©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hormone-dependent Transactivation by the Human Androgen Receptor Is Regulated by a dnaJ Protein (*)

(Received for publication, August 31, 1994; and in revised form, December 23, 1994)

Avrom J. Caplan (1)(§) Elizabeth Langley (2) Elizabeth M. Wilson (2) Johanna Vidal (1)

From the  (1)Department of Cell Biology and Anatomy, Mount Sinai Medical Center, New York, New York 10029 and the (2)Department of Biochemistry and Biophysics and Department of Pediatrics, University of North Carolina, Chapel Hill, North Carolina 27599

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Genetic studies were performed to examine the role of eukaryotic dnaJ protein, Ydj1p, in the regulated activation of human androgen receptor (hAR) after heterologous expression in Saccharomyces cerevisiae. Hormone-dependent activation of hAR was measured as a function of lacZ reporter gene expression, which was defective in ydj1-151 and ydj1-2 null mutant strains compared to the wild type. This defect was not due to receptor misfolding, since hAR in both wild type and mutant strains had a similar capacity to bind hormone. The target for Ydj1p action was determined to be the hAR hormone binding domain since an N-terminal fragment lacking this region was constitutively active in both wild type and ydj1-151 mutant strains. These data correlate hormone dependence of hAR activation with a requirement for Ydj1p function and are consistent with a role for dnaJ proteins in signal transduction by steroid hormone receptors.


INTRODUCTION

Steroid hormones effect profound physiological changes in animal systems by signaling cells to activate or inhibit a variety of genes. These signaling events are mediated by intracellular receptors which become active transcription factors when bound with ligand. The five most studied receptors of this class are androgen, estrogen, glucocorticoid, mineralocorticoid, and progesterone receptors. In addition, there are more than 50 so-called orphan receptors for which no ligand or responsive genes have been characterized (O'Malley and Connely, 1992).

Molecular chaperone proteins have been reported to have a role in the activation of steroid hormone receptors. For example, the inactive form of the glucocorticoid receptor is a 9 S complex that contains two molecules of Hsp90, a heat shock protein whose precise function remains unclear. Other proteins that bind to Hsp90 in this complex include immunophilins (e.g. Hsp56) and p23. The molecular chaperone Hsp70 has also been described as a component, but unlike Hsp90, its role appears to be transitory since its stoichiometry is less than one molecule per complex (Diehl and Schmidt, 1993). Molecular chaperones dissociate from steroid hormone receptors after hormone treatment (see Pratt(1993) for review).

Recent studies indicated that Hsp70 was required for assembly of the rat glucocorticoid receptor with Hsp90. This was demonstrated in reconstitution experiments between immunopurified glucocorticoid receptor and Hsp90 in rabbit reticulocyte lysates; depletion of Hsp70 from the lysates inhibited reconstitution, but its readdition facilitated complex formation (Hutchison et al., 1994). Using a similar approach, Smith et al.(1992) demonstrated that antibodies specific to Hsp70 inhibited reconstitution of the progesterone receptor with Hsp90. The role of Hsp70 in protein assembly events, however, is not limited to steroid hormone receptor complexes. In Escherichia coli, Hsp70 (dnaK) and its partner, the dnaJ protein, function together in the assembly of pre-primosomal complexes for phage replication (Georgopoulos et al., 1990), as well as in disassembly of the inactive dimer form of phage P1 repA proteins (Wickner et al., 1991).

Together, dnaJ and Hsp70 participate in a variety of co-translational and post-translational events that mediate the fate of nascent polypeptide chains. Eukaryotic homologues of E. coli dnaJ have only recently been characterized, and they constitute a large protein family with specific members present in different organelles (for reviews, see Caplan et al.(1993) and Cyr et al.(1994)). The events in which dnaJ and Hsp70 proteins function together include transfer of polypeptides to chaperonins or foldases (e.g. GroEL and TriC) (Langer et al., 1992; Frydman et al., 1994) and transport across biological membranes (Chirico et al., 1988; Deshaies et al., 1988; Atencio and Yaffe, 1992; Caplan et al., 1992a). The relationship between these two proteins is based, at least in part, on the ability of dnaJ to interact with Hsp70 and stimulate its ATPase activity (Liberek et al., 1991a; Cyr et al., 1992; Brodsky and Schekman 1993; Scidmore et al., 1993; Cheetham et al., 1994). This affects the conformation of Hsp70, its affinity for polypeptide, and, presumably, its role in protein assembly events (Liberek et al., 1991b).

The Saccharomyces cerevisiaeYDJ1 gene encodes a functional homologue of E. coli dnaJ (Caplan et al., 1992a). Ydj1 protein (Ydj1p) is localized to the cytosol and post-translationally modified by farnesylation at its C terminus (Caplan and Douglas, 1991; Caplan et al., 1992b). Ydj1p that was purified after overexpression in E. coli interacts specifically with Hsp70 proteins of the SSA subfamily and stimulates their ATPase activity (Cyr and Douglas, 1994). The ydj1-151 mutant protein has a reduced ability to stimulate this ATPase, and yeast strains expressing this mutant allele are defective for polypeptide translocation across both endoplasmic reticulum and mitochondrial membranes (Caplan et al., 1992a).

This report examines the role of Ydj1p in the activation of heterologously expressed human androgen receptor. Like glucocorticoid receptor, human androgen receptor (hAR) (^1)interacts with Hsp90 (Mariovet et al., 1992; Veldscholte et al., 1992) and is regulated in yeast by hormone (Purvis et al., 1991), suggesting conservation in the cellular machinery responsible for maintaining the apo-receptor inactive. The results shown below indicate that Ydj1p performs a regulatory function in the activation of hAR, specifically via the hormone binding domain.


EXPERIMENTAL PROCEDURES

Genetic Methods

Yeast cells were cultured in minimal medium containing 0.67% yeast nitrogen base, 2% dextrose, and appropriate purine, pyrimidine, and amino acid supplements depending on auxotrophy. Yeast cells were transformed by the lithium acetate procedure essentially as described by Ito et al.(1983). Integrative transformations were performed after linearization of pPGKareLacZI with NcoI and pPGKhARI with EcoRV. Construction of the ydj1 deletion mutant and ydj1-151 strain ACY17b have been described (Caplan and Douglas, 1991; Caplan et al., 1992a). JC2LZ strain was constructed by transforming JC2 with pPGKareLACZI (Purvis et al., 1991). ACY68 is a diploid strain of JC2 and ACY45. Sporulation and dissection of ACY68 tetrads yielded strains ACY72-ACY75.

Plasmid Constructions

pG1-hAR was constructed after ligating a 3-kilobase pair BglII fragment containing the hAR cDNA (from pPGKhARI, Purvis et al., 1991) into BamHI-digested pG1 (the gift of Dr. M. G. Douglas). pARVP-16 was constructed by digesting pG1-hAR with TthIII and SalI releasing a 1.2-kilobase pair DNA fragment encoding the hAR hormone binding domain. In its place was ligated a similarly digested 300-base pair fragment encoding the VP-16 activating domain that was amplified from pVP16 (the gift of Dr. J. Licht) using the primers 5`-GCGCGCGACAGTGTCAGCCCCCCCGACCGATGTC and 3`-GCGCGCGTCGACCGAACCGGGGACGGGAGG by PCR (20 cycles at 55 °C annealing temperature). pABC was constructed by ligating together the blunted overhangs left after TthIII/SalI digestion of pG1hAR.

Yeast Extracts and beta-Galactosidase Assays

Yeast cultures were grown in minimal medium with and without the agonist R1881 (a synthetic androgen stored in ethanol at -20 °C) to log phase. The cells (typically from 10-ml cultures) were harvested at 3000 times g and washed once in extract buffer (20 mM Hepes-KOH, pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/ml each aprotinin, chymostatin, leupeptin, and pepstatin). Cells were resuspended in 200 µl of extract buffer in 0.5-ml Eppendorf tubes and broken with glass beads (0.45 µ) in a mini-bead beater with a single 50-s burst on medium speed at 4 °C. The tubes were pierced with a 25-gauge needle and the extract eluted by centrifugation into capless 1.5-ml centrifuge tubes. The protein content was quantitated by the method of Bradford (as described by Stoscheck(1990)). Assays for beta-galactosidase activity were performed using of 1 ml of protein extract diluted (typically to 1 µg/ml) in Z buffer (60 mM Na(2)HPO(4)bullet7H(2)O, 40 mM NAH(2)PO(4)bulletH(2)O, 10 mM KCl, 1 mM MgSO(4)bullet7H(2)O, and 50 mM beta-mercaptoethanol. Reactions were started by the addition of 200 µl of a 4 mg/ml solution of o-nitrophenyl beta-D-galactopyranoside (in 0.1 M sodium phosphate buffer, pH 7), incubated for 15-60 min at 30 °C, and terminated by the addition of 0.5 ml of 1 M Na(2)CO(3). beta-Galactosidase activity was quantitated after measuring the absorbance of the reactions (performed in duplicate) at A in a Milton Roy Spectronic Genesys 5 spectrophotometer. beta-Galactosidase units are defined as nMo-nitrophenol generated/min/mg extract using the molar extinction coefficient of o-nitrophenol (4500 at A; described by Miller(1972)).

Extracts for the Western blot shown in Fig. 2were prepared as described above but using lysis buffer (50 mM Tris-HCl, pH 7.5, 1% SDS, 1 mM phenylmethylsulfonyl fluoride) instead of extract buffer. Also, the extracts were boiled prior to the quantitation of protein using the BCA assay (Pierce).


Figure 2: Characterization of hAR expression in wild type and ydj1-151 mutant yeast. A, Northern blot analysis of wild type (ACY40, lane1; ACY44, lane3) and ydj1-151 mutant (ACY41, lane2; ACY45, lane4) strains with (ACY44, lane3; ACY45, lane4) or without (ACY40, lane1; ACY41, lane2) pG1hAR, which expresses hAR from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Duplicate samples from the same gel were probed for actin mRNA as shown. B, Western blot analysis. Protein extracts from strains ACY44 (lanes1 and 2) and ACY45 (lanes3 and 4) as well as recombinant hAR from baculovirus (B)-infected SF9 cells (lane5) were probed the AR52 (IgG fraction at 0.9 µg/ml). Strains ACY44 and ACY45 were grown overnight in the absence (lanes1 and 3) or in the presence of 100 nM R1881 (lanes2 and 4). Molecular size markers are indicated at left: 97, 68, and 45 kDa. C, binding of R1881 to hAR in yeast cells. The specific binding of hormone is expressed as pmol/mg extract protein for the following strains: wild type not expressing hAR (ACY40, lane1), wild type expressing hAR (ACY44, lane2), ydj1-151 mutant not expressing hAR (ACY41, lane3), and ydj1-151 expressing hAR (ACY45, lane 4).



R1881 Binding Assay

The binding of R1881 to wild type and ydj1-151 yeast cells ± hAR was quantitated in a saturation binding assay as follows. Yeast cells from overnight cultures were adjusted to 2 times 10^6 cells/ml in fresh medium. One ml cultures were incubated at 30 °C for 2 h prior to addition of saturating amounts (36-72 nM; n = 5) of [^3H]R1881 (DuPont) ± 100-fold excess of unlabeled R1881. After an additional 2-h incubation, the cells were washed three times in water then lysed in 200 µl of lysis buffer (20 mM Hepes-KOH, pH 7.4, 1% SDS) using glass beads as described above. Extracts from these cells were quantitated for protein concentration and samples counted in a scintillation counter. The amount of specific [^3H]R1881 bound was calculated by subtracting the counts from the samples containing the excess cold hormone (corresponding to the nonspecific binding) and converting them to pmol/mg extract protein.

Northern Blot

RNA was extracted from growing yeast cultures according to the method of Schmitt et al.(1990) with the exception that three cycles of heating (at 65 °C) and freezing (on dry ice) were performed. RNA was resolved on formaldehyde-agarose gels (1.2% according to Sambrook et al.(1989)) and transferred to Amersham Hybond-N membrane. Hybridizations were performed in Denhardt's solution (Sambrook et al., 1989) at 65 °C using P-labeled probes generated by the random priming method of Feinberg and Vogelstein(1984). Filters were washed two times for 15 min in 2 times SSC, 0.1% SDS and two times for 15 min in 0.1 times SSC, 0.1% SDS at 65 °C.

Western Blot

Western blots were performed after resolving protein extracts in SDS-polyacrylamide gels and transferring to nitrocellulose membrane (0.45 µm, Micron Separations) using a semi-dry apparatus (Bio-Rad). Filters were processed by standard methods; briefly, filters were washed in TTBS (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.05% Tween 20) and blocked for 16 h in TTBS plus 5% nonfat dry milk solution. Primary antibody incubations with AR52 (an IgG fraction at 0.9 µg/ml) were for 1-2 h in 1 times phosphate-buffered saline, 3% bovine serum albumin, 0.05% Tween 20, 0.1% thimerosal. Filters were washed three times for 10 min in TTBS before incubation in secondary antibody (conjugated with horseradish peroxidase) at 1:10,000 dilution for subsequent detection by chemiluminescence (Renaissance, DuPont). Filters were washed three times for 5 min prior to treating with detection reagent.


RESULTS

In order to examine the role of S. cerevisiae Ydj1p in hAR activation, yeast strains were constructed that constitutively express the hAR gene from a 2-µm multi-copy plasmid. In addition, the E. colilacZ gene, itself under control of androgen response elements was integrated into the yeast genome (at chromosome V, linked to the URA3 gene) and served as a reporter for hAR activation (Purvis et al., 1991). Strains containing multiple copies of the hAR gene and a single copy of the lacZ gene under control of hAR were constructed in both wild type (YDJ1) and mutant (ydj1-151) backgrounds (see Fig. 1for graphic representation of strains and Table 1for strain genotypes). The hAR gene was constitutively expressed using the promoter from the glyceraldehyde-3-phosphate dehydrogenase gene.


Figure 1: Schematic representation of wild type (ACY44) and ydj1-151 mutant (ACY45) yeast strains. Roman numerals denote chromosome assignments. ydj1-2::HIS3 denotes a gene deletion allele where the wild type YDJ1 gene has been replaced by HIS3 on chromosome XIV. The mutant ydj1-151 gene is integrated at the LEU2 locus on chromosome III (Caplan et al., 1992a). The lacZ gene (under control of androgen response elements; ARE in the figure) was integrated at the URA3 locus on chromosome V. Both strains have the hAR gene under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (G3PDH) on a multi-copy (2 µm) plasmid.





Characterization of hAR in Wild Type and ydj1-151 Mutant Yeast

The relative level of expression of the hAR gene in wild type and ydj1-151 backgrounds was determined by Northern blot analysis. In the experiment shown in Fig. 2A, the wild type strain exhibits 1.5-fold more hAR mRNA than the ydj1-151 mutant after normalization to the levels of actin mRNA. The level of hAR mRNA is 4.5-fold greater than was found in wild type cells expressing hAR from a single gene integrated into the yeast genome (data not shown). Note that two bands are observed in the lanes corresponding to hAR mRNA. The smaller of the two (denoted with an asterisk) corresponds in size to actin mRNA and may result from internal priming within hAR cDNA (Simental et al., 1991). Neither band is observed when the hAR probe is hybridized to RNA from similar strains not expressing hAR (Fig. 2A, lanes1 and 2).

The hAR protein was detected in both wild type and ydj-151 mutant strains using a specific polyclonal antibody in a Western blot experiment (AR52; Fig. 2B). The hAR protein expressed in yeast comigrates with the 118-kDa recombinant hAR protein expressed in insect cells (see Fig. 2B, lane5; Wong et al.(1993)). The level of hAR in the ydj-151 mutant was usually slightly higher than that found in the wild type (compare lanes1 and 3 of Fig. 2B), but the steady state levels in both strains decreased when the cells were grown at 30 °C with 100 nM R1881, a synthetic androgen. This appears to contrast with the effect of hormone on hAR levels in transfected COS cells, where treatment with hormone stabilized hAR and increased its half-life from 1 to 6 h at 37 °C (Kemppainen et al., 1992). The level of hAR mRNA was unaffected by growing either wild type or mutant cells in the presence of 100 nM R1881 (data not shown). Quantitative hormone binding studies (Fig. 2C) indicated hAR to have a similar capacity for R1881 in both wild type and ydj1-151 mutant strains, consistent with hAR being folded correctly. The mean 1.6-fold increase in binding seen with the mutant strain (maximum difference observed was 3-fold) correlates with the increased steady state level of protein observed by Western blot (Fig. 2B). There was negligible specific binding of R1881 to cells not expressing hAR (Fig. 2C, lanes1 and 3).

Hormone-dependent Activation of hAR Is Defective in ydj1 Mutant Strains

The ability of R1881 to activate hAR in wild type and mutant strains was determined as a function of lacZ gene induction by measuring beta-galactosidase present in soluble whole cell extracts. In the experiment shown in Fig. 3, liquid cultures of wild type and ydj1-151 cells were titrated with R1881 and lacZ reporter gene activity measured after 6 (Fig. 3A) and 16 h (Fig. 3B). The level of beta-galactosidase protein induced in the wild type strain in the presence of 100 nM R1881 varied in range from 6- to over 20-fold above the background (uninduced) levels (average from seven independent experiments, induction ratio of 14.5). These induced levels were typically 2500-5000 beta-galactosidase units (nMo-nitrophenol/min/mg extract). By contrast, induced levels of beta-galactosidase in the ydj1-151 strain was not more than 3-fold above background, even at 100 nM R1881 after incubation times of 6 (Fig. 3A) or 16 h (Fig. 3B) under permissive growth conditions for this strain (30 °C; Caplan et al., 1992a). Northern blot analysis of lacZ mRNA revealed strong expression within 1 h of R1881 treatment in wild type but not mutant cells (data not shown), confirming that the defect observed in the ydj1-151 mutant reflects an induction phenotype rather than lability in the beta-galactosidase reporter protein. Furthermore, constitutive expression of lacZ results in similar levels of beta-galactosidase protein in both wild type and mutant cells (see below and Fig. 5). The defective induction phenotype also appears to be general since a similar defect was observed with the rat glucocorticoid receptor expressed in the ydj1-151 strain. (^2)


Figure 3: Induction of lacZ gene expression by hAR in R1881-treated wild type and ydj1-151 mutant strains. A, 6-h induction; B, 16-h induction. Filledcircles, wild type (ACY44); opentriangles, ydj1-151 (ACY45).




Figure 5: Transactivation by hAR deletion mutants in wild type and ydj1-151 strains. Wild type and ydj1-151 strains expressing hAR (ACY44, ACY45), hAR (ACY62, ACY63), and ARVP-16 (ACY70, ACY71) were assayed for steady state levels of beta-galactosidase in the absence of hormone. Data are from three independent experiments.



To confirm that the low induction phenotype resulted from mutation in the YDJ1 gene, the strain ACY45 was back-crossed to a wild type strain (JC2LZ, see Table 1). The resulting diploid strain (ACY68) was sporulated, and tetrads were dissected to yield four haploids. Since ACY45 carried both the ydj1 null allele (ydj1-2::HIS3, referred to as ydj1-2 hereafter) and ydj1-151 alleles on different chromosomes (see Fig. 1), it was possible to isolate haploids, from tetrads segregating as tetratypes, that contained one wild type (YDJ1), one heterozygous wild type (YDJ1 and ydj1-151), one mutant strain equivalent to ACY45 (i.e.ydj1-151 and ydj1-2), and one complete null strain (ydj1-2). Three colonies from each strain (derived from three different tetratypes) were analyzed for their ability to express the lacZ reporter gene in the presence of R1881. In each case (see Fig. 4), maximal response to hormone was observed in the wild type (YDJ1) strain. The lowest activity was by the ydj1-2 null strain (<3-fold activation of the lacZ gene). Intermediate levels of lacZ gene induction were observed in strains expressing the ydj1-151 allele, either by itself or in the presence of the wild type gene. This heterozygous strain (i.e.YDJ1 and ydj1-151) had a lower activity than the wild type strain (YDJ1), suggesting that the ydj1-151 phenotype is dominant negative. Note also that background (uninduced) levels of beta-galactosidase were on average 2-fold higher in ydj1-151 and ydj1-2 strains than was observed in wild type strains (see also Fig. 5). Whether this reflects deregulation of hAR or another aspect of lacZ gene transcription is unclear.


Figure 4: Defective induction phenotype co-segregates with mutant ydj1 alleles. Haploids from three complete tetrads (each a tetratype in A, B, and C) dissected from sporulated ACY68 diploid cells were tested for lacZ gene induction after overnight growth in the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of 100 nM R1881. The genotypes for each strain are given below the graph and correspond to the strains listed in Table 1as follows: YDJ1 (ACY72), YDJ1 and ydj1-151 (ACY73), ydj1-151 and ydj1-2 (ACY74), and ydj1-2 (ACY75).



Similar results were recorded when the original ACY45 mutant strain was transformed by a plasmid overexpressing wild type YDJ1. In these experiments, overexpression of wild type YDJ1 in the ydj1-151 strain largely suppressed the low induction phenotype of the mutant alone, stimulating hAR-dependent lacZ gene expression 2.7-fold above values typically obtained with the mutant, but at levels that were still 72% of the wild type value (data not shown). Overexpression of wild type YDJ1 itself has little effect on hAR activation in the wild type ACY44 strain, but a similar strain overexpressing the mutant ydj1-151 gene exhibited an average 20% decrease in beta-galactosidase levels (data not shown).

Ydj1p Functions via the hAR Hormone Binding Domain

Hsp90 forms a complex with hAR via the hormone binding domain at the C-terminal end of the protein (Mariovet et al., 1992). To determine whether Ydj1p also functions via this domain, an experimental strategy was devised to take advantage of the constitutive activation that occurs in hAR truncation mutants lacking the C terminus (Simental et al., 1991, Zhou et al., 1994). As with other steroid hormone receptors, loss of the steroid binding region relieves the negative regulatory function of this domain in the absence of ligand. For these experiments, truncated versions of hAR were expressed in yeast and their constitutive trans-activating function measured by the steady state levels of beta-galactosidase. If Ydj1p function is via the steroid binding domain, then its removal might also eliminate the need for Ydj1p action. If this occurred, then the defect manifest in ydj1-151 might also be suppressed and the hAR truncation mutant would be similarly active in both wild type and mutant strains. If, however, Ydj1p function is via another domain still present in the truncation mutant, then the hAR trans-activation defect in ydj1-151 should still manifest. This would result in greatly different steady state levels of beta-galactosidase in the wild type and mutant strains.

Two deletion mutants of hAR were constructed for these experiments: one in which the C-terminal 259 amino acids of hAR (including the entire hormone binding domain) was deleted (hAR) and a second where the hormone binding domain was replaced with the 78-amino acid C-terminal activation domain of the viral transcription factor VP-16 (termed ARVP-16). Wild type and mutant strains were transformed with multi-copy plasmids (Table 2) encoding hAR and the ARVP-16 chimeric gene under control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. The results of this experiment, shown in Fig. 5, reveal that hAR and ARVP-16 behave as high level constitutive activators in both wild type and ydj1-151 strains. The steady state level of beta-galactosidase activity was at least 10-fold greater than was observed in either strain expressing full-length hAR in the absence of hormone (the data for constitutive levels of hAR in wild type and ydj1-151 strains are comparable to the background levels found in other experiments shown in Fig. 3and Fig. 4). The defect associated with the ydj1-151 mutation, therefore, is relieved when the hAR hormone binding domain is deleted. This is consistent with a specific role for Ydj1p in hAR activation through interaction with the hormone binding domain.




DISCUSSION

The results described in this paper are consistent with the Ydj1p molecular chaperone playing a role in the regulation of hAR expressed in yeast. The hAR protein was barely activated by hormone in the ydj1-151 strain (and the ydj1-2 null strain), yet was fully active after deletion of the steroid binding domain. Furthermore, since hAR had a similar binding capacity for hormone in both wild type and ydj1-151 mutant strains, the role of Ydj1p in receptor activation is apparently independent of any function it may have in folding of nascent polypeptide chains (see below).

Ydj1p thus joins other molecular chaperones such as Hsp90 and Hsp70 that appear to function in hormone-regulated activation of steroid receptors via the hormone binding domain. Unlike these other Hsps, however, a dnaJ protein has not previously been described as a component of the 9 S complex nor involved in steroid-dependent activation. There is evidence, however, for unidentified factors present in rabbit reticulocyte lysates that function in the formation of Hsp70-Hsp90 complexes and Hsp90-hormone receptor complexes. One example is from the recent work by Czar et al.(1994), who propose the existence of an Hsp70-Hsp90 complex forming factor. This is based on the observation that complex formation between Hsp70 and Hsp90 is stimulated by the presence of additional factors in rabbit reticulocyte lysate. Other studies from the laboratory of Pratt suggest that factors in addition to Hsp70 are required for heterocomplex assembly between Hsp90 and glucocorticoid receptor (Hutchison et al., 1994). The dissociation of Hsp90 from the progesterone receptor is energy-dependent and requires factors other than hormone (Kost et al., 1989 and Smith et al., 1992). Although a dnaJ protein was not identified in any of these studies, the known functions of dnaJ correlates well with the activities of these factors, that is, co-operation with Hsp70 in protein assembly and disassembly (Georgopoulos et al., 1990). If the proteins required for these events prove to be dnaJ proteins, then the function of Ydj1p in hAR activation might also involve assembly and/or disassembly of the receptor-Hsp90 complex.

Little is known of how Hsp90 assembles with steroid hormone receptors except that it appears to involve Hsp70 (Hutchison et al., 1994; Smith et al., 1992) in a post-translational event. Several lines of evidence are consistent with this assembly being post-translational rather than co-translational. First, receptors that have been isolated after immunoadsorption from animal cell cytosols are competent for reconstitution with Hsp90 in rabbit reticulocyte lysates (see Pratt(1993) for review). Second, Hsp90 will only bind to full-length glucocorticoid receptors after in-vitro translation (Dalman et al., 1989). Third, Hsp70 and Hsp40 (a dnaJ protein) but not Hsp90 are associated with polysomes (Frydman et al., 1994).

The binding of dnaJ and Hsp70 to nascent polypeptide chains is thought to reflect the first step in a chaperone-mediated protein folding pathway (Langer et al., 1992; Hendrick et al., 1993; Frydman et al., 1994). This binding is followed by transfer of the nascent polypeptide chain to a chaperonin for folding. The sequential binding of molecular chaperones also occurs as polypeptides are imported into mitochondria (Manning-Krieg et al., 1991), and in the endoplasmic reticulum immunoglobulin light chains bind sequentially, first to Bip (an Hsp70 protein located in the lumen) and then to grp94 (a similarly located Hsp90 protein; Melnick et al.(1994)).

Whether Ydj1p participates with Hsp70 in similar events for assembly of hAR-Hsp90 complexes has yet to be addressed. However, a common link between Hsp90 and Ydj1p is via their specific association with Hsp70 but not Hsp70 subfamilies (Chang and Lindquist, 1994; Cyr and Douglas, 1994). This specificity provides indirect evidence that Ydj1p could affect Hsp90 function via its interaction with Hsp70. The defect for hAR induction in the ydj1-151 strain might then be explained by the failure of the mutant ydj1-151 protein to assist in Hsp70-dependent assembly (or perhaps disassembly, see below) of the receptor-Hsp90 complex. This is supported by the previous observation that purified ydj1-151 protein was only 16% as effective as wild type Ydj1p for stimulating the ATPase activity of Hsp70 (Caplan et al., 1992a).

Previous studies using yeast have established a physiological role for Hsp90 in the hormone-dependent activation of several steroid hormone receptors (Picard et al., 1990; Bohen and Yamamoto, 1993). In the study by Picard et al.(1990), decreasing levels of Hsp90 reduced the hormone inducible activation of the glucocorticoid, estrogen, and mineralcorticoid receptors. In a similar study, Xu and Lindquist(1993) discovered that a yeast mutant having substantially reduced levels of Hsp90 remained viable when pp60 is expressed, which results in lethality in wild type yeast cells (Brugge et al., 1987). This genetic study revealed a physiological basis for the pp60-Hsp90 interaction that was previously observed in animal cells (Brugge, 1986). Recent genetic studies also revealed a role for Ydj1p in the activation of pp60 in yeast cells, since mutation of YDJ1 also suppresses the lethal phenotype resulting from pp60 expression. (^3)In immunoprecipitation experiments using the ydj1-151 strain, much higher levels of Hsp90 were coimmunoprecipitated with antibodies specific to pp60 than were found for the wild type strain after inducible expression. These data appear to confirm that mutation in the YDJ1 gene affects the interaction of Hsp90 with other proteins. Whether this is true for hAR in the ydj1-151 mutant strain awaits further investigation.

It seems likely that Ydj1p function in hAR activation will be conserved in animal cells since a human counterpart, HDJ2 (47% identity), has recently been described (Chellaiah et al., 1993; Oh et al., 1993). As shown recently by Chang and Lindquist(1994), proteins that form a stable complex with Hsp90 are also conserved in yeast and animal cells. Whether all events in the hAR activation pathway are conserved in yeast, however, is open to question. For example, hormone stabilizes hAR in animal cells (Kemppainen et al., 1992), yet reduces steady state hAR levels in yeast (Fig. 2B). While the basis for this is unclear, such differences warrant a cautious interpretation when considering data obtained using yeast and extrapolating its significance to higher animal systems. The similarities between chaperone components, on the other hand, point to a conservation in the mechanism of activation as it relates to these proteins. This is supported by the finding of a fungus-like water mold, Achyla ambisexualis, which uses steroids as both pheromones and hormones. Significantly, the unliganded receptors for these steroids form 9 S complexes that contain Hsp90 (Riehl et al., 1985, Brunt et al., 1990).


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 212-241-6563; Fax: 212-860-1174; caplan{at}msvax.mssm.edu.

(^1)
The abbreviations used are: hAR, human androgen receptor; R1881, methyltrienolone.

(^2)
M. Kenna, M. G. Douglas, and A. J. Caplan, unpublished results.

(^3)
B. Dey, A. J. Caplan, and F. Boschelli, submitted for publication.


ACKNOWLEDGEMENTS

We thank Elena Gorn (Mt. Sinai School of Medicine) for technical assistance, Dr. Marilyn Y. McGinnis for the generous gift of [^3H]R1881, and Dr. John Licht for advice on the VP-16 activating domain. We also thank Dr. J. Licht and Dr. M. G. Douglas for the gift of plasmids and Dr. Serafín Piñol Roma for critical reading of the manuscript.


REFERENCES

  1. Atencio, D. P., and Yaffe, M. P. (1992) Mol. Cell. Biol. 12, 283-291 [Abstract]
  2. Bohen, S. P., and Yamamoto, K. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11424-11428 [Abstract]
  3. Brodsky, J. L., and Schekman, R. (1993) J. Cell Biol. 123, 1355-1363 [Abstract]
  4. Brugge, J. S. (1986) Curr. Top. Microbiol. Immunol. 123, 1-22 [Medline] [Order article via Infotrieve]
  5. Brugge, J. S., Jarosik, G., Andersen, J., Queral-Listig, A., Fedor-Chaiken, M., and Broach, J. R. (1987) Mol. Cell. Biol. 7, 2180-2187 [Medline] [Order article via Infotrieve]
  6. Brunt, S. A., Riehl, R., and Silver, J. C. (1990) Mol. Cell. Biol. 10, 273-281 [Medline] [Order article via Infotrieve]
  7. Caplan, A. J., and Douglas, M. G. (1991) J. Cell Biol. 114, 609-621 [Abstract]
  8. Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1992a) Cell 71, 1143-1155 [Medline] [Order article via Infotrieve]
  9. Caplan, A. J., Tsai, J., Casey, P. J., and Douglas, M. G. (1992b) J. Biol. Chem. 267, 18890-18895 [Abstract/Free Full Text]
  10. Caplan, A. J., Cyr, D. M., and Douglas, M. G. (1993) Mol. Biol. Cell 4, 555-563 [Medline] [Order article via Infotrieve]
  11. Cheetham, M. E., Jackson, A. P., and Anderton, B. H. (1994) Eur. J. Biochem. 226, 99-107 [Abstract]
  12. Chellaiah, A., Davis, A., and Mohanakumar, T. (1993) Biochim. Biophys. Acta 1174, 111-113 [Medline] [Order article via Infotrieve]
  13. Chang, H.-C., and Lindquist, S. (1994) J. Biol. Chem. 269, 24983-24988 [Abstract/Free Full Text]
  14. Chirico, W. J., Waters, M. G., and Blobel, G. (1988) Nature 332, 805-810 [CrossRef][Medline] [Order article via Infotrieve]
  15. Craig, E. A., Baxter, B. K., Becker, J., Halladay, J., and Ziehelhoffer, T. (1994) in The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R. I., Tissi è res, A., and Georgopoulos, C., eds) pp. 31-52, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  16. Cyr, D. M., Lu, X., and Douglas, M. G. (1992) J. Biol. Chem. 267, 20927-20931 [Abstract/Free Full Text]
  17. Cyr, D. M., and Douglas, M. G. (1994) J. Biol. Chem. 269, 9798-9804 [Abstract/Free Full Text]
  18. Cyr, D. M., Langer, T., and Douglas, M. G. (1994) Trends Biochem. Sci. 19, 176-181 [CrossRef][Medline] [Order article via Infotrieve]
  19. Czar, M. J., Owens-Grillo, J. K., Dittmar, K. D., Hutchison, K. A., Zacharek, A. M., Leach, K. L., Diebl, M. R., Jr., and Pratt, W. B. (1994) J. Biol. Chem. 269, 11155-11161 [Abstract/Free Full Text]
  20. Dalman, F. C., Bresnick, E. H., Patel, E. D., Perdew, G. H., Watson, S. J., Jr., and Pratt, W. B. (1989) J. Biol. Chem. 264, 19815-19821 [Abstract/Free Full Text]
  21. Deshaies, R. J., Sanders, S. L., Feldheim, D. A., and Schekman, R. (1991) Nature 349, 806-808 [CrossRef][Medline] [Order article via Infotrieve]
  22. Diehl, E. E., and Schmidt, T. J. (1993) Biochemistry 32, 13510-13515 [Medline] [Order article via Infotrieve]
  23. Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267 [Medline] [Order article via Infotrieve]
  24. Frydman, J., Nimmwafwen, E., Ohtsuka, K., and Hartl, F. U. (1994) Nature 370, 111-117 [CrossRef][Medline] [Order article via Infotrieve]
  25. Georgopoulos, C., Ang, D., Liberek, K., and Zylicz, M. (1990) Stress Proteins in Biology and Medicine , pp. 191-221, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Hendrick, J. P., Langer, T., Davis, T. A., Hartl, F. U., and Wiedmann, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10216-10220 [Abstract]
  27. Hutchison, K. A., Dittmar, K. D., Czar, M. J., and Pratt, W. B. (1994) J. Biol. Chem. 269, 5043-5049 [Abstract/Free Full Text]
  28. Ito, H., Fukada, Y., Murata, K., and Kimura. A. (1983) J. Bacteriol. 153, 163-168 [Medline] [Order article via Infotrieve]
  29. Kemppainen, J. A., Lane, M. V., Sar, M., and Wilson, E. M. (1992). J. Biol. Chem. 267, 968-974 [Abstract/Free Full Text]
  30. Kost, S. L., Smith, D. F., Sullivan, W. P., Welch, W. J., and Toft, D. O. (1989) Mol. Cell Biol. 9, 3829-3838 [Medline] [Order article via Infotrieve]
  31. Langer, T., Lu, C., Echols, H., Flanagan, J., Hayer, M. K., and Hartl, F.-U. (1992) Nature 356, 683-689 [CrossRef][Medline] [Order article via Infotrieve]
  32. Liberek, K., Marzalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991a) Proc. Natl. Acad. Sci. U. S. A. 8, 2874-2878
  33. Liberek, K., Skowyra, D., Zylicz, M., Johnson, C., and Georgopoulos, C. (1991b) J. Biol. Chem. 266, 14491-14496 [Abstract/Free Full Text]
  34. Luke, M. M., Sutton, A., and Arndt, K. A. (1991) J. Cell Biol. 114, 623-638 [Abstract]
  35. Manning-Krieg, U. C., Scherer, P. E., and Schatz, G. (1991) EMBO J. 10, 3273-3280 [Abstract]
  36. Mariovet, S., Van Dijck, P., Verhoeven, G., and Heyns, W. (1992) Mol. Cell. Endocrinol. 88, 165-174 [CrossRef][Medline] [Order article via Infotrieve]
  37. Melnick, J., Dul, J. L., and Argon, Y. (1994) Nature 370, 373-375 [CrossRef][Medline] [Order article via Infotrieve]
  38. Miller, J. (1972) Experiments in Molecular Genetics , pp. 352-355, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  39. Oh, S., Iwahori, A., and Kato, S. (1993) Biochim. Biophys. Acta 1174, 114-116 [Medline] [Order article via Infotrieve]
  40. O'Malley, B. W., and Connely, O. M. (1992) Mol. Endocrinol. 6, 1359-1361 [Medline] [Order article via Infotrieve]
  41. Picard, D., Khursheed, B., Garabedian, M. J., Fortin, M. G., Lindquist, S., and Yamamoto, K. R. (1990) Nature 348, 166-168 [CrossRef][Medline] [Order article via Infotrieve]
  42. Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458 [Free Full Text]
  43. Purvis, I. J., Chotai, D., Dykes, C. W., Lubahn, D. B., French, F. S., Wilson, E. M., and Hobden, A. N. (1991) Gene (Amst.) 106, 35-42 [Medline] [Order article via Infotrieve]
  44. Riehl, R. M., Sullivan, W. P., Vroman, B. T., Bauer, V. J., Pearson, G. R., and Toft, D. O. (1985) Biochemistry 24, 6586-6591 [Medline] [Order article via Infotrieve]
  45. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY
  46. Schmitt, M. E., Brown, T. A., and Trumpower, B. L. (1990) Nucleic Acids Res. 18, 3091-3092 [Medline] [Order article via Infotrieve]
  47. Scidmore, M. A., Okamura, H. H., and Rose, M. D. (1993) Mol. Biol. Cell 4, 1145-1159 [Abstract]
  48. Simental, J. A., Sar, M., Lane, M. V., French, F. S., and Wilson, E. M. (1991) J. Biol. Chem. 266, 510-518 [Abstract/Free Full Text]
  49. Smith, D. F., Stensgard, B. A., Welch, W. J., and Toft, D. O. (1992) J. Biol. Chem. 267, 1350-1356 [Abstract/Free Full Text]
  50. Stoscheck, C. M. (1990) Methods Enzymol. 182, 50-68 [Medline] [Order article via Infotrieve]
  51. Veldscholte, J., Berrevoets, C. A., Zegers, N. D., van-der-Kwast, T. H., and Grootegoed, J. A. (1992) Biochemistry 31, 7422-7430 [Medline] [Order article via Infotrieve]
  52. Wickner, S., Hoskins, J., and McKenney, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7903-7907 [Abstract]
  53. Wong, C., Zhou, Z., Sar, M., and Wilson, E. M. (1993) J. Biol. Chem. 268, 19004-19012 [Abstract/Free Full Text]
  54. Xu, Y., and Lindquist, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7074-7078 [Abstract]
  55. Zhou, Z., Sar, M., Simental, J. A., Lane, M. V., and Wilson, E. M. (1994) J. Biol. Chem. 269, 13115-13123 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.