosols prepared from hormone-free mammalian cells, steroid receptors exist in multiprotein complexes that contain hsp90, 1 an immunophilin (either FKBP52/hsp56 or CyP-40), a 23-kDa acidic protein, p23, and, often, substoichiometric amounts of hsp70 (for review see Refs. 1 and 2). This multiprotein receptor heterocomplex can be assembled under cell-free conditions by incubating immunoadsorbed, hormone-free receptors (preincubated with salt to strip them free of associated proteins) with rabbit reticulocyte lysate (3,4). This heterocomplex assembly system is versatile in that reticulocyte lysate has been used to assemble heterocomplexes containing the oncogenic protein kinases Src or Raf with hsp90 and p50 (5,6). The heterocomplex assembly system appears to be ubiquitous, in that concentrated lysates from other mammalian, insect, and even plant cells are able to assemble the glucocorticoid receptor (GR) into a complex with hsp90 (7). The GR must be associated with hsp90 for it to bind steroid (8), and conversion of the GR to a steroid binding form constitutes a rapid assay for heterocomplex assembly (4,9).
Over the past five years, several conditions required for steroid receptor⅐hsp90 heterocomplex assembly by reticulocyte lysate have been defined. Assembly is an ATP/Mg 2ϩ -dependent process that requires the presence of a monovalent cation, such as K ϩ or NH 4 ϩ (9, 10). Removal of hsp70 (11) or its inactivation with antibody (10) inactivates the assembly process, and the activity is restored by purified hsp70 (11), proving that hsp70 is required to form complexes with hsp90. p23, a conserved, widely distributed protein that is neither a heat shock protein nor an immunophilin (12), binds directly to hsp90 in an ATPdependent manner (13) and is required for assembly of a functional GR⅐hsp90 heterocomplex (14,15). It is thought that a 60-kDa protein of reticulocyte lysate is also required for heterocomplex assembly. This p60 was originally observed in reconstituted progesterone receptor complexes when ATP was limiting (10) or at early stages of assembly (16). p60 is a protein that was identified by Honoré et al. (17) to be up-regulated by viral transformation, and it is a homolog of the nonessential yeast heat shock protein, Sti1 (18). It is established that p60, hsp90, and hsp70 interact with each other (19), and they are thought to act in a cooperative manner in receptor heterocomplex assembly.
The immune adsorption of hsp90 from reticulocyte lysate and other cell extracts copurifies several proteins, including hsp70, p60, p23, and the immunophilins FKBP52/hsp56 (also called p59) and CyP-40 (19 -21). The immunoadsorbed hsp90 with its coimmunoadsorbed proteins contains all the components of reticulocyte lysate required to reassociate the GR with hsp90 and to restore the receptor to its steroid binding state (22). The immunophilin components of this foldosome machinery are not required for GR⅐hsp90 heterocomplex assembly (23), but a loosely associated component that is easily washed away is required to form a functional (i.e. steroid binding) GR⅐hsp90 complex (22). This weakly bound component has been identified as p23 (15).
It is thought that the core complex for receptor heterocomplex assembly may be the hsp90⅐p60⅐hsp70 unit and that p60 dissociates after hsp70 and hsp90 bind to the receptor, with p23 becoming tightly bound to the receptor heterocomplex at that time. To define the mechanism of heterocomplex assembly in greater detail, the system must first be reconstituted from purified components. In this work, we have separated rabbit reticulocyte lysate into three fractions that contain the four identified components of the heterocomplex assembly system. When the fraction containing hsp70 and p60 is reconstituted with two other fractions containing hsp90 and p23, respectively, the heterocomplex assembly activity of reticulocyte lysate is reconstituted. Substitution of the reticulocyte lysate fractions with purified rabbit hsp90 and hsp70 and bacterially expressed human p23 and p60 yields a system that assembles a GR⅐hsp90 complex with steroid binding activity. Although it is likely that other proteins (e.g. rDnaJ, p48) are required for optimal heterocomplex assembly in intact cells, this work identifies a minimal system for receptor heterocomplex assembly under cell-free conditions.

Materials
[6,7-3 H]Triamcinolone acetonide (42.8 Ci/mmol) and 125 I-conjugated goat anti-mouse and anti-rabbit IgGs were obtained from DuPont NEN. Untreated rabbit reticulocyte lysate was from Green Hectares (Oregon, WI). Protein A-Sepharose, Sepharose CL-6B, and goat anti-mouse and anti-rabbit IgG horseradish peroxidase conjugates were from Sigma. Phenyl-Sepharose was purchased from Pharmacia Biotech Inc. The rabbit antiserum against hsp70 and hsp90 (24) was a generous gift from Dr. Ettore Appella (National Cancer Institute). The BuGR2 monoclonal IgG antibody against the GR and the anti-cyclophilin 40 (C-Terminal Peptide) rabbit antiserum were from Affinity Bioreagents (Neshanic Station, NJ). The JJ3 monoclonal IgG against p23 (12) and purified, bacterially expressed human p23 were gifts from Dr. David Toft (The Mayo Clinic). The DS14F5 monoclonal IgG against p60 (19) was kindly provided by Dr. David Smith (University of Nebraska Medical School). The IgM monoclonal antibody against p50 (25) and the UPJ56 rabbit antiserum against FKBP52/hsp56 (26) were generous gifts from Drs. Gary Perdew (Pennsylvania State University) and Karen Leach (The Upjohn Co., Kalamazoo, Michigan), respectively. Rabbit antiserum (anti-YDJ1) against the yeast DnaJ protein (27) also recognizes rabbit DnaJ (rDnaJ) and was kindly provided by Dr. Avrom Caplan (Mount Sinai Medical Center).

Methods
Receptor Immunoadsorption-Prior to immunoadsorption, the BuGR antibody was prebound to protein A-Sepharose pellets by incubating 40 l of a 20% slurry of protein A-Sepharose for 1 h at 4°C with 40 l of antibody at a concentration of 100 g/ml and 150 l of TEG buffer (10 mM TES, 50 mM NaCl, 4 mM EDTA, 10% glycerol, pH 7.6), followed by centrifugation and washing with TEG. Glucocorticoid receptors were immunoadsorbed from 250 -300-l aliquots of L cell cytosol (11) by rotation for 2 h at 4°C with 8 l of protein A-Sepharose prebound with BuGR antibody. Prior to incubation with reticulocyte lysate, immunoadsorbed receptors were stripped of associated hsp90 by incubating the immunopellet an additional 2 h at 4°C with 0.5 M NaCl followed by one wash with 1 ml TEG and a second wash with 1 ml of HEPES buffer (10 mM HEPES, pH 7.4).
Glucocorticoid Receptor Heterocomplex Reconstitution-BuGR immune pellets (8 l of protein A-Sepharose) containing GR stripped of hsp90 were incubated with 50 l of rabbit reticulocyte lysate, with combinations of purified proteins, or with fractions A-C of reticulocyte lysate (each at 10 l) and adjusted to 50 l with HKD buffer (10 mM HEPES, 100 mM KCl, 5 mM dithiothreitol, pH 7.35). Dithiothreitol (1 l) was added to each incubation to a final concentration of 5 mM, and 5 l of an ATP-regenerating system (50 mM ATP, 250 mM creatine phosphate, 20 mM MgOAc and 100 units/ml creatine phosphokinase) were added to all assays to yield a final assay volume of 56 l. The assay mixtures were incubated for 20 min at 30°C with resuspension of the pellets by shaking the tubes every 5 min. At the end of the incubation, one-fourth of the suspension was removed for assay of steroid binding as described previously (9), and the remainder was used for Western blotting of receptor and associated proteins. The portion of the immunopellet used for steroid binding assay was washed one time with 1 ml of iced TEGM (TEG buffer plus 20 mM sodium molybdate), whereas the portion used for Western blotting was washed four times with 1 ml of TEGM. As noted previously (9), 100 l of L cell cytosol contains ϳ60,000 cpm of [ 3 H]triamcinolone acetonide binding capacity, and with 1 g of BuGR/100 l of L cell cytosol (the binding conditions employed here), we immunoadsorb about 50% of the glucocorticoid receptor. Thus, ϳ30,000 cpm represents 100% of receptors reactivated to the steroid binding form.
Western Blotting of Receptor and Associated Proteins-For assay of GR and associated proteins, immune pellets were boiled in SDS sample buffer with 10% ␤-mercaptoethanol, and proteins were resolved on 7% SDS-polyacrylamide gels (12% for resolving p23) as described previously (22). Proteins were then transferred to Immobilon-P membranes and probed with 2 g/ml BuGR monoclonal antibody for the glucocorticoid receptor, 0.05% rabbit antiserum for hsp70 and hsp90, 0.1% F5 anti-p60 mouse ascites for p60, 0.1% UPJ56 rabbit antiserum for FKBP52/hsp56, 0.1% anti-CyP-40 antiserum for CyP-40, 0.1% anti-YDJ1 antiserum for rDnaJ, and 0.1% JJ3 mouse ascites for p23. The immunoblots were then incubated a second time with the appropriate 125 I-labeled or horseradish peroxidase-conjugated counterantibody to visualize the immunoreactive bands.
DE52 Chromatography of Reticulocyte Lysate-Rabbit reticulocyte lysate (25 ml) was adsorbed to a 2.5 ϫ 20-cm column of DE52 equilibrated with HE buffer (10 mM HEPES, 1 mM EDTA, pH 7.35), the column was washed with 150 ml of HE buffer with 20 mM sodium molybdate followed by 150 ml of HE buffer alone, and the proteins were eluted with a 400-ml gradient of 0 -0.5 M KCl. hsp90, hsp70, p60, FKBP52/hsp56, CyP-40, and p23 were detected by resolving an aliquot of each fraction by SDS-PAGE and Western blotting with appropriate antibodies. Fractions were combined in three pools designated A-C (see Fig. 1). Pooled fractions were dialyzed against HKD buffer, concentrated to 1 ml ( 1 ⁄25 the original volume of lysate), and flash-frozen in small aliquots. ATP-agarose-treated lysate, which does not contain hsp70, was made as described previously (11).
Protein Purification-The bacterial expression of human p23 and its purification have been described (14). Briefly, p23 is soluble in bacterial lysates, and its abundance and high affinity for DEAE-cellulose allowed purification to 90% purity by chromatography on DEAE-cellulose. The protein was concentrated by precipitation with ammonium sulfate at 80% of saturation. It was dissolved and dialyzed into 10 mM Tris, 100 mM KCl, and 10% glycerol, pH 7.4 and stored at Ϫ70°. Rabbit p23 was purified by adding solid ammonium sulfate to fraction pool C (see Fig.  1) from DE52 chromotography of rabbit reticulocyte lysate to achieve a final concentration of 1.5 M. After centrifugation, the p23-containing supernatant was applied to a small column containing 5 ml of phenyl-Sepharose, which was washed with 50 ml of 1.5 M ammonium sulfate in 10 mM phosphate buffer, pH 7.4. The column was then eluted with 150 ml of a descending gradient of 1.5-0 M ammonium sulfate in phosphate buffer. Fractions containing p23 were identified by SDS-PAGE and Western blotting with JJ3 antibody. p23-containing fractions were pooled, contracted to 1 ml, dialyzed against HKD buffer, flash frozen, and stored at Ϫ70°C.
hsp70 was purified from the DE52 fraction pool A of rabbit reticulocyte lysate by chromatography on ATP-agarose and elution with ATP followed by ammonium sulfate precipitation exactly as described previously (11). hsp90 was purified from the DE52 fraction pool B of either rabbit reticulocyte lysate or rabbit brain cytosol by chromatography on hydroxylapatite followed by chromatography over ATP-agarose exactly as described in Hutchison et al. (11). After removal of hsp70 by ATPagarose, the hsp90-containing drop-through fractions were pooled, contracted to 1.0 ml, dialyzed against HKD buffer, flash frozen, and stored at Ϫ70°C.
Expression of p60 -For bacterial lysates containing p60, cDNA for the 60-kDa human protein (IEF SSP 3521) cloned by Honoré et al. (17), which is the homolog of the rabbit p60 (19), was subcloned into a pET 23C vector (Novagen) using the EcoRI and NotI sites. 2 This construct was used to transform (Escherichia coli strain BL21 (DE3), which harbors an integrated T7 polymerase gene. Control E. coli and bacteria expressing p60 (kindly provided by Dr. David Smith) were grown to an A 600 of 0.6, induced with isopropyl-1-thio-␤-D-galactopyranoside for 3 h at 25°C, and harvested. Bacterial lysates were prepared by sonication in phosphate-buffered saline, and aliquots were flash frozen and stored at Ϫ70°C.

Reconstitution of GR⅐hsp90 Heterocomplex Assembly Activity with DE52
Fractions of Reticulocyte Lysate-The elution of hsp90 and its associated proteins from a DE52 column is shown in Fig. 1. Fractions were combined into three pools designated A, B, and C, as indicated by the brackets shown under the Western blots of the individual proteins. The distribution of proteins in DE52 fraction pools A-C are shown in Fig. 2A, and the ability of each pool and combination of pools to assemble GR⅐hsp90 heterocomplexes is shown in Fig. 2B. When the stripped GR (lane 1) was incubated with DE52 pool A, B, or C (lanes 3, 4, and 5, respectively), there was no heterocomplex assembly, and very little assembly was seen with combinations of two fractions (lanes 6 -8). However, when the stripped GR immune pellet was incubated with the combination of DE52 pools A, B, and C (lane 9), there was substantial GR⅐hsp90 heterocomplex assembly, comparable with the unfractionated lysate control in lane 2. It should be mentioned that immunoblots of larger volumes of the DE52 pools than that analyzed in Fig. 2A show that pool B always contains some hsp70 and trace amounts of p23, although as shown in Fig. 2A, the great majority of each of these proteins segregates to pools A and C, respectively. Also, DE52 pool C contains small amounts of hsp90.
Replacement of DE52 Pool A with Purified Rabbit hsp70 and Bacterially Expressed p60 -To determine the components of DE52 pool A required for reconstitution of GR⅐hsp90 heterocomplex assembly, we first asked whether the activity of pool A could be replaced by hsp70. As shown in Fig. 3A, removal of hsp70 by passing reticulocyte lysate through ATP-agarose inactivates heterocomplex assembly activity (cf. lane 3 with lane 2). The addition of the ATP-agarose-retained material (lane 5) or purified rabbit hsp70 (lane 4) restores the heterocomplex assembly activity. We have demonstrated previously that the hsp70 is purified to near homogeneity and that neither the purified hsp70 nor the ATP-agarose-retained fraction of lysate have any heterocomplex assembly activity when present alone (11). In the experiment of Fig. 3B, the addition of purified hsp70 to the the combination of DE52 pools B and C (lane 9) produced a small increment in activity over that seen with the combination of B and C alone (lane 8), whereas addition of DE52 pool A to the combination of B and C (lane 6) yielded nearly the same heterocomplex assembly activity as unfractionated lysate (lane 2). Because the addition of larger amounts of purified hsp70 to the combination of B and C does not yield any greater activation than that seen in lane 9 (data not shown), it seems likely that another component (or components) of DE52 pool A is required for heterocomplex assembly.
A likely candidate for this additional factor is p60, and we wanted to determine the effect of p60 depletion on the activity of pool A. To facilitate depletion, selected DE52 fractions were combined such that fractions from the half of pool A containing the majority of p60 were in one subfraction designated A 1 , and selected fractions from the other half of pool A containing a minority of p60 were in a subfraction designated A 2 . Half of A 1 and A 2 was extracted with nonimmune antibody, and the other half of A 2 was extracted with protein A-Sepharose prebound with F5 antibody to extract the remaining p60. The distributions of selected proteins in the subfractions of pool A are shown in Fig. 4A, where it can be seen that the amount of p60 in A 2 is low relative to A 1 and p60 is eliminated from A 2 by immunoextraction with F5 antibody. As shown in Fig. 4B, the combination of subfraction A 1 with B and C (lane 9) yields the same GR heterocomplex assembly as unfractionated reticulocyte lysate (lane 2), whereas subfraction A 2 is less active (lane 10) and F5-extracted A 2 (lane 11) has only a low activity comparable with that of hsp70 alone added to B and C (Fig. 3, lane  9).
The experiment of Fig. 5 was performed to determine if bacterially expressed human p60 would restore heterocomplex assembly activity to the p60-depleted DE52 A 2 subfraction of reticulocyte lysate. Fig. 5A shows the presence of p60 by Coo-  . However, addition of extract from p60-expressing bacteria to the mixture yielded a concentration-dependent increase in heterocomplex assembly (lanes 8 -10). Fig. 6 shows that the receptor activating activity of the DE52 pool A of reticulocyte lysate can be replaced by the combination of highly purified rabbit hsp70 and lysate from p60-expressing E. coli. It can be seen that purified hsp70 alone (lane 6), bacterial lysate containing p60 alone (lane 7), and the combination of hsp70 and p60 (lane 8) were inactive at heterocomplex assembly. Also, the addition of either p60 alone (lane 9) or hsp70 alone (lane 10) to the combination of DE52 pools B and C was without effect. However, the addition of both purified rabbit hsp70 and bacterial lysate containing human p60 to the combined DE52 pools B and C (lane 11) yielded steroid binding activity that was close to that achieved with the combination of DE52 pools A, B, and C (lane 5).
Replacement of DE52 Pool B with Purified Rabbit hsp90 -In the experiment shown in Fig. 7, DE52 pool B was substituted with purified rabbit hsp90. It can be seen in Fig. 7B that purified hsp90 does not bind to the GR or activate steroid binding activity when incubated alone with stripped receptors (lane 6). However, in the presence of DE52 pools A and C, which together yield only a small amount of heterocomplex reconstitution (lane 4), the addition of purified hsp90 (lane 7) yields GR⅐hsp90 complex formation and steroid binding activity comparable with that achieved with the combination of DE52 pools A, B, and C (lane 5). The addition of purified hsp90 to pool A alone does not yield steroid binding activity (lane 9).
Replacement of DE52 Pool C with Purified p23- Fig. 8 shows that the activity of DE52 pool C can be replaced with purified p23. In preliminary experiments (data not shown) we found that rabbit p23 purified from the DE52 pool C of reticulocyte FIG. 3. Purified hsp70 is required for heterocomplex assembly, but it alone does not replace DE52 fraction pool A of reticulocyte lysate for reconstitution of receptor steroid-binding activity. A, purified hsp70 promotes reconstitution of steroid binding activity in ATP-agarose-extracted (i.e. hsp70-depleted) lysate. Reticulocyte lysate was depleted of hsp70 by extraction of hsp70 onto a matrix of ATP-agarose exactly as described by Hutchison et al. (11). Immunoadsorbed, stripped receptors were incubated with mock-extracted lysate, ATP-agarose extracted lysate, or ATP-agarose-extracted lysate plus either highly purified hsp70 or the ATP-agarose-retained material that was eluted from the column with ATP. Samples were then assayed for steroid binding activity.

FIG. 4. Immunoadsorption of p60 from DE52 pool A of reticulocyte lysate inactivates reconstitution of receptor steroid binding activity.
Stripped GR immunopellets were incubated in the presence of 100 mM KCl and an ATP-generating system with normal unfractionated reticulocyte lysate or with various combinations of reticulocyte lysate DE52 pools B and C and subfractions of the DE52 pool A that had been extracted twice with either nonimmune ascites (A 1 and A 2 ) or with an F5 (i.e. anti-p60-bound) immunopellet (A 2-extr ) to extract p60. Samples were then assayed for steroid binding activity. A, Western blot of selected proteins in DE52 subfractions A 1 , A 2 , and p60-depleted A 2 (A 2-extr 5. Bacterially expressed human p60 replaces rabbit p60 for reconstitution of receptor steroid binding activity. Stripped GR immunopellets were incubated in the presence of 100 mM KC1 and an ATP-generating system with normal unfractionated reticulocyte lysate or various combinations of DE52 fraction pool A 2 , pools B and C together, p60-depleted A 2 , and increasing amounts of lysate from bacteria expressing p60, or control bacterial lysate. Samples were then assayed for steroid binding activity. lysate could substitute for the activity of pool C in the recombined system. As shown in Fig. 8A, purified human p23 (shown in Fig. 8B, lane 1), which does not itself activate steroid binding activity (lane 6) produces a concentration-dependent activation when added to the combination of rabbit reticulocyte lysate DE52 pools A and B (lane 7-10). Fig. 8C shows that the incubation of GR with purified p23 and lysate pools A and B (lane 7) yields more GR⅐hsp90 heterocomplex (see Western blot) than pools A and B without p23 (lane 4), with both steroid binding activity and heterocomplex formation being equivalent to that achieved with the combination of DE52 pools A, B, and C (lane 5).
Reconstitution with Individual Components of the System-To optimize the concentrations of the various components of the assembly system, concentration curves like that shown for p23 in Fig. 8A were performed for p60 (Fig. 9A), hsp70 (Fig.  9B), and hsp90 (Fig. 9C). The heterocomplex assembly system was then reconstituted in the experiment of Fig. 10, utilizing the optimal concentrations of components determined in Figs.

DISCUSSION
The components of the rabbit reticulocyte receptor⅐hsp90 heterocomplex assembly system were identified by analyzing the proteins that were bound to the chicken progesterone receptor (PR) at early and late stages of assembly of the PR⅐hsp90 heterocomplex (10,12,14,19). Although there is evidence that hsp70, p60, and p23 are each required for PR⅐hsp90 heterocomplex assembly (10,14,19), the proteins have not previously been reconstituted into a receptor⅐hsp90 heterocomplex assembly system.
Herein, we have divided rabbit reticulocyte lysate into three DE52 fraction pools (Fig. 1), each of which must be present in the incubation mixture for substantial assembly of GR⅐hsp90 heterocomplexes and reactivation of steroid binding activity (Fig. 2). The ability of DE52 fraction pool A to activate the steroid binding activity of the mouse GR was replaced with purified rabbit hsp70 and bacterial lysate containing human p60 (Figs. 3-6), and pools B and C were replaced with purified rabbit hsp90 and purified human p23, respectively (Figs. 7 and 8). When these four preparations are combined, the mixture assembles GR⅐hsp90 heterocomplexes and restores the receptors to the steroid binding conformation (Fig. 10).
Reconstitution of the system with these p60, hsp70, hsp90, and p23 preparations does not establish that these are the only proteins required for receptor⅐hsp90 heterocomplex assembly. The Smith laboratory has shown that a 48-kDa protein of reticulocyte lysate is recovered with PR⅐hsp90 complexes at early times of assembly (16) or when assembly is blocked at an intermediate stage by geldanamycin (28), an hsp90-binding benzoquinone ansamycin (29). This 48-kDa protein appears to be the same as Hip, the hsc70-interacting protein identified by Hohfeld et al. (30), and it could be present as a contaminant in the purified rabbit hsp70 and/or hsp90. Whether Hip is required for GR⅐hsp90 heterocomplex assembly or whether it facilitates assembly has not yet been determined.
It is known that mammalian cells contain homologs of the bacterial DnaJ (31). The DnaJ homolog Ydj1 has been found in GR⅐hsp90 heterocomplexes from yeast, and genetic experiments show that Ydj1 is required for the GR to be hormoneresponsive in yeast (32). DnaJ homologs could also be present as contaminants in our purified rabbit hsp70 and/or hsp90 preparations and might facilitate assembly.
Two FK506-binding immunophilins, FKBP52 and p54, as well as a cyclosporin A-binding immunophilin, CyP-40, are present in receptor⅐hsp90 heterocomplexes reconstituted with reticulocyte lysate (33)(34)(35). We established by immunoblotting (data not shown) that the purified preparations of hsp70, hsp90, and p23 used in our reconstitution of the GR⅐hsp90 heterocomplex assembly system do not contain any of these mammalian immunophilins. These immunophilins all have peptidylprolyl isomerase activity (36), but that activity does not seem to be required for GR⅐hsp90 heterocomplex assembly. Reticulocyte lysate also assembles the oncogenic protein kinases Src and Raf into heterocomplexes that contain hsp90 and a 50-kDa protein, p50 (5,6). We have also demonstrated by immunoblotting (data not shown) that p50 is not present in the purified proteins we have used for GR⅐hsp90 heterocomplex reconstitution. Thus, like the immunophilins, it is not required for heterocomplex assembly.
This work presents the first demonstration of p60 activity in a biochemical assay and the first reconstitution of the GR⅐hsp90 heterocomplex assembly system. The availability of a reconstituted system should greatly facilitate study of the mechanism of the assembly process.