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Originally published In Press as doi:10.1074/jbc.M414245200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 10, 8906-8911, March 11, 2005
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Functional Comparison of Human and Drosophila Hop Reveals Novel Role in Steroid Receptor Maturation*

Patricia E. Carrigan{ddagger}, Daniel L. Riggs{ddagger}, Michael Chinkers§, and David F. Smith{ddagger}

From the {ddagger}Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259 and §Department of Pharmacology, University of South Alabama, Mobile, Alabama 36688

Received for publication, December 17, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes. Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear. Human Hop and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting the efficient maturation of glucocorticoid receptor (GR) function. To gain a better understanding of Hop structure/function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1. Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop failed to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding. Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional maturation of steroid receptors requires multistep assembly with molecular chaperones (1), and Hop, which binds both Hsp70 and Hsp90, can facilitate the progression through the intermediate stages of assembly (2). The early steps in assembling receptor/chaperone complexes are the binding of Hsp40 to a receptor monomer (3) followed by recruitment of Hsp70. Hop-Hsp90 complexes are then recruited to bind the receptor-associated Hsp70. In a transition that remains poorly understood, Hsp90 becomes directly associated with the receptor, and Hsp70 and Hop leave the complex. The Hsp90 co-chaperone p23 binds to Hsp90, stabilizing its association with the receptor, and immunophilin-related co-chaperones bind Hsp90 at a site vacated by Hop. Only when the receptor has achieved assembly with Hsp90 and p23 does it attain full hormone binding ability (4, 5).

Through its ability to simultaneously bind both Hsp70 and Hsp90, Hop serves as an adaptor to coordinate the recruitment of Hsp90 to intermediate receptor complexes containing Hsp70 (6). Hop can also inhibit Hsp90 ATPase, but it acts somewhat differently toward yeast or vertebrate Hsp90. Yeast Hsp90 has a much higher basal ATPase activity, and Sti1p, the Hop ortholog in Saccharomyces cerevisiae (7), inhibits this activity (8, 9). In contrast, human Hsp90 has very low basal ATPase activity that can be stimulated by binding to substrate, and Hop is able to inhibit the client-stimulated ATPase (10). In some settings Hop can also affect Hsp70 ATPase. For example, Sti1p dramatically stimulates activity of the yeast Ssa family of Hsp70 proteins (11), although Hop was not observed to stimulate the ATPase activity of vertebrate Hsp70 (12). Unlike most other receptor-associated chaperone components, Hop does not possess independent chaperone activity by as determined by in vitro refolding assays (13, 14), so it is unlikely to act directly on a receptor.

In addition to cell-free assembly studies, yeast models have been used to show that deletion of Sti1p inhibits steroid receptor function (15). Yeast lacking functional Sti1p are viable but have retarded growth at elevated temperature or in the presence of minimal media (7). We have shown previously (6) that Sti1p can functionally replace human Hop (hHop)1 using an in vitro receptor assembly system. We have also shown (16) that hHop can fully rescue the growth phenotype in a yeast strain (sti1{Delta}0) lacking the gene for Sti1p and that hHop can fully restore steroid receptor function in the sti1{Delta}0 background. Thus, it appears that Sti1p and hHop are functionally interchangeable.

Sti1p and hHop share over 50% amino acid sequence similarity and have a common domain structure consisting of three tetratricopeptide repeat domains (TPR1, TPR2a, and TPR2b) and two small domains (DP1 and DP2) containing a characteristic DP repeat motif (17). The domain arrangement is TPR1-DP1-TPR2a-TPR2b-DP2. Two of the Sti1p TPR domains have been individually crystallized (18) and have overall folds similar to each other and to the TPR domains from several other Hsp90-binding co-chaperones (reviewed in Ref. 19). Several lines of evidence suggest that the highly conserved EEVD sequence that terminates many eukaryotic Hsp90 and Hsp70 proteins directly interacts with a TPR binding pocket in co-chaperones.

Hop TPR2a is required for Hsp90 binding (20, 21); the Hsp90-specific peptide MEEVD co-crystallizes with TPR2a (18), and site-directed mutation of the EEVD terminus of Hsp90 is sufficient to disrupt binding with Hop (22–24). Therefore, EEVD-TPR interaction is critical for Hsp90 binding to Hop. Hop TPR1 is required for Hsp70 binding (20, 21), and the Hsp70-specific peptide PTIEEVD co-crystallizes with TPR1 (18). On the other hand, deletion of PTIEEVD and up to 40 additional amino acids from the C terminus of Hsp70 do not prevent binding of truncated Hsp70 to Hop (16). In contrast to Hsp90, therefore, there must be one or more additional interaction modes between Hop and Hsp70, although the EEVD-TPR1 interaction is probably functionally important.

Based on analysis of Hop co-crystal structures (18), positively charged amino acids in the TPR binding pocket form a carboxylate clamp that interacts with the negatively charged EEVD. Point mutations of carboxylate clamp residues have been shown to disrupt binding of TPR-containing proteins to Hsp90 (25, 26) or Hsp70 (27). However, we also observed that carboxylate clamp mutations in Hop TPR domains had complex, unexpected effects on Hsp70 binding (16). We have further observed that mutation of the C-terminal DP2 domain of Hop disrupts Hsp70 binding (6). Our working model is that multiple Hop domains contribute to Hsp binding and that domain-domain interactions within Hop are likely important.

To further characterize the role of individual Hop domains in Hsp binding and in functional maturation of steroid receptors, we have undertaken a functional comparison between hHop and a Hop ortholog from Drosophila melanogaster (dHop), which shares ~65% sequence similarity. One unique feature of dHop is the absence of DP1, a domain of poorly understood function found in hHop and Sti1p (16), but there are sequence dissimilarities in other domains as well. In a variety of functional assays presented here, dHop is found to be similar to hHop and Sti1p, yet dHop fails to support steroid receptor maturation in vivo, and the deficiency maps to DP2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies—The following mouse monoclonal antibodies were used in these studies: F5 (28), which recognizes vertebrate Hop; St2 (15), which is specific for yeast Sti1p; PR22 (29), which recognizes the progesterone receptor (PR); BuGR2 (Affinity Bioreagents, Golden, CO), which recognizes the glucocorticoid receptor (GR); and anti-L3, which reacts with a yeast ribosomal protein (30). Novel mouse monoclonal antibodies specific for dHop were prepared by standard procedures in the Mayo Clinic Scottsdale Immunology Core Facility. Mice were immunized with purified recombinant dHop, and hybridoma supernatants were initially screened by Western immunostaining for antibodies cross-reacting with dHop. Five monoclonal hybridoma cell lines producing IgG specific for dHop were established. The antibody selected for this study was termed DH1.

Yeast Strains and Plasmids—All S. cerevisiae strains were generated in the W303a background (MATa leu2–112 ura3–1 trp1–1 his3–11, 15 ade2–1 can1–100 GAL SUC2). Plasmids were introduced into wild type or the previously described sti1{Delta}0 yeast strain (16) using a lithium acetate/polyethylene glycol protocol. For each new strain construction, 2–4 independent transformants were compared for consistent properties. Strains were propagated in selective media consisting of 0.67% (w/v) yeast nitrogen base without amino acids, 2% (w/v) glucose, the appropriate SC supplement mixture (Qbiogene, Carlsbad, CA), and 1.6% (w/v) agar for the plates.

As described previously (31), the GR expression strain contained the plasmid pG/N795, which constitutively expresses rat GR, and pUC{Delta}SS-26X, a reporter plasmid in which lacZ expression is regulated by glucocorticoid-responsive elements. Hop cDNA was ligated into the constitutive expression plasmid p425GPD, which contains a LEU2 nutritional marker, a 2µ origin of replication, and a constitutive GPD promoter (32).

Yeast Assays for Hormone-induced Reporter Gene Expression—Hormone induction assays were conducted as described previously (31). Briefly, yeast strains were grown in selective media at 25 °C to an absorbance at 600 nm (A600) of 0.05–0.12 units. Growth was monitored by spectrophotometry for 30 min before hormone addition to ensure that the culture was in exponential phase. Deoxycorticosterone was added to the culture at 50 nM final concentration. To assay for {beta}-galactosidase activity 100 µl of culture was withdrawn and immediately added to 100 µl of the Gal-ScreenTM substrate (Applied Biosystems, Bedford, MA) in 96-well microtiter plates at room temperature. Five samples were taken at 10-min intervals starting 70–80 min after hormone addition and read in a luminometer. To determine the rate of reporter expression, {beta}-galactosidase induction curves were first generated by plotting relative light units against the A600 of the culture sample. Regression analysis of the linear portion of each data set yielded a best fit line (typically, R2 > 0.98) the slope of which (1000) is the growth-normalized rate of {beta}-galactosidase expression.

Preparation of Yeast Lysates—Yeast strains were cultivated in 4 liters of minimal medium at 30 °C. At an A600 of 1.0–1.5 units, cells were harvested and the cell pellet (~20 g) was resuspended in 20 ml of ice-cold breakage buffer (20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl2, 10 mM {beta}-mercaptoethanol plus Complete Mini protease inhibitor mixture (Roche Applied Science)). After the addition of 0.5-mm glass beads, cells were disrupted by five 30-s pulses (2 min of cooling between pulses) in a BeadBeater (Biospec Products, Bartlesville, OK) or until ~50% of the cells were broken as determined by phase contrast microscopy. The cell slurry was maintained in an ice/ethanol bath during the disruption process. After centrifugation at 100,000 x g for 1 h, the lysate protein concentration was 3–5 mg/ml as determined by Coomassie Plus protein assay (Pierce).

Co-immunoprecipitations and Immunoblots of Yeast Lysates—Mouse monoclonal antibodies specific for hHop (F5), dHop (DH1), and Sti1p (St2) were preadsorbed to protein G-Sepharose (1 µg of antibody/µl of packed resin). For each IP reaction, yeast lysate containing 5 mg of protein was incubated with 10 µl of immunoresin for 4 h at 4 °C. Resin-bound complexes were washed three times in 1 ml of wash buffer (20 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.5% Tween 20), resuspended in 10 µl of 2x SDS-PAGE sample buffer, and separated by gel electrophoresis. For immunoblots of yeast protein, aliquots of lysate (3 µg of protein) were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunostained for Sti1p, hHop, dHop, GR, or L3.

Construction of Hop Chimeras—Chimeric cDNAs combining human and Drosophila Hop cDNA sequences were constructed by PCR. Primers for the two fragments were designed to contain the complementary sequences that surrounded the desired fusion site. The resulting DNA products were gel-purified and used as megaprimers in a reaction with the appropriate 5'-3' primers to generate the full-length chimeric cDNA. The final PCR product was ligated into pSPUTK (Stratagene) for in vitro expression or into a yeast expression vector.

In Vitro Protein Binding Assays—Radiolabeled Hop forms were generated by in vitro expression (TNT kit, Promega, Madison, WI) in the presence of [35S]methionine. A 5-µl aliquot of each synthesis mixture was separated by SDS-PAGE; following autoradiography, protein bands were quantified by densitometry (Fluor-S Multi-Imager, Bio-Rad, Hercules, CA). For each co-IP, molar equivalents of each radiolabeled chimera were separately added to 100 µl of rabbit reticulocyte lysate (RL) (1:1 lysate; Green Hectares, Oregon, WI) supplemented with an ATP regenerating system (33). The RL mixture was added to the immunoresin (a 10-µl pellet preadsorbed with 10 µg of antibody) and incubated for 30 min at 30 °C. Washed, resin-bound complexes were separated by SDS-PAGE, Coomassie-stained, and autoradiographed.

PR complexes were assembled in vitro as described previously (Chen and Smith (6)). Briefly, recombinant chicken PR-A was immunopurified from Sf9 insect cell extracts using the monoclonal antibody PR22 preadsorbed to protein A-Sepharose. Each assembly reaction contained 10 µl of PR resin (representing 1 µg of PR) plus an equimolar amount of radiolabeled Hop or Hop chimera in 200 µl of RL containing an ATP regenerating system. The assembly mixture, which was supplemented with the Hsp90 inhibitor geldanamycin (20 µg/ml) to promote recovery of intermediate PR complexes containing Hop (34), was incubated for 30 min at 30 °C, and the resin-bound complexes were washed and then extracted into SDS sample buffer. Proteins were separated by SDS-PAGE, Coomassie-stained, and autoradiographed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Yeast that lack Sti1p grow normally at 30 °C but more slowly at 37 °C (7). We determined previously that hHop could functionally substitute for Sti1p as it restores normal growth at elevated temperature (16). To initiate an examination of dHop function, we tested whether dHop can rescue the temperature-sensitive growth phenotype of the sti1{Delta}0 yeast strain. As shown in Fig. 1, dHop and hHop both restore growth at 37 °C in a sti1{Delta}0 strain; thus, it appears that dHop is fully functional in this growth assay.



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  		FIG. 1.
Rescue of sti1{Delta}0 growth defect. 5-fold serial dilutions of WT or sti1{Delta}0 yeast were spotted on minimal media plates and incubated at 30 or 37 °C for 5 days. The sti1{Delta}0 strain was used without addition or was transformed with a plasmid expressing either hHop or dHop as indicated on the left. Growth at 37 °C was retarded for sti1{Delta}0 cells but not for WT or sti1{Delta}0 cells expressing exogenous Hop.

 
Further functional analysis of dHop was performed using the well established yeast model for steroid receptor function. The Yamamoto laboratory (35) first showed that yeast expressing a vertebrate steroid receptor contain the minimal cellular factors needed to support hormone-inducible activation of an appropriate reporter gene. The Lindquist laboratory (15) showed that Sti1p is necessary for full receptor activity in the yeast background, and we subsequently demonstrated (16) that hHop will fully rescue GR function in sti1{Delta}0. Here we extend those studies by demonstrating the ability of dHop to restore GR function (Fig. 2). In sti1{Delta}0 transformed with empty vector, hormone-induced activity remains near the uninduced level, but wild-type activity is restored by expressing hHop. In contrast to the human protein, dHop was unable to restore GR function even though dHop can rescue the temperature-sensitive growth phenotype in sti1{Delta}0. Western immunostains confirmed that Sti1p is absent in sti1{Delta}0 cells (Fig. 2B), that hHop and dHop are expressed in transformed yeast (Fig. 2C), and that the level of GR protein is similar in all strains (Fig. 2D).



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  		FIG. 2.
Functional comparison of hHop and dHop in yeast GR reporter strains. Either WT or sti1{Delta}0 yeast were transformed with a plasmid constitutively expressing rat GR and a reporter plasmid in which lacZ expression is regulated by hormone-responsive DNA elements. The sti1{Delta}0 reporter strain was co-transformed with an empty plasmid vector (vec.) or with a plasmid constitutively expressing hHop or dHop. A, liquid cultures of each strain (n = 3) were induced (solid bars) or not (open bar) with 50 nM deoxycorticosterone, and the rate of increase in {beta}-galactosidase activity was measured as described under "Materials and Methods." B, extracts from WT and sti1{Delta}0 yeast were Western immunoblotted for Sti1p. C, extracts were prepared from sti1{Delta}0 cells transformed with one of three plasmids (indicated above the gel) and Western immunoblotted with antibodies specific for hHop, dHop, or L3 (indicated on the left). L3 is an endogenous ribosomal protein that serves as a loading control. D, GR and L3 protein levels were compared by Western immunoblotting of WT or sti1{Delta}0 extracts.

 
To gain insight into the nature of the defect that prevents dHop support of GR function, we examined protein interactions of dHop. A critical feature of Hop-mediated assembly of receptor complexes is the binding of Hop to both Hsp70 and Hsp90. To examine whether dHop and hHop interact differently with Hsp70, Hsp90, or steroid receptor complexes, co-immunoprecipitation assays were performed in rabbit RL, a medium commonly used for cell-free assembly of receptor complexes. As seen in Fig. 3, radiolabeled hHop and dHop co-immunoprecipitate equally with either Hsp70 or Hsp90 (Fig. 3A). Furthermore, hHop and dHop seemed to assemble equally well in PR complexes (Fig. 3B). The co-IP analysis was extended to yeast extracts, as shown in Fig. 4. Sti1p, hHop, and dHop complexes were individually targeted using specific monoclonal antibodies. To control for nonspecific binding of chaperones to immune complexes, IP was performed at low ionic strength, which preserves Hsp70-Hop-Hsp90 complexes, and at elevated ionic strength, which dissociates Hsp binding to Hop. Further controls included IP reactions using extracts from yeast lacking the targeted Hop forms. Yeast Hsp90 (Hsc82, confirmed by Western immunostaining) and Hsp70 forms (Ssa1 and Ssa2 as determined by mass spectrometry) were specifically recovered with each of the three Hop orthologs.



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  		FIG. 3.
Interaction of Hop orthologs with Hsp70, Hsp90, and PR. Hop orthologs were expressed in vitro to generate radiolabeled protein products, and molar equivalents of either hHop or dHop were separately added to rabbit reticulocyte lysate. A, chaperone complexes were immunoprecipitated with antibodies specific for Hsp70 or Hsp90 (as indicated above the lanes) and separated by SDS gel electrophoresis. The gel was Coomassie-stained to detect total proteins (upper panel) and autoradiographed to detect radiolabeled Hop forms in each complex (small panel labeled Bound). Aliquots of each radiolabeled Hop synthesis mixture were electrophoretically separated and autoradiographed to demonstrate equal loading (Input). Major proteins in the chaperone complex and antibody heavy chain (HC) are indicated to the left of the stained gel. B, immunoadsorbed recombinant PR was incubated in lysate mixtures to assemble complexes. Recovered complexes were electrophoretically separated, Coomassie-stained (upper panel), and autoradiographed for associated Hop forms (Bound).

 



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  		FIG. 4.
Sti1p, hHop, and dHop complexes in yeast. Extracts were prepared from WT or sti1{Delta}0 cells transformed with the indicated plasmid and immunoprecipitated with antibodies specific for Sti1p, hHop, or dHop (IP). One aliquot of each extract was adjusted to 500 mM NaCl (Salt) prior to the immunoprecipitations. Recovered complexes were electrophoretically separated and Coomassie-stained to detect bound proteins. The identity of Hsc82 was confirmed by a separate Western immunoblot. The band labeled Ssa was confirmed by mass spectrometry to contain Ssa1 and Ssa2 but not other yeast Hsp70 forms.

 
To better understand why dHop fails to support GR maturation, the deduced amino acid sequences for hHop, Sti1p, and dHop were aligned to identify regions of dissimilarity among the orthologs (Fig. 5). The most striking difference is the apparent absence of DP1 in dHop. Other domains are intact, although amino acid differences within these domains might be critical. As an experimental approach to map the relevant differences in hHop and dHop, a series of chimeric constructs was generated in which domains were exchanged between orthologs. The exchange boundaries are indicated by arrowheads in Fig. 5, and the resulting chimeras are illustrated in Fig. 6.



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  		FIG. 5.
Amino acid sequence alignment for Hop orthologs. Deduced amino acid sequences for hHop (human, GenBankTM accession number M86752 [GenBank] ), Sti1p (yeast, GenBankTM accession number P15705 [GenBank] ), and dHop (fly, GenBankTM accession number AF056198 [GenBank] ) were aligned using PileUp (GCG Wisconsin Package). The TPR domains (single overline), DP domains (double overline), DP repeat sites (underline) within the DP domains, and junction sites (arrowheads) for the chimeric constructs are indicated within the alignment.

 



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  		FIG. 6.
Schematic of chimeric proteins. A series (A–J) of chimeric cDNA encoding a combination of hHop (open) and dHop (filled) domains was constructed as shown.

 
Radiolabeled wild-type and chimeric proteins were generated in vitro and tested for co-IP with Hsp70 (Fig. 7A), Hsp90 (Fig. 7B), and PR (Fig. 7C). In addition, wild-type and chimeric Hop forms were functionally compared after separately transforming the sti1{Delta}0 GR reporter strain with an expression plasmid for each Hop form. Results for the 4 chimeras that generated reporter signals above background are plotted in Fig. 8. The results from all chimera assays are summarized in Fig. 9. One of the clear findings is that transfer of the dHop C-terminal DP2 domain to hHop (chimera I) is sufficient to block Hop-mediated GR activation even though Hsp binding and receptor assembly in vitro are unaffected. Another potentially important observation is that placing the human DP1 domain into dHop (chimera C) completely disrupts Hsp binding. The converse hHop construct in which DP1 has been removed (chimera D) retains Hsp binding and GR activation.



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  		FIG. 7.
Recovery of chimeric proteins with Hsp70 (A), Hsp90 (B), and PR (C) complexes. Wild-type and chimeric cDNA were expressed in vitro to obtain the radiolabeled protein products. Molar equivalents of each product (Input) were separately added to rabbit RL prior to the immunoprecipitations, as described for Fig. 3 and under "Materials and Methods."

 



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  		FIG. 8.
Ability of chimeric Hop forms to stimulate GR activity. The sti1{Delta}0 GR reporter strain was separately transformed with an empty vector (vec.) or plasmid constitutively expressing the indicated Hop form or chimera (D, E, H, J). Replicate samples (n = 3) of each were induced with 50 nM deoxycorticosterone and assayed for induced {beta}-galactosidase activity.

 



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  		FIG. 9.
Summary of chimera interactions and GR activity. Relative levels of co-precipitation from Fig. 7 and GR activity from Fig. 8 were quantitated as shown for hHop, dHop, and each of the chimeric constructs (A–J).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A comparative approach has uncovered unique features of Hop function that relate to its role in GR maturation and function. The fact that dHop, similar to human and yeast orthologs, can support yeast growth at elevated temperatures, and yet dHop alone fails to support GR activity, provides a unique demonstration that Hop functions differently in these roles. The molecular targets of Hop that are relevant to temperature-sensitive growth have not been identified, but multiple clients of Hsp70 and Hsp90 could be adversely affected by the loss of Hop and collectively contribute to growth retardation. GR, however, must be affected by Hop in a distinct manner since dHop fails to support GR activity.

For Hop to support steroid receptor assembly and functional maturation it must bind both Hsp70 and Hsp90 (20). Because Hop does not directly interact with a receptor, it has been considered an adaptor protein that helps recruit Hsp90 to pre-existing Hsp70-receptor complexes (6). However, in light of our results with chimera I in which binding to Hsp70 and Hsp90 remains maximal, whereas recovery of GR function is minimal, there must be an additional Hop activity other than passive binding to Hsp70 and Hsp90 that is critical for GR function in vivo. This additional activity maps most closely with DP2.

We have shown that DP2 forms an independent domain that is readily disrupted by point mutations in the conserved DP repeat motif (36). A consequence of DP2 disruption is abrogation of Hsp70 binding, which provides evidence that DP2 and TPR1 interact in some manner to support Hsp70 binding ability. The DP2 domain of dHop retains DP repeat sites and shares greater sequence similarity to hHop (70% similarity) than does the DP2 domain of Sti1p (60% similarity). A key observation in this study is that exchanging DP2 between hHop and dHop does not grossly affect Hsp70 binding; nonetheless, chimera I (hHop with Drosophila DP2) fails to support GR activity, and the converse chimera J (dHop with human DP2) gains the ability to enhance GR activity. Therefore, DP2 contributes something other than Hsp70 binding to promote GR function.

It is intriguing that Hop has a second DP-related domain, DP1, inserted between TPR1 and TPR2a, and that DP1 is absent from dHop. DP1 does not appear to be critical for hHop function because mutations in the DP repeat motif had no effect on Hsp binding or GR activity (16), and deletion of DP1 from hHop (chimera D) only partially reduced Hsp binding and GR activity. On the other hand, insertion of human DP1 into dHop (chimera C) abolished binding to either Hsp, suggesting that the Drosophila protein has adapted to interact appropriately with Hsp70 or Hsp90 only when DP1 is missing.

A complete understanding of how Hop promotes functional maturation of steroid receptors will likely require a better knowledge of domain-domain interactions within Hop and how these interactions are affected by Hsp binding, by the presence of a receptor bound to Hsp, by additional Hsp co-chaperones that participate in the full assembly pathway, and perhaps by post-translational modification of Hop (37, 38). Changes in Hop structure could contribute to the poorly defined transition of the receptor from intermediate complexes most closely associated with Hsp70 to functionally mature complexes most closely associated with Hsp90.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant R01-DK44923. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: S. C. Johnson Research Bldg., Mayo Clinic Scottsdale, 13400 E. Shea Blvd., Scottsdale, AZ 85259. Tel.: 480-301-6595; Fax: 480-301-9162; E-mail: smith.david26{at}mayo.edu.

1 The abbreviations used are: hHop, human Hop; dHop, Drosophila Hop; TPR, tetratricopeptide repeat; RL, reticulocyte lysate; PR, progesterone receptor; GR, glucocorticoid receptor; IP, immunoprecipitation; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Patricia Roberts for expert technical assistance, D. O. Toft for providing antibody St2, and J. R. Warner for providing the anti-L3 antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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