(Received for publication, August 31, 1994; and in revised form, December 23, 1994)
From the
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.
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) ()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.
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).
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.
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).
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
-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
-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 -galactosidase levels (data not shown).
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
-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.
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. (
)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).