©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The 23-kDa Acidic Protein in Reticulocyte Lysate Is the Weakly Bound Component of the hsp Foldosome That Is Required for Assembly of the Glucocorticoid Receptor into a Functional Heterocomplex with hsp90 (*)

(Received for publication, April 17, 1995; and in revised form, May 30, 1995)

Kevin A. Hutchison (1) Louis F. Stancato (1)(§),   Janet K. Owens-Grillo (1)(¶),   Jill L. Johnson (2) Priti Krishna (3) David O. Toft (2) William B. Pratt (1)(**)

From the (1)Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, the (2)Department of Biochemistry and Molecular Biology, Mayo Graduate School, Rochester, Minnesota 55905, and the (3)Department of Plant Sciences, The University of Western Ontario, London, Ontario N6A 5B7, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified ``foldosome'' directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K. A., Dittmar, K. D., and Pratt, W. B.(1994) J. Biol. Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte lysate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directed folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography. Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.


INTRODUCTION

In cytosols prepared from hormone-free mammalian cells, steroid receptors exist in multiprotein complexes containing hsp90, (^1)hsp70, two immunophilin chaperones (hsp56 and CyP-40), and a 23-kDa acidic protein, p23 (see (1) and (2) for review). These multiprotein receptor-hsp90 heterocomplexes can be formed by incubating immunoadsorbed, hormone-free receptors (pretreated with salt to strip them free of associated proteins) with rabbit reticulocyte lysate(3, 4) . Some of the steroid receptors, for example the glucocorticoid receptor (GR), must be bound to hsp90 for the hormone binding domain to be in a steroid binding conformation(5) , and incubation of the immunoadsorbed GR with reticulocyte lysate not only forms the receptor heterocomplex but restores the receptor to the steroid binding state(4) . Until recently, all of the experiments involving cell-free assembly of receptor-hsp90 heterocomplexes have been carried out with the protein folding system in rabbit reticulocyte lysate, but this system appears to be ubiquitous in that concentrated lysates of human, monkey, mouse, insect (Sf9), and plant (wheat germ) cells fold the GR into a functional (i.e. steroid binding) heterocomplex with hsp90. (^2)A number of requirements for the heterocomplex assembly/receptor folding system of reticulocyte lysate have been defined. Formation of the receptor-hsp90 complex is an ATP/Mg-dependent process(6, 7) that requires the presence of a monovalent cation, such as K or NH(4)(7) . hsp70 is required for the binding of hsp90 to steroid receptors (6, 8) and for simultaneous conversion of the GR to the steroid binding conformation (8) .

In reticulocyte lysate, as well as in a variety of mammalian cell lysates, hsp90 and hsp70 exist in multiprotein complexes containing the immunophilins hsp56 (FKBP52) or CyP-40(9, 10) , (^3)a 60-kDa stress-related protein(11) , the p50 protein that was originally identified as a member of the pp60-hsp90 complex(12, 13) , and the conserved and widely distributed p23(14) . When these multiprotein complexes are immunoadsorbed from reticulocyte lysate, they retain protein chaperoning activity as assayed by renaturation of thermally denatured luciferase (15) and by formation of a functional GR-hsp90 heterocomplex (16) . Thus, the multiprotein hsp90 heterocomplex acts as a self-sufficient protein folding machine that we refer to as a ``foldosome''(16) .

The foldosome can be immunoadsorbed to protein A-Sepharose from rabbit reticulocyte lysate using the 3G3 monoclonal IgM directed against hsp90. When this immunopellet is incubated at 30 °C with immunoadsorbed GR, ATP/Mg, and KCl, the receptor is bound to hsp90 and restored to the steroid binding conformation(16) . But, if the immunoadsorbed multiprotein hsp90 complex is washed extensively with a low salt buffer, it loses a weakly bound protein (not hsp90 or hsp70) that is required for receptor activation to the steroid binding state. This factor is contained in a Centricon C-100 filtrate of lysate that reconstitutes the receptor activating activity of the washed foldosome(16) .

In this work we show that the Centricon C-100 filtrate of rabbit reticulocyte lysate also stimulates GR-hsp90 heterocomplex assembly by wheat germ lysate. Using this stimulation of the wheat germ heterocomplex assembly system as a simple and rapid assay for the factor, we show by ammonium sulfate fractionation and ion exchange chromatography of reticulocyte lysate that the activity segregates with p23 and not with any other component of the foldosome (i.e. hsp90, hsp70, p60, p50, hsp56, or CyP-40). Johnson and Toft (10) have shown recently that immunoadsorption of reticulocyte lysate with the JJ3 monoclonal antibody against p23 reduces its ability to form a progesterone receptor-hsp90 heterocomplex. Here we show that immunoadsorption of the Centricon C-100 filtrate of reticulocyte lysate with the similar JJ5 monoclonal antibody eliminates its ability to reconstitute the receptor activating activity of the washed foldosome, and addition of purified bacterially expressed p23 restores the ability of the system to refold the receptor to the steroid binding conformation. This work provides the first assay of p23 activity in a substantially purified hsp90-associated chaperone system, providing an important step toward the long term goal of reconstituting a biologically active foldosome from purified protein components.


EXPERIMENTAL PROCEDURES

Materials

[6,7-^3H]Triamcinolone acetonide (42.8 Ci/mmol) and I-conjugated goat anti-mouse and anti-rabbit IgGs were obtained from DuPont NEN. Untreated rabbit reticulocyte lysate was from Green Hectares (Oregon, WI). Wheat germ lysate was purchased from Promega. Protein A-Sepharose and goat anti-mouse and anti-rabbit IgG horseradish peroxidase conjugates were from Sigma. The rabbit antiserum against hsp70 and hsp90 (17) was a generous gift from Dr. Ettore Appella (National Cancer Institute). The rabbit R2 antiserum against plant hsp90 was raised against a fusion protein comprising a portion of Pharbitis nil (Japanese morning glory) hsp90 fused to the trpE protein of Escherichia coli, and a detailed description of the antibody will be published elsewhere. (^4)The BuGR2 monoclonal IgG antibody against the GR, the 3G3 monoclonal IgM against hsp90, and the anti-cyclophilin 40 (C-terminal peptide) rabbit antiserum were from Affinity Bioreagents (Neshanic Station, NJ). The JJ5 monoclonal IgG against p23 (14) was described previously. The DS14F5 monoclonal IgG against p60 (11) and the IgM monoclonal antibody against p50 (12) were kindly provided by Drs. David Smith (University of Nebraska) and Gary Perdew (Purdue University), respectively. The EC1 monoclonal IgM against hsp56 (18) was a gift from Dr. Lee Faber (Medical College of Ohio).

Methods

Receptor Immunoadsorption

Prior to immunoadsorption, the BuGR antibody was prebound to protein A-Sepharose pellets by incubating 40 µl of a 20% slurry of protein A-Sepharose for 1 h at 4 °C with 40 µl of antibody at a concentration of 10 µg/ml and 150 µl 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 400-µl aliquots of L cell cytosol (8) by rotation for 2 h at 4 °C with 8 µl of protein A-Sepharose prebound with BuGR antibody. Prior to incubation with wheat germ lysate, reticulocyte lysate, or 3G3 immunoadsorbates of 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 of TEG and a second wash with 1 ml of HEPES buffer (10 mM HEPES, pH 7.4).

hsp90 Immunoadsorption

Protein A-Sepharose beads were precoated with goat IgG against mouse IgM by incubating 72 µl of a 20% suspension of protein A-Sepharose with 100 µg of anti-IgM, followed by rotation in the cold for 1 h and washing the pellet with 1 ml of TEG. The precoated pellet was then rotated an additional 1 h with 10 µl of 3G3 or nonimmune ascites fluid and washed once with 1 ml of TEG. hsp90 and its associated proteins in the hsp heterocomplex were then immunoadsorbed from 400-µl aliquots of rabbit reticulocyte lysate by rotating for 1 h with 3G3-bound Sepharose pellets followed by two washes with 1 ml of HEPES buffer. In the experiment of Fig.1A, the washed 3G3 immunopellets were incubated three times for 10 min at 30 °C in HEPES buffer containing 100 mM KCl to release hsp90 from the 3G3 antibody as described previously(16) . Supernatants from the three sequential elutions were combined, contracted, and incubated with BuGR immunopellets containing stripped glucocorticoid receptors.


Figure 1: The Centricon C-100 filtrate of reticulocyte lysate stimulates reconstitution of the glucocorticoid receptor-hsp90 heterocomplex by both the washed 3G3-immunoadsorbed rabbit hsp complex and wheat germ lysate. Glucocorticoid receptors were immunoadsorbed from aliquots of L cell cytosol to protein A-Sepharose prebound with BuGR or nonimmune IgG. Receptor-associated proteins were stripped from the immunopellets with salt and incubated for 20 min at 30 °C with the additions noted under the indicated conditions. Receptor, hsp90, and hsp70 were assayed in each sample by Western blotting, and a portion of the immunopellet was incubated with [^3H]triamcinolone acetonide to determine steroid binding activity. A, reconstitution of GR-hsp90 heterocomplexes with the washed 3G3-immunoadsorbed hsp complex. B, reconstitution of GR-hsp90 heterocomplexes with wheat germ lysate. In both panels condition 1 is a single lane showing the stripped glucocorticoid receptor, and condition 2 is divided into two lanes of stripped nonimmune (no receptor) and immune (GR-containing) pellets incubated with whole rabbit reticulocyte lysate. Condition 3, stripped nonimmune and immune pellets incubated with the proteins released from the washed 3G3 immunopellet of reticulocyte lysate with 100 mM KCl (A) or with whole wheat germ lysate (B). Condition 4 is condition 3 plus the Centricon C-100 filtrate of reticulocyte lysate. Condition 5, stripped nonimmune and immune pellets incubated with only the Centricon C-100 filtrate. The solid bars represent the steroid binding for the samples presented in the upper panel.



Glucocorticoid Receptor Heterocomplex Reconstitution

BuGR immune pellets (8 µl of protein A-Sepharose) containing GR stripped of hsp90 were incubated with 50 µl of wheat germ lysate or rabbit reticulocyte lysate. For reconstitution of GR by the immunoadsorbed hsp heterocomplex, stripped receptors were suspended in 50 µl of an assay mix consisting of HEPES buffer with 100 mM KCl and then the whole GR immunopellet suspension was pipetted onto the 3G3 immunopellets containing the immunoadsorbed hsp90 and its associated complex. Dithiothreitol was added to each incubation to a final concentration of 5 mM, and 5 µl of an ATP-regenerating system (50 mM ATP, 250 mM creatine phosphate, 20 mM MgCl(2) and 100 units/ml creatine phosphokinase) were added to all assays to yield a final assay volume of 56 µl. When additions, such as Centricon C-100 filtrate or ammonium sulfate fractions of reticulocyte lysate, were made to the wheat germ system, the addition volume was 50 µl, and the final incubation volume was 106 µl. When additions were made to the resuspended 3G3 immunopellet or to the hsp heterocomplex released from the 3G3 pellet with salt, the addition replaced the HEPES buffer and final incubation volume was maintained at 56 µl. 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(7) , 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(7) , 100 µl of L cell cytosol contains 60,000 cpm of [^3H]triamcinolone acetonide binding capacity, and with 10% BuGR we immunoadsorb about 50% of the glucocorticoid receptor. Thus, 30,000 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% beta-mercaptoethanol, and proteins were resolved on 7% SDS-polyacrylamide gels (12% for resolving p23) as described previously(16) . Proteins were then transferred to Immobilon-P membranes and probed with 2 µg/ml BuGR monoclonal antibody for the glucocorticoid receptor, 0.05% rabbit antiserum for hsp70 and hsp90, or 0.2% R2 rabbit antiserum for plant hsp90. The hsp70 and hsp90 regions of the Western blot were probed with anti-hsp70/hsp90 serum to detect rabbit hsp90 and hsp70 and wheat hsp70 and they were also probed with R2 antibody to detect wheat hsp90. p60 was probed with 0.1% F5, hsp56 with 1 µg/ml EC1, CyP-40 with 0.1% anti-CyP-40, and p23 with 0.1% JJ5. The immunoblots were then incubated a second time with the appropriate I-labeled or horseradish peroxidase-conjugated counterantibody to visualize the immunoreactive bands.

Centricon Filtration of Reticulocyte Lysate

The Centricon C-100 filtrate of rabbit reticulocyte lysate was prepared as described previously(16) . Briefly, 1 ml of lysate was centrifuged in a Centricon C-100 concentrator at 2000 g until 700 µl of filtrate was present. This filtrate was then passed through a second C-100 concentrator, and the second filtrate was concentrated 10-fold using a Centricon C-50 concentrator, which does not permit the passage of the active factor(16) . The concentrated Centricon C-100 filtrate was stored in aliquots at -20 °C. After dilution in the final GR heterocomplex reconstitution mix, the Centricon C-100 filtrate is at a concentration of 5-fold.

Ammonium Sulfate Fractionation of Reticulocyte Lysate

Rabbit reticulocyte lysate was fractionated by the addition of solid ammonium sulfate to the desired percent of saturation followed by mixing at 4 °C for 1 h. Precipitated proteins were resuspended in HEPES buffer and dialyzed overnight against HEPES buffer plus 100 mM KCl and 5 mM dithiothreitol. Fractions were then concentrated to one-half the original volume of lysate using a Centricon C-10 concentrator.

DE52 Chromatography of Reticulocyte Lysate

Rabbit reticulocyte lyate (20 ml) was chromatographed on a 2.5 20-cm column of DE52 equilibrated with HE buffer, and the proteins were eluted with a 400-ml gradient of 0-0.5 M KCl. hsp90, hsp70, p23, and other proteins in the multiprotein hsp90 heterocomplex were detected by resolving an aliquot of each fraction by SDS-PAGE and Western blotting with appropriate antibodies. Fractions were combined in four pools designated A-D in Fig.5, concentrated to the original volume of lysate, and dialyzed against 10 mM HEPES, 25 mM KCl, and 2 mM dithiothreitol. The final preparations were flash-frozen in small aliquots.


Figure 5: DE52 fractionation of activity from reticulocyte lysate. A, rabbit reticulocyte lysate (20 ml) was chromatographed on a column of DE52 as described under ``Methods'' (solid line, absorbance at 280 nm; dotted line, KCl gradient). Fractions were pooled (A-D) as indicated by the bars under the graph, concentrated to the original volume of lysate and dialyzed against 10 mM HEPES, 25 mM KCl, and 2 mM DTT for further analysis. Aliquots of each fraction pool were Western blotted for the proteins in the hsp heterocomplex (shown below graph). B, each fraction was assayed for its ability to stimulate the GR-hsp90 reconstituting activity of wheat germ lysate. Condition 1, stripped receptor; condition 2, stripped nonimmune and immune pellets, respectively, incubated with wheat germ lysate; condition 3, stripped nonimmune and immune pellets incubated with wheat germ lysate and 1 µg of purified, bacterially expressed p23; conditions 4-7, stripped receptors incubated with wheat germ lysate and DE52 fractions A-D, respectively.



Protein Purification

The bacterial expression of human p23 and its purification have been described(10) . 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 80% saturated ammonium sulfate. It was dissolved and dialyzed into 10 mM Tris, 1 mM EDTA, 100 mM KCl, and 10% glycerol, pH 7.4, and stored at -70 °C.


RESULTS

The Centricon C-100 Filtrate Stimulates GR-hsp90 Heterocomplex Reconstitution by Wheat Germ Lysate

The effect of the Centricon C-100 filtrate on GR-hsp90 heterocomplex assembly by the immunoadsorbed foldosome from reticulocyte lysate is shown in Fig.1A. For this experiment, hsp90 and its associated proteins in the hsp heterocomplex were immunoadsorbed from reticulocyte lysate with the 3G3 monoclonal IgM against hsp90, and after the immunopellet was washed, hsp90 was released from the 3G3 antibody by treatment with salt as described previously(16) . As shown in condition 3, the salt-released hsp heterocomplex reassembles a GR-hsp90 complex, but it has little ability to reactivate steroid binding activity (cf. condition 3 with condition 2). However, incubation of stripped receptors with both the released hsp heterocomplex and the Centricon C-100 filtrate, which has no receptor heterocomplex reconstituting activity of its own (condition 5), results in both GR-hsp90 complex assembly and substantial generation of steroid binding activity (condition 4).

Fig.1B shows the effect of the same Centricon C-100 filtrate on assembly in wheat germ lysate of a complex between the GR and plant hsp90. As shown in condition 3, incubation of stripped receptors with wheat germ lysate produces a small amount of complex with the plant hsp90. The plant hsp90 is the band migrating slightly faster than the rabbit hsp90, which is shown in condition 2. Addition of the Centricon C-100 filtrate to the wheat germ lysate yields more complexes between the GR and plant hsp90 and is accompanied by substantial reactivation of steroid binding activity. The same amount of wheat hsp70 binds to the receptor with (condition 4) and without (condition 3) the Centricon C-100 filtrate. As we have noted previously(8) , the binding of hsp90 is hsp70-dependent, but it is only GR-associated hsp90 (not hsp70) that is associated with steroid binding activity. As shown in Fig.2, the factor in the Centricon C-100 filtrate produces a concentration-dependent increase in both the amount of GR-plant hsp90 complex that is recovered and steroid binding activity.


Figure 2: The Centricon C-100 filtrate of reticulocyte lysate produces a concentration-dependent stimulation of glucocorticoid receptor-plant hsp90 heterocomplex assembly by wheat germ lyate. Immunoadsorbed, stripped receptors were incubated with wheat germ lysate in the presence of the indicated concentrations of Centricon C-100 filtrate of reticulocyte lysate. After the 20-min incubation, the immunopellets were washed and assayed for steroid binding activity (bullet) and receptor-associated plant hsp90 (Western blot). Lanes 1 and 2, stripped nonimmune and immune pellet, respectively, incubated with wheat germ lysate alone; lane 3, nonimmune pellet incubated with wheat germ lysate and a 10-fold concentration of Centricon C-100 filtrate; lanes 4-7, immune pellets containing stripped GR incubated with wheat germ lysate and 1-, 2.5-, 5-, and 10-fold concentrated Centricon C-100 filtrate, respectively.



We have shown previously that GR-plant hsp90 complexes formed in wheat germ lysate are less stable than GR-rabbit hsp90 complexes formed in reticulocyte lysate. A rapid dissociation of hsp90 from receptors during the incubation with wheat germ lysate at 30 °C would account for the fact that fewer GR-hsp90 complexes are recovered from wheat germ lysate than reticulocyte lysate and that there is very little steroid binding activity in the immunopellet after lysate removal. It is possible, however, to trap the steroid binding state by having [^3H]triamcinolone acetonide present during the 20-min incubation with wheat germ lysate at 30 °C. This effect is shown in Fig.3A. When [^3H]steroid is present during the incubation with wheat germ lysate (condition 1, open bar), the amount of steroid binding is about two-thirds that seen with reticulocyte lysate (condition 1, solid bar). However, if the wheat germ reconstitution mix is simply cooled on ice after the 20-min incubation at 30 °C and [^3H]steroid is added to the cooled mixture, then there is very little steroid binding activity (condition 2, open bar). Essentially the same steroid binding activity is seen under the two conditions with rabbit reticulocyte lysate. As shown by the slashed bars, the presence of the Centricon C-100 filtrate does not affect the amount of steroid binding activity seen under condition 1, but it increased it under condition 2. The GR that is reactivated in the wheat germ lysate in the presence of the Centricon C-100 filtrate can be washed free of lysate with both retention of steroid binding activity (Fig.3A, condition 3) and the expected increased yield of GR-hsp90 complex (Fig.3B, cf. lanes 7 and 3).


Figure 3: The Centricon C-100 filtrate of reticulocyte lysate stabilizes the GR-hsp90 complex during the 30 °C incubation with wheat germ lysate. A, effect of C-100 filtrate on steroid binding activity. Immunoadsorbed, stripped receptors were incubated with rabbit reticulocyte lysate (solid bars), wheat germ lysate (open bars), or wheat germ lysate plus Centricon C-100 filtrate of reticulocyte lysate (hatched bars), and steroid binding was assayed under three conditions. Condition 1, [^3H]triamcinolone acetonide was present during the 20 min reconstitution at 30 °C, samples were centrifuged, washed, and radioactivity in the immunopellet was assayed. Condition 2, following the 20-min reconstitution, the entire reconstitution mix was placed on ice for 20 min, [^3H]triamcinolone acetonide was then added and the mix was incubated overnight at 0 °C. Condition 3, after the 20-min reconstitution, samples were washed once with 1 ml of cold TEGM buffer and then incubated overnight at 0 °C with [^3H]triamcinolone acetonide. B, effect of C-100 filtrate on the amount of plant hsp90 recovered with the GR in the same experiment. Lane 1, stripped receptors; lanes 2 and 3, stripped nonimmune and immune pellet, respectively, incubated with wheat germ lysate; lanes 4 and 5, stripped nonimmune and immune pellets incubated with the Centricon C-100 filtrate of reticulocyte lysate; lanes 6 and 7, stripped nonimmune and immune pellets incubated with both wheat germ lysate and Centricon C-100 filtrate. The conditions of lanes 3 and 7 in B are the same as the open bar and hatched bar, respectively, shown in section 3 of A.



The Factor in Reticulocyte Lysate Segregates with p23

At this time, we had no evidence that the effect of the Centricon C-100 filtrate on reconstitution of GR binding activity by wheat germ lysate was due to the same factor that restored receptor heterocomplex reconstituting activity to the washed 3G3 immunopellet of reticulocyte lysate as shown in Fig.1A. However, reconstitution assays like that shown in Fig.1A are both expensive and difficult to perform, whereas the assay in wheat germ lysate is inexpensive, rapid, and permits easy screening of multiple samples. We have shown previously that a factor precipitated from rabbit reticulocyte lysate between 55 and 85% of saturation with ammonium sulfate could replace the Centricon C-100 filtrate of reticulocyte lysate in restoring GR-hsp90 heterocomplex reconstitution activity to the washed 3G3 immunopellet(16) . Thus, we first fractionated rabbit reticulocyte lysate stepwise with ammonium sulfate and assayed the effect of each fraction on GR-hsp90 heterocomplex reconstitution by wheat germ lysate.

Fig.4A shows the fractional precipitation of rabbit reticulocyte lysate with ammonium sulfate and the distribution of the hsp heterocomplex proteins in each fraction. Fig.4B shows the ability of each fraction to stimulate formation of a GR-hsp90 complex when added to wheat germ lysate. As we have reported previously (16, 19) , the ammonium sulfate fractions of reticulocyte lysate do not form complexes between the GR and rabbit hsp90. However, the 60-85% fraction of reticulocyte lysate stimulates GR-hsp90 heterocomplex assembly by wheat germ lysate, and a small amount of activity is present in the 45-60% fraction (Fig.4B, lanes 8 and 7, respectively). Two-thirds of the hsp90 and 90% of the hsp70 are precipitated below 60% ammonium sulfate, with the remainder of each being present in the 60-85% fraction (Fig.4A). In addition, the 60-85% fraction contains a trace amount of p60 and almost all of the p23 (a small amount of p23 that can be seen in the 45-60% fraction in the original blot is not seen in the photograph).


Figure 4: Ammonium sulfate fractionation of activity from reticulocyte lysate. Rabbit reticulocyte lysate was sequentially fractionated with ammonium sulfate at 30, 45, 60, and 85% of saturation, and each fraction was dialyzed and contracted to one-half the original lysate volume. A, aliquots of each fraction were resolved by SDS-PAGE and either stained with Coomassie blue (left) or immunoblotted with antibodies (right) to detect the components of the hsp heterocomplex. B, each fraction was then assayed for its ability to stimulate GR-hsp90 reconstituting activity by wheat germ lysate. Lane 1, stripped receptor; lane 2, stripped receptor plus rabbit reticulocyte lysate; lane 3, stripped receptor plus wheat germ lysate; lane 4, stripped receptor plus wheat germ lysate and Centricon C-100 filtrate; lane 5, stripped receptor plus wheat germ lysate plus 0-30% ammonium sulfate fraction; lane 6, plus lysate and 30-45% fraction; lane 7, plus lysate and 45-60% fraction; lane 8, plus lysate and 60-85% fraction; lane 9, plus lysate and 85% supernatant.



p23 is more acidic than the other components of the hsp heterocomplex and can be separated from them by chromatography on DE52. As with ammonium sulfate fractionation, the GR-hsp90 assembly/receptor activating activity of reticulocyte lysate is destroyed by ion exchange chromatography. However, a fraction obtained by DE52 chromatography has the ability to stimulate GR-hsp90 heterocomplex assembly by wheat germ lysate. In the experiment shown in Fig.5A, reticulocyte lysate was fractionated on a DE52 column, each fraction was assayed for each member of the hsp heterocomplex by Western blotting (not shown), and fractions were combined into four pools designated A-D indicated by the bars under the figure. As shown in Fig.5B, addition of fraction C to wheat germ lysate (condition 6) stimulates GR-hsp90 heterocomplex assembly by wheat germ lysate. The only component of the hsp heterocomplex in fraction C is p23 (Fig.5A), and the rabbit p23 is recovered with the GR-plant hsp90 complex (Fig.5B, condition 6) formed in the wheat germ lysate.

Because the ability to stimulate GR-hsp90 heterocomplex assembly by wheat germ lysate segregated with p23 on fractionation of reticulocyte lysate, we asked if purified, bacterially expressed human p23 would stimulate GR-hsp90 heterocomplex formation. As shown in condition 3 of Fig.5B, the purified p23 stimulated formation of GR-plant hsp90 heterocomplexes by wheat germ lysate and it is a component of receptor heterocomplexes formed in wheat germ lysate. The bacterially expressed p23 is greater than 90% pure by Coomassie Blue staining of a gel-resolved sample.

p23 Accounts for the Wheat Germ Stimulating Activity of the Centricon C-100 Filtrate

When we originally found that the Centricon C-100 filtrate contained an activity that restores receptor heterocomplex assembly activity to the washed foldosome, we felt that the factor in the Centricon C-100 filtrate was not likely to be a previously identified component of the hsp heterocomplex(16) . This was based on the demonstration that our Centricon C-100 filtrate did not contain hsp90, hsp70, p60, hsp56, or p23 by Western blotting. In the experiment of Fig.6A, we again assayed by Western blotting for the presence of p23 in unconcentrated Centricon C-100 filtrate as before (lane 2) and also in filtrate concentrated 10-fold (lane 4). In the concentrated filtrate, we do not detect hsp90 but we do detect p23 (Fig.6A, lane 4).


Figure 6: p23 accounts for the wheat germ stimulating activity of the Centricon C-100 filtrate. A, p23 is present in the Centricon C-100 filtrate. One ml of reticulocyte lysate was centrifuged in a Centricon C-100 concentrator, both the retentate (0.3 ml) and the filtrate (0.7 ml) were diluted to 1 ml with buffer, and a portion of the filtrate was concentrated 10-fold. Aliquots of each fraction were resolved by SDS-PAGE, and hsp90 and p23 were assayed by Western blotting. Lane 1, whole reticulocyte lysate; lane 2, Centricon C-100 filtrate, lane 3, Centricon C-100 retentate; lane 4, 10-fold concentrated Centricon C-100 filtrate. B, depletion of p23 from Centricon C-100 filtrate with the JJ5 antibody. Aliquots of 10-fold concentrated Centricon C-100 filtrate were immunoadsorbed with JJ5 or nonimmune IgG prebound to protein A-Sepharose, and p23 was assayed in the supernatant by Western blotting. Lane 1, Centricon C-100 filtrate; lane 2, Centricon C-100 filtrate extracted with nonimmune IgG; lane 3, Centricon C-100 filtrate extracted with JJ5. C. Activity of p23-depleted Centricon C-100 filtrate. The Centricon C-100 filtrate preparations shown in B were assayed for their ability to stimulate GR-hsp90 reconstitution in wheat germ lysate. Stripped nonimmune and immune pellets (left and right lanes, respectively) were incubated with wheat germ lysate in all conditions. Condition 1, wheat germ lysate alone; condition 2, lysate plus C-100 filtrate; condition 3, lysate plus 1 µg purified p23; condition 4, lysate plus C-100 filtrate extracted with nonimmune IgG; condition 5, lysate plus C-100 filtrate extracted with JJ5; condition 6, lysate plus JJ5-extracted C-100 filtrate and 1 µg purified p23.



Although more than 90% of the p23 is retained by the filter (Fig.6A, lane 3), it seems that a sufficient amount passes through it to account for the activity of the filtrate. Extraction of the Centricon C-100 filtrate with the JJ5 antibody depletes it of p23 (Fig.6B, cf. lanes 1 and 3). As shown in Fig. 6C, Centricon C-100 filtrate that is depleted of p23 does not stimulate GR-hsp90 heterocomplex formation by wheat germ lysate (cf. condition 5 with condition 1) and addition of purified, bacterially expressed p23 to the p23 depleted lysate restores activity (condition 6). The effect of p23 on receptor heterocomplex assembly by wheat germ lysate is concentration-dependent. As shown in Fig.7, peak activity is achieved with addition of 0.5-1 µg of purified p23 to the 56 µl wheat germ reconstitution mix. At higher concentrations, there is less activity, and an addition of 20 µg p23 yields no stimulation of the wheat germ lysate.


Figure 7: Concentration dependence of p23 effect in wheat germ lysate. Immunoadsorbed, stripped receptors were incubated with wheat germ lysate plus various concentrations of p23 and steroid binding was assayed. Lane 1, stripped receptor plus wheat germ lysate alone; lanes 2-6, receptors plus lysate and 0.1 µg (lane 2), 0.5 µg (lane 3), 1 µg (lane 4), 2.5 µg (lane 5), 10 µg (lane 6), 20 µg (lane 7) of purified bacterially expressed p23.



p23 Accounts for the Activity That Is Eliminated by Washing the 3G3 Immunoadsorbed Foldosome

In the experiment of Fig.8A, the hsp heterocomplex was immunoadsorbed from rabbit reticulocyte lysate and the immunopellet was washed with buffer to deplete it of the weakly bound component. The washed foldosome immunopellet was then incubated with stripped receptors in the presence of Centricon C-100 filtrate or filtrate depleted of p23 by immunoadsorption with the JJ5 antibody. As shown in lane 5, immunoadsorption of the Centricon C-100 filtrate with nonimmune IgG does not affect its ability to restore GR activating activity to the washed foldosome (cf. lane 5 with lane 4). However, immunoadsorption with the JJ5 antibody (lane 6) eliminated the activity of the Centricon C-100 filtrate. Fig.8B shows the ability of purified human p23 to stimulate the GR activating activity of the washed foldosome. Again, at higher concentrations, p23 is less active.


Figure 8: p23 accounts for the ability of the Centricon C-100 filtrate to restore receptor activating activity to the washed foldosome. A, depletion of p23 inactivates the Centricon C-100 filtrate. Aliquots of Centricon C-100 filtrates were immunoadsorbed with JJ5 or nonimmune IgG prebound to protein A-Sepharose, and samples were incubated with stripped receptors and the washed 3G3 immunopellet of reticulocyte lysate. Lane 1, stripped receptor; lane 2, stripped receptor plus Centricon C-100 filtrate alone; lane 3, stripped receptor plus washed 8D3 immunopellet of reticulocyte lysate; lanes 4-6, condition of lane 3 plus Centricon C-100 filtrate (lane 4), filtrate extracted with nonimmune IgG (lane 5), filtrate extracted with JJ5 (lane 6). B, addition of purified human p23 to the washed foldosome increases receptor activating activity. Stripped receptors (lane 1) were incubated with the washed 8D3 immunopellet of reticulocyte lysate plus filtrate extracted with JJ5 (lane 2) or these conditions plus 0.1 µg (lane 3), 0.3 µg (lane 4), 1 µg (lane 5), or 3 µg (lane 6) of purified human p23.




DISCUSSION

The fact that human p23 interacts with the components of the wheat germ lysate to stimulate receptor heterocomplex assembly and folding attests to the conservation of function and ubiquitous distribution of the hsp heterocomplex chaperone system. Although the anti-p23 antibodies recognize a 23-kDa protein in a broad range of species from yeast to man, they do not react with a similar protein in wheat germ. The fact that rabbit and human p23s stimulate the wheat germ system could reflect either the lack of an animal p23 homolog in the plant lysate, or it could reflect different properties between the animal p23s and an as yet undetectible plant homolog.

The effect of p23 in the wheat germ lysate might involve either acceleration of GR-hsp90 heterocomplex assembly or stabilization of newly assembled complexes to dissociation during the reconstitution incubation at 30 °C (Fig.3). The effect of p23 is such that the number of GR-hsp90 complexes present in wheat germ lysate at the end of 20 min is higher (Fig.3, condition 2), but the total number of GR-hsp90 complexes generated (i.e. those that can be ``trapped'' by steroid binding during the 20 min incubation) remains the same (Fig.3, condition 1). At the moment, it seems quite reasonable to assume that p23 is required for receptor-hsp90 heterocomplex assembly and its presence in the receptor heterocomplex may contribute to, or even be necessary for, maintaining the particularly high affinity interaction between the steroid receptors and hsp90. In both the wheat germ system (Fig.7) and the washed foldosome (Fig.8B), p23 is less active when present at high concentrations. It is possible that p23 aggregates at high concentration, but it is also possible that the stoichiometry between p23 and hsp90 and/or other components of the system must be optimal for efficient receptor heterocomplex assembly.

The identification of p23 as an essential component that can restore GR-activating activity to the washed hsp heterocomplex is an important step toward total reconstitution of a functional foldosome. We now have an immunopurified hsp90 heterocomplex (the washed foldosome) to which purified p23 and the required dialyzable components of the receptor heterocomplex assembly system, ATP/Mg and K(6, 7) , can be added. The essential elements of the washed foldosome must now be identified and reconstituted into a functional chaperone system. The washed 3G3 immunopellet contains hsp90, hsp70, p60, hsp56, p50, and CyP-40. The peptidylprolyl isomerase activity of the two immunophilins, hsp56 and CyP-40 is apparently not required for GR-hsp90 heterocomplex assembly, because assembly is not affected by the inhibitors FK506 and cyclosporin A(20) .^3 Also, hsp56 can be removed from the system without affecting assembly activity.^3 hsp70 is required for receptor binding to hsp90 (6, 8) , but hsp70 and hsp90 do not form a complex with each other unless they are incubated with a third factor (or factors) from reticulocyte lysate and ATP/Mg(9) . It seems this third factor must be required for generating an active foldosome, and it may or may not be present in the hsp90 complexes we have immunoadsorbed with the 3G3 antibody. Given the possibility that the immunophilins are not required for assembly of a receptor-hsp90 complex, it is reasonable to focus on potential requirements for p60 and p50 in promoting association of hsp90 and hsp70 and in reconstitution of a core foldosome complex that is active at receptor heterocomplex assembly when it is supplemented with ATP/Mg, K, and p23.

Although the Centricon C-100 filtrate and bacterially expressed p23 facilitate reconstitution of the GR-hsp90 complex, the addition of either preparation never gives more than about 55% of the binding activity seen with reticulocyte lysate. It is possible that factors other than those discussed above are required to achieve the full activity of the reticulocyte lysate. As we have noted before(16) , eukaryotic homologs of the bacterial DnaJ and GrpE proteins may be required for full folding activity.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK31573 (to W. B. P.) and HDO9140 (to D. O. T.) and a grant from the National Sciences and Engineering Research Council of Canada (to P. K.). 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.

§
Trainee under Pharmacological Sciences Training Program Training Grant GM07767 from the National Institutes of Health.

Trainee of Cancer Biology Training Program Grant T32 CA09676 from the National Cancer Institute.

**
To whom correspondence should be addressed: Dept. of Pharmacology, 1301 Medical Science Research Bldg. III, The University of Michigan Medical School, Ann Arbor, MI 48109-0632. Tel.: 313-764-5414; Fax: 313-763-4450.

^1
The abbreviations used are: hsp, heat shock protein; GR, glucocorticoid receptor; FKBP, FK506-binding protein; CyP-40, the 40-kDa cyclosporin A-binding protein; TES, 2-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]aminoethane sulfonic acid; PAGE, polyacrylamide gel electrophoresis.

^2
L. F. Stancato, K. A. Hutchison, P. Krishna, and W. B. Pratt, submitted for publication.

^3
J. K. Owens-Grillo, K. Hoffmann, K. A. Hutchison, A. W. Yem, M. R. Deibel, R. E. Handschumacher, and W. B. Pratt, submitted for publication.

^4
M. Sacco, J. R. H. Frappier, R. F. Felsheim, and P. Krishna, submitted for publication.


ACKNOWLEDGEMENTS

We thank David Smith, Ettore Appella, and Lee Faber for providing antibodies.


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