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Two tetratricopeptide repeat proteins facilitate human aryl hydrocarbon receptor signalling in yeast
Cellular Signalling 14 (2002) 615 – 623 www.elsevier.com/locate/cellsig
Two tetratricopeptide repeat proteins facilitate human aryl hydrocarbon receptor signalling in yeast Charles A. Miller* Environmental Health Sciences Department, Center for Bioenvironmental Research, Tulane University School of Public Health and Tropical Medicine, 1430 Tulane Avenue New Orleans, LA 70112, USA Received 30 September 2001; accepted 4 December 2001
Abstract A human aryl hydrocarbon (Ah) receptor signalling pathway was constructed in yeast and used to identify regulatory proteins that may be related to those present in mammalian cells. The sequence similarity of human hepatitis B protein X-associated protein 2 (XAP2) protein to yeast Cpr7 and Cns1 proteins suggested that these proteins might be involved in Ah receptor signalling in this model system. Ah receptor signalling from a lacZ reporter gene was reduced by 60% in cells that lacked Cpr7. In vitro interaction experiments indicated that a Cpr7 – GST fusion protein and Ah receptor formed a complex. Expression of Cpr7, Cns1 and the isolated tetratricopeptide repeat (TPR) region of Cpr7 from plasmids restored Ah receptor signalling function in the Cpr7-deficient strain. Thus, Cpr7 and Cns1 proteins facilitate the signalling of human Ah receptor expressed in yeast, perhaps in the same manner as the TPR-containing XAP2 protein and related chaperone proteins in mammalian cells. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Aryl hydrocarbon receptor; Dioxin; Chaperone; Yeast; Tetratricopeptide repeat
1. Introduction Aryl hydrocarbon (Ah) receptor is a ligand-activated component of a heterodimeric transcription factor called the Ah receptor complex (AHRC) [1– 3]. Ah receptor nuclear translocator (Arnt) protein makes up the other component of the AHRC. The most notable and best-characterized Ah receptor ligand is the highly toxic environmental contaminant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Ah receptor and Arnt are structurally related members of a family of proteins that contain PAS motifs, a domain that is involved in both protein – protein interactions and regulatory functions [4]. The Ah receptor is activated upon binding of an aromatic ligand to a region that spans its second PAS domain [5]. Prior to activation by ligand, Ah receptor exists
Abbreviations: Ah receptor, aryl hydrocarbon receptor; Arnt, aryl hydrocarbon nuclear translocator; TCDD, 2,3,7,8 tetrachlorodibenzop-dioxin; AHRC, aryl hydrocarbon receptor complex; Hsp, heat shock protein; TPR, tetratricopeptide repeat; XAP2, hepatitis B protein X-associated protein 2 * Tel.: +1-504-585-6942; fax: +1-504-584-1726. E-mail address: [email protected] (C.A. Miller).
in a cytoplasmic complex with heat shock protein-90 (Hsp90) and other proteins that regulate its function. Ligand binding studies, receptor sedimentation rates in sucrose density gradients, coprecipitation studies, and genetic evidence from yeast expressing either Ah receptor– Arnt or Ah receptor chimeras have provided evidence that Hsp90 complexes play a key role in Ah receptor signalling [5 –10]. In addition to Hsp90, other proteins may participate in the initial steps that prepare the nuclear receptors for ligand interaction and signal transduction. Several proteins have been identified as components of the eukaryotic Hsp90 chaperone machinery. These factors either facilitate or are required for the function of specific steroid hormone receptors, protein kinases and other substrates [11,12]. Thus, the Hsp90 complexes participate in the regulation of diverse but specific signalling pathways and enzymes. Steroid hormone receptors are among the bestcharacterized substrates of the Hsp90 complexes. Early events in the Hsp90 chaperone pathway involve steroid receptor interaction with DnaJ-like proteins, Hsp70 and Hsp90 [13]. Sti1 (also called p60 and HOP), a protein that contains several tetratricopeptide repeat (TPR) motifs, is thought to function early in the receptor maturation process
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by mediating receptor interactions with Hsp70 and Hsp90 [14]. The TPR motifs of Sti1 (also called p60 or hop) are thought to provide a scaffold for the formation of multiprotein interactions [15,16]. A group of proteins, which includes the immunophilins and an acidic 23-kDa protein ( p23), participates in the latter steps of steroid receptor maturation [11,12,17 – 21]. Some immunophilin family members have prolyl isomerase activity and may direct protein structure. Cofactors of Hsp90 complexes are important for signal transduction through the Ah receptor, but the role(s) of these proteins is not well studied. A 40-kDa protein that tightly associated with Ah receptor– Hsp90 complexes was noted by Chen and Perdew [6]. Subsequently, the 40-kDa protein was identified using a reverse genetic approach [22] and was identified independently by two other groups using the yeast two-hybrid methods [23,24]. The 40-kDa factor associated with the Ah receptor– Hsp90 complex is hepatitis B protein X-associated protein 2 (XAP2) and is also known as ARA9 (Ah receptor-associated protein 9) and AIP (Ah receptor-interacting protein). XAP2 is a 37-kDa TPRcontaining protein that resembles the molecular chaperone FKBP52 in structure. Like FKBP52, the related XAP2 protein is associated with Hsp90 complexes but it preferentially associates with the Ah receptor and interacts poorly with steroid hormone receptors. Unlike FKBP52, XAP2 does not bind to the immunosuppressive drug FK506 [25]. XAP2 enhances Ah receptor stability and facilitates the association with Hsp90 [26,27]. Thus, the XAP2 and FKBP52 proteins are structurally similar but display unique regulatory functions and interaction specificities. Studies with XAP2 implicate its TPR region in Ah receptor interactions and signalling [25,26]. Receptors and enzymes of metazoans usually function and display appropriate regulation when expressed in Saccharomyces cerevisiae. Such yeast model systems provide a means to elucidate signal transduction pathways [28] and are especially applicable to studying receptors that function through Hsp90-regulated processes [29]. This is possible due to the strong structural and functional conservation of Hsp90s and cofactors that extend across the eukaryotic phyla [29,30]. A model system that expresses functional human Ah receptor and Arnt proteins has been constructed in yeast as a means to study ligand interactions and signal transduction [9,31,32]. This model Ah receptor system is ligand dependent and responds to known ligands such as TCDD, benzo[a]pyrene, b-napthoflavone and hexachlorobenzene at concentrations that have biological activity in mammalian cells. Ah receptor requires sufficient levels of intact Hsp90 for signal transduction when expressed in this model system. Thus, the Ah receptor pathway expressed in yeast faithfully duplicates the regulation observed in vertebrate cells. Since Ah receptor signalling functions in yeast, it was reasoned that yeast factors related to human XAP2 might regulate human Ah receptor function in yeast. This possibility seemed probable in light of the findings that such
Hsp90-associated factors were shown to govern steroid hormone receptor function in yeast [17,33]. Here it is reported that two TPR-containing yeast proteins, Cpr7 and Cns1, are important for human Ah receptor signalling in a model yeast system.
2. Experimental procedures 2.1. Reagents Reagent grade chemicals used in this study were purchased from Fisher Scientific (Fairlawn, NJ) and Sigma (St. Louis, MO). Enzymes used for subcloning and other genetic manipulations were from New England Biolabs (Beverley, MA). Taq DNA polymerase, transcription and translation reaction mixes were from Promega (Madison, WI) and radioisotopes were purchased from New England Nuclear (Boston, MA). 2.2. Bacterial and yeast strains The DH5a strain of Escherichia coli was used for DNA manipulations and plasmid DNA amplification via routine techniques that have been described elsewhere [34]. The BL21 strain of E. coli was used to express GST fusion proteins from plasmids. W303a (MATa, ade2-1, can1-100, his3-11,15, leu2-3,112, trp1-1, ura3-1) is the parental yeast strain for all the experiments reported here. The cpr6,7- and cpr7-deficient strains have been described previously, along with the Cpr6 and Cpr7 expression plasmids used in this study [17,35,36]. The pJM88 and pAADB97 centromeric vectors were used to express Cpr6 and Cpr7, respectively. The pAADB158 vector expressed the Cpr7 derivative with three point mutations in the immunophilin-like domain and pAADB163 expressed the TPR region of Cpr7. The pJM4 and pAADB66 were used to express the Cpr7 – Gst and Cpr6– Gst fusion proteins in E. coli. The pD16 vector (a gift from Linda Hyman, Tulane University) expresses the lacZ gene under the control of a galactose-inducible promoter. Yeast was transformed with these various plasmids using a lithium acetate protocol [37]. Transformants were plated on synthetic complete medium containing 2% glucose as a carbon source that lacked the essential amino acids or nucleosides [38]. Cells were propagated in synthetic complete liquid medium that contained glucose, and galactose-containing synthetic medium was used to induce expression of genes under control of the GAL1,10 (galactose inducible) promoter. 2.3. DNA manipulations The CNS1 gene was amplified by polymerase chain reaction (PCR) using genomic DNA derived from the W303a strain as the template and the primers 50-CCGTCGACAAATACTCTGGTATTCCCCCTG and 50-GGGGATCCGCAGCATTGAATTAAAAGTGAGGTC. After an initial
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denaturation at 95 C for 2 min, the reaction was cycled twice at an annealing temperature of 42 C for 45 s, followed by 2 min extension at 72 C and denaturation at 95 C for 30 s, then 28 more cycles were performed with a higher annealing temperature of 55 C. The CNS1 PCR product was digested with BamHI and SalI restriction enzymes and subcloned between the BamHI and SalI sites of the GAL1,10 promoter of the high-copy pBEVY-GA yeast expression vector [9], to create pBEVY-GA – CNS1. The human XAP2 (ARA9) sequence was a gift from Christopher Bradfield, University of Wisconsin, and was provided in the vector pI613. The XAP2 sequence was excised using BspE1 and XmaI and inserted into the pBEVY-GA plasmid at the XmaI site to make pBEVY-GA – ARA9. To make a low-copy expression vector, the XAP2 gene expression construct in pBEVY-GA was excised with SalI and XhoI and inserted into the SalI site of the low-copy centromeric vector YCplac33 [39]. The pIGAHRC plasmid [31] was used to introduce the bidirectional Ah receptor – GAL1,10 – Arnt expression construct into chromosome III of W303a, cpr6,7-deficient and cpr7deficient strains. A two-step recombination methodology using the URA3 vector followed by negative selection on 5-fluoroorotic acid-containing plates was used for this insertion [40]. This integrated genomic construct provided for the proper expression levels of human Ah receptor and Arnt proteins when cells were grown in medium that contained galactose as the inducing carbon source. 2.4. lacZ expression assays The functional lacZ (b-galactosidase) assay to detect ligand-activated gene expression by AHRC has been described previously. A modified lacZ assay was scaled to a 96-well plate format and used to assess AHRC activation in yeast [41]. Cells from saturated cultures grown overnight in synthetic minimal glucose medium were inoculated into tubes containing 1 ml of synthetic minimal galactose medium supplemented with essential amino acids and nucleosides as required by the genotype of the yeast strain. Polystyrene culture tubes were used for exposure of yeast to the aromatic hydrocarbon b-napthoflavone. It is important to note that for some aromatic hydrocarbons other than b-napthoflavone, the type of culture tube used can determine Ah receptor ligand efficacy in yeast [31]. Dimethyl sulfoxide (DMSO) with or without the ligand b-napthoflavone (Acros Organics) was added to a final concentration of 1% for all exposures. This concentration of solvent did not affect cell growth or signalling from the reporter plasmid. All dilutions of ligands were made in DMSO rather than by serial dilution in medium to avoid loss of aromatic hydrocarbons due to the adsorption on plastic surfaces. Triplicate cultures were exposed to ligand for 18 h with shaking at 30 C. Incubations for more than 1 h during lacZ assays were avoided due to attrition of enzyme activity over time. Readings of lacZ assays and cell density were taken in the linear ranges of spectrophotometric measurement. The data
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reported here are means and standard deviations of representative experiments that have given consistent results in at least three independent trials. 2.5. In vitro interaction experiments The cDNA sequences of Ah receptor, Arnt (gifts from Drs. Christopher Bradfield, University of Wisconsin and Oliver Hankinson, UCLA, respectively) and firefly luciferase (Promega) were transcribed and translated in vitro from plasmids that contained the SP6 promoter. The TNT protocol (Promega) was used to generate 35S-radiolabelled proteins with minor modifications. Purification of the Ah receptor and Arnt plasmids by CsCl gradient centrifugation and supplementation of the TNT reaction mix with an additional 10 mM KCl and 0.25 mM MgCl2 was necessary to make sufficient amounts of 35S-labelled Ah receptor and Arnt proteins. Following synthesis, phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 mM to prevent proteolysis. The Cpr6 – GST and Cpr7 – GST expression plasmids (pAADB66 and pJM4, respectively) were expressed in BL21 cells. Isopropylthio-b-D-galatopyranoside was added to a final concentration of 0.1 mM when cultures reached an optical density of 0.6 at a wavelength of 600 nm. The cells were then cultured for an additional 3 h and harvested. Cultures were washed once in water and once in interaction buffer (20 mM Tris HCl, 100 mM NaCl, 2 mM EDTA and 0.5% Nonidet P40, pH 7.8) supplemented with PMSF (1 mM) to reduce proteinase activity. The cells were resuspended in 1 ml ice-cold interaction buffer containing 1 mM dithiothreitol (DTT) and PMSF and lysed with three pulses of 30 s each from a Vibra Cell sonicator (Sonics and Materials) with a microtip at a power setting of 10%. The lysate was clarified by centrifugation at 14,000 g for 15 min at 4 C. The supernatant (500 ml) was reacted with glutathione – sepharose beads (Sigma) to immobilize the GST fusion proteins. The beads were washed five times with interaction buffer containing 1 mM DTT to liberate unassociated proteins. The beads were blocked by a wash in interaction buffer containing 1% nonfat dry milk followed by a wash in buffer with 5% bovine serum albumin and then washed three times in interaction buffer alone. Twenty microliters packed volume of the beads was reacted with 15 ml of the 35S-labelled translated protein reaction described above in a final volume of 100 ml of interaction buffer. Protein binding reactions were incubated at room temperature with gentle mixing for 30 min and then the beads were washed five times with interaction buffer to remove unassociated proteins. Supernatants were removed from the beads and 20 ml of 2 SDS polyacrylamide gel (SDS-PAGE) sample buffer was added. Samples were heated to 70 C for 5 min and then loaded onto a 6% SDS-PAGE and resolved. The resulting gels were dried and subjected to autoradiography. The images were obtained using a phosphorimager and were compared using densitometric software (MacBAS version 2.5, Fuji).
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2.6. Western blotting and immunodetection of Ah receptor and Arnt Yeast strains expressing Ah receptor and Arnt proteins were grown to late log phase in 50-ml cultures of synthetic medium containing galactose as the inducing carbon source. The cells were sedimented, washed once in water, resuspended in disruption buffer supplemented with proteinase inhibitors and broken with glass beads. The cell lysates were clarified by centrifugation at 14,000 g for 10 min. Supernatants were mixed with an equal volume of 2 concentration of SDS-PAGE sample buffer [42], heated to 70 C for 5 min and separated on 7.5% SDS-PAGE. Gels were either fixed and stained with Coomassie Blue dye to determine protein concentrations or blotted to nitrocellulose as described previously [43]. Blots were blocked with 5% nonfat dry milk in a solution of 0.1 M NaCl and 0.1% Tween 20 detergent buffered with 25 mM Tris HCl to pH 7.8 to prevent nonspecific interaction of antibodies. Rabbit polyclonal antisera to Ah receptor and Arnt (provided by Dr. Richard Pollenz, University of South Florida) were used as the primary antibodies at a dilution of 1:100. Following antibody binding, blots were washed with five changes of the buffer above lacking nonfat dry milk. Secondary goat antirabbit peroxidase conjugated antibodies were used with an ECL detection system (Amersham) according to the manufacturer’s instructions and the resulting blot was exposed to X-ray film to visualize the Ah receptor and Arnt proteins. It was necessary to absorb primary and secondary antisera with yeast lysates bound to nitrocellulose to remove high background reactivity before detection of Ah receptor and Arnt.
3. Results 3.1. Relationship between XAP2 and yeast proteins Three groups have independently identified XAP2 as a conserved protein that interacts with Ah receptor in murine, simian and human cells (see Introduction). The relatively ubiquitous expression of XAP2 and its physical interaction with Ah receptor and Hsp90 suggest that a regulatory relationship exists between these proteins. Since Ah receptor functions in yeast, it was hypothesized that related proteins with analogous function to XAP2 might exist. A search of the Saccharomyces Database with the Blast program hwww.ncbi.nlm.nih.govi indicated that human XAP2 contains a 150 amino acid region of similarity to the yeast Cpr7 (19% identity and 39% similarity) and Cns1 (25% identity and 41% similarity) proteins. The human XAP2 sequence matched most of the carboxyl terminal half of the Cpr7 protein and a central region of the Cns1 protein to a similar extent (Fig. 1). The common region shared by these proteins contains TPR motifs. The significance of the similarity between XAP2 and Cpr7 was intriguing since
Fig. 1. Structures of TPR-containing proteins. The human XAP2 protein is compared to the yeast Cpr6, Cpr7 and Cns1 protein structures. The dotted lines denote the common multiple TPR region that is shared by the proteins. The FKBP12- and CyP18-like domains indicate sequences that resemble prolyl isomerase domains of other chaperones. Note that Cns1 has no apparent FKBP12- or CyP18-like domains and is not related to the other proteins except for the sequence similarity in the TPR region.
both are immunophilins. Furthermore, Cpr7 regulates signalling of vertebrate steroid hormone receptors expressed in yeast [17]. Another surprising result was that Cpr6, a protein that shows considerable sequence conservation ( 40% identity and 55% similarity) with Cpr7, did not match the human XAP2 sequence. Thus, the relationship among XAP2, Cpr7 and Cns1 appeared specific and spanned the regions of the proteins that included TPRs. 3.2. A specific role for Cpr7 in Ah receptor signalling A strain that contained deletions in both the CPR6 and CPR7 genes and coexpressed the human Ah receptor and ARNT genes was constructed in order to test the role of these proteins in signal transduction. An expression construct for Ah receptor and Arnt was inserted into chromosome III and a lacZ reporter plasmid, pTXRE5-Z, was transformed into the cpr6,7-deficient strain. The response to the aromatic hydrocarbon ligand, b-napthoflavone, was determined in this strain and compared to that of an isogenic strain with intact CPR6 and CPR7 genes (Fig. 2A). Ligand dependent signalling through the Ah receptor pathway was reduced by 50– 70% in the cpr6,7 strain. Expression of Cpr7 protein from a plasmid introduced into the cpr6,7 strain completely restored Ah receptor signalling to levels that were equal to that of wild-type cells. Thus, Cpr7 expression is needed for efficient signalling in yeast expressing human Ah receptor and Arnt. In contrast, expression of Cpr6 had no effect on the impaired Ah receptor signalling in the cpr6,7 strain. Note that wild-type Cpr7 strains responded well at 10 nM b-napthoflavone, whereas a high ligand concentration (1 mM) produced only modest signalling in the cpr7 strains. Thus, the Cpr7 protein modulated the apparent potency and efficacy of the Ah receptor ligand. It was surprising that the Cpr6 protein, which showed considerable conservation of primary sequence structure with Cpr7, had no apparent role in Ah receptor signalling. The effect of the cpr7 deletion on Ah receptor signalling was not due to a general (nonspecific) reduction in transcriptional or translational processes. Evidence for the specificity
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Fig. 2. Elimination of Cpr7 specifically disrupts Ah receptor signalling. Yeast strains expressing human Ah receptor and Arnt with or without disrupted CPR6 and CPR7 genes were constructed. These strains also carried a b-galactosidase reporter plasmid directed by Ah receptor – Arnt responsive DNA elements, thus permitting assessment of Ah receptor signalling. (A) The strain with no mutations that coexpresses Ah receptor and Arnt proteins is indicated as the wild-type (wt). The strain lacking both proteins is indicated as cpr6,7. Strains lacking a single protein were generated by transforming the cpr6,7 strain with a plasmid that expressed either Cpr6 (labelled cpr7) or Cpr7 proteins (labelled cpr6). Cells were treated with 0 (black bars), 10 (darker grey bars) or 1000 nM (lighter grey bars) b-napthoflavone (Ah receptor ligand) for 18 h and assessed for reporter plasmid expression. Results are reported as the mean b-galactosidase activity in units along with the standard deviation (error bars). (B) The results from wild-type and a cpr6,7-deficient strain that were transformed with a Gal4-regulated b-galactosidase expression plasmid (pD16) and assessed for enzyme activity. (C) Immunoblot analysis of Ah receptor and Arnt proteins in yeast after 18 h induction in galactose without ( ) or with (+) 1000 nM b-napthoflavone. Asterisks indicate immunoreactivity at the expected molecular weights of 96 and 82 kDa for the Ah receptor and Arnt proteins, respectively. The lane labelled ‘‘NR’’ is the parent strain of yeast that has no Ah receptor or Arnt genes, the lanes labelled ‘‘CPR7’’ express Ah receptor and Arnt in a wild-type genetic background and lanes labelled ‘‘cpr7’’ indicate expression in the mutant cells.
of the cpr7 deletion effect on Ah receptor signalling was provided by assessing the activity of b-galactosidase from a plasmid-born lacZ gene expressed under the control of a Gal4-regulated promoter (Fig. 2B). The cpr6,7 strain had no impairment of signalling from the galactose-inducible b-galactosidase expression vector pD16. Thus, the effect of the cpr7 deletion on Ah receptor signalling was not due to a general depression of transcriptional or translational processes and had no apparent effect on b-galactosidase levels, stability or activity. This result also indicates that signalling through the Gal4 transcription factor is not affected by the absence of Cpr6 and Cpr7 proteins. Expression of extra copies of either Cpr6 or Cpr7 from centromeric plasmids in the wild-type yeast strain that expressed Ah receptor and Arnt had no effect on signalling (data not shown). Thus, elevated expression of the Cpr proteins did not perturb signal transduction in this system. The possibility of Ah receptor instab-
ility in a cpr7 background was investigated by using anti-Ah receptor and anti-Arnt sera to probe Western blots. There were no changes in the levels of Ah receptor and Arnt in the cpr7 strain as compared to those in wild-type yeast in the presence or absence of ligand (Fig. 2C). Thus, Cpr7 does not appear to enhance Ah receptor signalling by stabilizing Ah receptor levels. Whether Cpr7 interacted with Ah receptor and Arnt was examined using in vitro binding assays. Evidence for a physical interaction was supported by interactions observed between a glutathione-S-transferase – Cpr7 (Gst – Cpr7) fusion protein and 35S-labelled Ah receptor that was synthesized in vitro (Fig. 3). Additionally, Gst –Cpr6 – Ah receptor complexes were formed in vitro. Measurement of the Ah receptor recovered in the complexes (data not shown) indicated that Cpr6 – Gst and Cpr7 – Gst were approximately equivalent in their ability to associate with the Ah receptor
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Fig. 3. Ah receptor interacts with Cpr6 and Cpr7 in vitro. Ah receptor and Arnt genes were transcribed and translated in vitro to generate 35S-labelled proteins. The translated proteins were reacted with Cpr6 – and Cpr7 – GST fusion proteins immobilized on glutathione – sepharose beads. The translation reactions (TRANSLATED) were run to identify the mobility of the Ah receptor (AhR, lane 1) and Arnt (Arnt, lane 2) proteins (respective arrows) in the gel, which were visualized by autoradiography. The protein (Ah receptor) that remained tightly bound to the GST fusion proteins was dissociated by SDS-PAGE buffer and is seen in the lanes labelled Cpr6 BEADS and Cpr7 BEADS.
shown). Thus, Ah receptor interacted specifically with both Cpr6 and Cpr7 fusion proteins in this in vitro assay. This interaction may direct or may consist of a ternary complex that contains Hsp90 or other proteins that were present in the reticulocyte lysate. Which region of Cpr7 is involved in the interaction with Ah receptor? The two significant features of Cpr7 are the cyclophilin-like region in the amino-terminal half of the protein that strongly resembles a prolyl isomerase domain and the multiple TPR domains that reside in the carboxylterminal half of the protein. Expression of a cpr7 derivative that contained three point mutations in the putative prolylisomerase fully restored Ah receptor signalling in a cpr7 genetic background. Additionally, a deletion derivative that expressed only the TPR-containing half of Cpr7 from a plasmid sufficed to restore full Ah receptor signalling activity in the cpr7-deficient yeast strain (Fig. 4). Thus, the carboxylterminal half of Cpr7 with its three TPR motifs played an important role in Ah receptor signalling in the yeast model system. Conversely, the immunophilin-like region in the amino-terminal half of the Cpr7 protein was fully dispensable with respect to Ah receptor signalling in yeast. 3.3. Cns1 and Ah receptor signalling
in vitro. The Gst – Cpr6 interaction with Ah receptor in vitro was surprising, considering that cells lacking Cpr6 (Fig. 2A) were competent in the Ah receptor signalling in the reporter gene assay. Additional results that demonstrated the specificity of the Cpr6 – and Cpr7 – Ah receptor interactions included the observations that Arnt and luciferase did not interact with Gst –Cpr6 and Gst – Cpr7 and that Ah receptor did not interact with GST or glutathione beads (data not
Fig. 4. Restoration of Ah receptor signalling by the TPR domain of Cpr7. Yeast expressing Ah receptor and Arnt were tested for activation of the b-galactosidase reporter plasmid under different genetic conditions. The signal from the wild-type (wt) strain is compared to cells lacking Cpr7 without (cpr7) and with an empty expression vector (cpr7 + vec.). Yeast transformed with vectors that either expressed a Cpr7 derivative with three point mutations targeted to functionally critical region of the putative prolyl isomerase domain (cpr7 + mut) or expressed the carboxyl terminal half of Cpr7 with the three TPRs (cpr7 + tpr) were capable of restoring Ah receptor signalling when transformed into the cpr7-deficient strain. Solid bars indicate the mean response from cells treated with no ligand and light grey bars indicate the response to 1-mM b-napthoflavone. Results are reported as in Fig. 2.
The sequence similarity between XAP2 and another yeast protein, Cns1, was noted in Fig. 1. Cns1 is an essential gene in S. cerevisiae and has been shown to form complexes with Hsp90, Hsp70 and Cpr7 proteins in yeast [33,44]. Cns1 is not a member of the immunophilin family, having no prolyl isomerase domain. However, Cns1 does contain three TPR motifs that are positioned in a similar manner to those in XAP2 and Cpr7. Given this similarity, Cns1 was tested for role in Ah receptor signalling. A vector designed to overexpress Cns1, pBEVY-GA – CNS1, was transformed into wild-type and cpr7-deficient yeast expressing Ah receptor
Fig. 5. Restoration of Ah receptor signalling by Cns1. Yeast expressing Ah receptor and Arnt were tested for activation of the b-galactosidase reporter in wild-type (wt) strain and in a strain lacking Cpr7 without (cpr7) or with a high-copy vector expressing the CNS1 gene (cpr7 + Cns1). Note that the chromosomal copy of the CNS1 gene is expressed in all the conditions shown, as CNS1 is an essential yeast gene. The solid black bars indicate the mean response from untreated controls and light grey bars indicate the response from cells that were treated with 1-mM b-napthoflavone.
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and Arnt and ligand-induced signalling was assessed. The Cns1 protein fully restored Ah receptor signalling in the cpr7-deficient background (Fig. 5). This result was consistent with the role of the multiple TPR regions as the relevant component needed for full activity of the Ah receptor signalling pathway.
icity as the yeast expressing this protein grew at a normal rate and its actions may be restricted to a direct effect on the Ah receptor pathway.
3.4. XAP2 and Ah receptor signalling in yeast
4.1. The role of TPR proteins in Ah receptor signalling
The restoration of full Ah receptor signalling by the TPR domain of Cpr7 and the TPR-containing Cns1 in cpr7deficient cells prompted testing of whether the human XAP2 protein could complement this signalling defect. In addition to specific effects on transcription factor signalling, strains lacking Cpr7 show a mild reduction in growth rate that is genetically complemented by the overexpression of Cns1 protein [33,44]. Expression of human XAP2 from both low- (centromere-containing) and high-copy (2 mm) vectors was assessed in cpr7-deficient and wild-type yeast strains that expressed Ah receptor and Arnt as described above. Expression of XAP2 did not genetically complement the impaired Ah receptor signalling pathway in the cpr7deficient strain and was incapable of complementing the mild growth defect as well (data not shown). In wild-type cells the expression of low XAP2 levels from the centromeric vector produced ligand-independent Ah receptor signalling and enhanced the maximal response to ligand (Fig. 6). Cells that overexpressed XAP2 from the high-copy plasmid ( 25 copies/cell) displayed enhanced ligand-independent activity that was approximately 20% of that induced by an optimal concentration of ligand (Fig. 6). Interestingly, there was an impaired ligand-dependent response in wild-type yeast expressing high amounts of XAP2. Thus, high levels of XAP2 blocked ligand-mediated Ah receptor signalling in the yeast model system, indicating that an alteration of the Ah receptor pathway had occurred. Overexpression of XAP2 had no apparent tox-
Comparison of the human XAP2 sequence with the S. cerevisiae proteome showed the existence of structural similarities with the yeast proteins Cpr7 and Cns1. The TPRcontaining Cpr7 protein was needed for efficient signalling by the human Ah receptor– Arnt complex expressed in yeast. Furthermore, the TPR-containing domain of Cpr7 sufficed in restoring full Ah receptor signalling in yeast in a cpr7 background. Cns1, another TPR-containing protein that is not an immunophilin, also restored full Ah receptor function in a cpr7-deficient background. Cpr7, Cns1 and XAP2 share the common feature of three TPR motifs (Fig. 1) and this appears to be the significant feature that influenced signalling in this model system (Figs. 4 and 5). The TPR motif is a loosely conserved 34 amino acid sequence that is involved in homotypic and heterotypic interactions among proteins [15,16]. The TPR-containing region of Cpr7, Cns1 and XAP2 physically interacts with both the MEEVD peptide sequence in the carboxyl-terminal of Hsp90 and with the ligand binding domains of receptors [26,33,44]. Furthermore, it was shown that Ah receptor signalling is enhanced when the XAP2 levels are artificially increased in cells [22,23,25]. Because Hsp90 complexes play an important role in signal transduction through the steroid and Ah receptor pathways, the TPR proteins are thought to facilitate receptor function by stimulating chaperone interactions that enhance responses to ligands. One mechanism by which XAP2 may achieve this effect is increasing receptor stability [26,27], although this effect was not apparent in the Ah receptor and Arnt immunoblotting experiments described here. Another proposed role of the receptor – chaperone complexes is facilitation of transport into the nucleus [45]. However, the chaperone – receptor shuttling hypothesis needs to be reconciled with the classical nuclear import and export processes that are directed by the localization signals within the amino terminal region of Ah receptor [46]. The association of Cpr7 with Ah receptor in vitro (Fig. 3) and the in vivo genetic effects (Figs. 2, 4 and 5) suggest that a direct regulatory interaction between these proteins occurs in the cell. However, more studies are required to verify this idea. The reticulocyte lysate used in the transcription and translation reactions contained Hsp90 chaperone complexes that may form indirect associations between TPRs and Ah receptor. Meyer and Perdew [26] have provided evidence that XAP2 can associate independently with Hsp90 and Ah receptor through different TPR domains, thus supporting the possibility of a direct interaction between a specific TPR of Cpr7 and the Ah receptor. Other possibilities that may
Fig. 6. Low XAP2 expression augments Ah receptor signalling in yeast, but high XAP2 levels block signalling. Wild-type cells expressing Ah receptor and Arnt (wt) and the same cells transformed with either a low- (low XAP2) or a high-copy vector (high XAP2) expressing XAP2 were tested for b-galactosidase expression from a reporter plasmid as described above. Solid black bars indicate the mean response from untreated cells and bars and light grey bars indicate the response to treatment with 1000 nM b-napthoflavone.
4. Discussion
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explain the results presented here include an indirect genetic effect of a Cpr7 deletion on this synthetic signal transduction pathway. The control experiment (Fig. 2B) showing equivalent b-galactosidase expression in wild-type and cpr7 mutant cells reduces the possibilities for an indirect genetic effect on Ah receptor signalling. 4.2. The relationship between Cpr6 and Cpr7 with respect to Ah receptor signalling The genetic data above indicated that Cpr7 is specifically required for full Ah receptor signalling, whereas the in vitro binding studies indicated that both Cpr6 and Cpr7 interact with Ah receptor. Cpr6 and Cpr7 are 40% identical and 55% similar in their protein sequences, are structurally related to the CyP-40 subfamily and contain the multiple TPR motifs (Fig. 1). Thus, given the in vitro binding results in Fig. 3 and the structural similarities, it is surprising that Cpr7, but not Cpr6, plays a specific role in Ah receptor signalling in yeast. In contrast, given that a Cpr7 deletion specifically impairs glucocorticoid receptor function in yeast [17], it was predicted and demonstrated that Cpr7, but not Cpr6, is involved in Ah receptor signalling in yeast (Fig. 2A). The reason why Cpr6 does not function in the synthetic Ah receptor signalling pathway is not known and is not clear from comparisons of protein sequences. However, it is interesting to note that the computer alignments matched the Cpr7 and Cns1 TPR domains to XAP2, but did not identify the TPR domain of Cpr6 as a match. Thus, some subtle difference in the TPRs or other sequences may cause Cpr7 to be active and Cpr6 to be inactive in the functional assays. This possibility could be tested using a detailed mutational analysis of the multiple TPR regions of these proteins to reveal which subportion(s) is significant for Ah receptor function. 4.3. The relationship between Cpr7 and Cns1 in Ah receptor signalling It is important to note that there was still appreciable ligand-dependent Ah receptor signalling in strains lacking Cpr7 (Fig. 2). What factor(s) could be responsible for the residual activity of Ah receptor in a cpr7-deficient strain? Given the relationships between the Cpr7 and Cns1 proteins described above, it is plausible that the residual activity is due to the basal level of Cns1 normally expressed in the yeast. Expression of Cns1 from a high-copy plasmid was capable of restoring full signalling function in a cpr7deficient strain (Fig. 5), suggesting that a sufficient amount of Cns1 can substitute for Cpr7. Thus, it may be that a low basal level of Cns1 provides for the residual Ah receptor function in the absence of Cpr7. The corollary experiment of examining Ah receptor signalling in a cns1-deficient strain cannot be readily performed because Cns1 is essential for viability. However, in future studies the generation of conditional cns1 alleles may permit this assessment to be made.
4.4. XAP2 and Ah receptor signalling in yeast Whether human XAP2 had an effect on Ah receptor signalling was examined in the model system. XAP2 failed to complement both the mild growth defect and the impaired Ah receptor signalling observed in cpr7-deficient strains. Thus, the human XAP2 may not play the same role as Cpr7 and Cns1 in the synthetic Ah receptor signalling pathway, despite the conservation of its three TPRs. Numerous reasons could explain this negative result and this issue cannot be resolved without significant experimentation. One obvious possibility for XAP2’s inability to complement the cpr7 defect is that the phylogenetic gap between organisms is too great. In support of this idea, database searches show that yeast may not contain true counterparts to the FKBP52 family of immunophilins [29]. However, XAP2 does have an effect on Ah receptor signalling in yeast. In the wild-type strain expressing Ah receptor, a relatively low level of XAP2 expression enhanced both ligand-independent and -dependent activity. This effect has been reported previously in both mammalian cells and in yeast model systems [22,23,25]. Since expression of XAP2 in yeast does not complement the cpr7 deletion’s effect on Ah receptor signalling, XAP2’s mechanism for augmenting Ah receptor signalling in the wild-type strain is unclear. It was also found that overexpression of XAP2 from a high-copy plasmid produced some ligand-independent activity in the yeast reporter assay, but eliminated the ligand-activated portion of the Ah receptor response (Fig. 6). The inhibitory effects of overexpressed XAP2 differed from a previous study that was derived using a yeast similar model system [25]. In the previous study, only augmentation of Ah receptor-directed reporter gene activation was observed and the authors concluded that XAP2 –Ah receptor complexes might be responsible for increased transactivation in the nucleus. Other interpretations of this result are possible and one explanation could reside in the differences between the experimental systems. Importantly, the Ah receptor– LexA DNA binding domain fusion protein constitutes the ligand-dependent transcription factor in the previously described study [25], whereas the Ah receptor– Arnt heterodimer constitutes the functional transcription factor in the model system presented here. In light of the finding that the PAS2/ligand binding region of the Ah receptor is a site where both XAP2 and Arnt associate with Ah receptor [26], it is possible that high XAP2 levels prevent Arnt from associating with Ah receptor and preclude the formation of a productive transcription factor (Fig. 6). This result is similar to the observation that ectopic expression of the TPR region from protein phosphatase 5 had a dominant negative function and inhibited glucocorticoid receptor signalling in mammalian cells [47]. It is important to note that XAP2 is not found in the ligand-activated Ah receptor– Arnt transcription complexes, which suggests that XAP2 must dissociate from Ah receptor to mediate gene expression under normal circumstances. Thus, high XAP2 expression levels may block signalling by preventing the formation of
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the Ah receptor –Arnt transcription factor, whereas lower XAP2 levels enhance Ah receptor activity by an unknown mechanism(s) that may include the enhancement of receptor stability [27]. In summary, the role that endogenous and human TPRcontaining proteins play in the Ah receptor– Arnt signalling pathway constructed in yeast has been investigated. Using bioinformatic, yeast genetic and biochemical approaches, Cpr7 and Cns1 were identified and shown to play a role in the Ah receptor signalling process. The genetic studies further implicate the multiple TPR regions of these proteins in Ah receptor function and provide the first evidence of impaired signalling in the absence of a TPR protein. The most likely role for these two yeast proteins appears to be the recruitment of Ah receptor to the Hsp90 chaperone complexes via their multiple TPR motifs. The yeast model system described here is useful for studying the signalling pathway of the human Ah receptor.
Acknowledgments I thank William Toscano, Sam Landry, and Marc Cox for helpful comments on this research. This publication was made possible by Grant ES 09055 from the National Institute of Environmental Health Sciences branch of the United States National Institute of Health and by the support from the Tulane-Xavier Center for Bioenvironmental Research.
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Two tetratricopeptide repeat proteins facilitate human aryl hydrocarbon receptor signalling in yeast Charles A. Miller* Environmental Health Sciences Department, Center for Bioenvironmental Research, Tulane University School of Public Health and Tropical Medicine, 1430 Tulane Avenue New Orleans, LA 70112, USA Received 30 September 2001; accepted 4 December 2001
Abstract A human aryl hydrocarbon (Ah) receptor signalling pathway was constructed in yeast and used to identify regulatory proteins that may be related to those present in mammalian cells. The sequence similarity of human hepatitis B protein X-associated protein 2 (XAP2) protein to yeast Cpr7 and Cns1 proteins suggested that these proteins might be involved in Ah receptor signalling in this model system. Ah receptor signalling from a lacZ reporter gene was reduced by 60% in cells that lacked Cpr7. In vitro interaction experiments indicated that a Cpr7 – GST fusion protein and Ah receptor formed a complex. Expression of Cpr7, Cns1 and the isolated tetratricopeptide repeat (TPR) region of Cpr7 from plasmids restored Ah receptor signalling function in the Cpr7-deficient strain. Thus, Cpr7 and Cns1 proteins facilitate the signalling of human Ah receptor expressed in yeast, perhaps in the same manner as the TPR-containing XAP2 protein and related chaperone proteins in mammalian cells. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Aryl hydrocarbon receptor; Dioxin; Chaperone; Yeast; Tetratricopeptide repeat
1. Introduction Aryl hydrocarbon (Ah) receptor is a ligand-activated component of a heterodimeric transcription factor called the Ah receptor complex (AHRC) [1– 3]. Ah receptor nuclear translocator (Arnt) protein makes up the other component of the AHRC. The most notable and best-characterized Ah receptor ligand is the highly toxic environmental contaminant 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). Ah receptor and Arnt are structurally related members of a family of proteins that contain PAS motifs, a domain that is involved in both protein – protein interactions and regulatory functions [4]. The Ah receptor is activated upon binding of an aromatic ligand to a region that spans its second PAS domain [5]. Prior to activation by ligand, Ah receptor exists
Abbreviations: Ah receptor, aryl hydrocarbon receptor; Arnt, aryl hydrocarbon nuclear translocator; TCDD, 2,3,7,8 tetrachlorodibenzop-dioxin; AHRC, aryl hydrocarbon receptor complex; Hsp, heat shock protein; TPR, tetratricopeptide repeat; XAP2, hepatitis B protein X-associated protein 2 * Tel.: +1-504-585-6942; fax: +1-504-584-1726. E-mail address: [email protected] (C.A. Miller).
in a cytoplasmic complex with heat shock protein-90 (Hsp90) and other proteins that regulate its function. Ligand binding studies, receptor sedimentation rates in sucrose density gradients, coprecipitation studies, and genetic evidence from yeast expressing either Ah receptor– Arnt or Ah receptor chimeras have provided evidence that Hsp90 complexes play a key role in Ah receptor signalling [5 –10]. In addition to Hsp90, other proteins may participate in the initial steps that prepare the nuclear receptors for ligand interaction and signal transduction. Several proteins have been identified as components of the eukaryotic Hsp90 chaperone machinery. These factors either facilitate or are required for the function of specific steroid hormone receptors, protein kinases and other substrates [11,12]. Thus, the Hsp90 complexes participate in the regulation of diverse but specific signalling pathways and enzymes. Steroid hormone receptors are among the bestcharacterized substrates of the Hsp90 complexes. Early events in the Hsp90 chaperone pathway involve steroid receptor interaction with DnaJ-like proteins, Hsp70 and Hsp90 [13]. Sti1 (also called p60 and HOP), a protein that contains several tetratricopeptide repeat (TPR) motifs, is thought to function early in the receptor maturation process
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by mediating receptor interactions with Hsp70 and Hsp90 [14]. The TPR motifs of Sti1 (also called p60 or hop) are thought to provide a scaffold for the formation of multiprotein interactions [15,16]. A group of proteins, which includes the immunophilins and an acidic 23-kDa protein ( p23), participates in the latter steps of steroid receptor maturation [11,12,17 – 21]. Some immunophilin family members have prolyl isomerase activity and may direct protein structure. Cofactors of Hsp90 complexes are important for signal transduction through the Ah receptor, but the role(s) of these proteins is not well studied. A 40-kDa protein that tightly associated with Ah receptor– Hsp90 complexes was noted by Chen and Perdew [6]. Subsequently, the 40-kDa protein was identified using a reverse genetic approach [22] and was identified independently by two other groups using the yeast two-hybrid methods [23,24]. The 40-kDa factor associated with the Ah receptor– Hsp90 complex is hepatitis B protein X-associated protein 2 (XAP2) and is also known as ARA9 (Ah receptor-associated protein 9) and AIP (Ah receptor-interacting protein). XAP2 is a 37-kDa TPRcontaining protein that resembles the molecular chaperone FKBP52 in structure. Like FKBP52, the related XAP2 protein is associated with Hsp90 complexes but it preferentially associates with the Ah receptor and interacts poorly with steroid hormone receptors. Unlike FKBP52, XAP2 does not bind to the immunosuppressive drug FK506 [25]. XAP2 enhances Ah receptor stability and facilitates the association with Hsp90 [26,27]. Thus, the XAP2 and FKBP52 proteins are structurally similar but display unique regulatory functions and interaction specificities. Studies with XAP2 implicate its TPR region in Ah receptor interactions and signalling [25,26]. Receptors and enzymes of metazoans usually function and display appropriate regulation when expressed in Saccharomyces cerevisiae. Such yeast model systems provide a means to elucidate signal transduction pathways [28] and are especially applicable to studying receptors that function through Hsp90-regulated processes [29]. This is possible due to the strong structural and functional conservation of Hsp90s and cofactors that extend across the eukaryotic phyla [29,30]. A model system that expresses functional human Ah receptor and Arnt proteins has been constructed in yeast as a means to study ligand interactions and signal transduction [9,31,32]. This model Ah receptor system is ligand dependent and responds to known ligands such as TCDD, benzo[a]pyrene, b-napthoflavone and hexachlorobenzene at concentrations that have biological activity in mammalian cells. Ah receptor requires sufficient levels of intact Hsp90 for signal transduction when expressed in this model system. Thus, the Ah receptor pathway expressed in yeast faithfully duplicates the regulation observed in vertebrate cells. Since Ah receptor signalling functions in yeast, it was reasoned that yeast factors related to human XAP2 might regulate human Ah receptor function in yeast. This possibility seemed probable in light of the findings that such
Hsp90-associated factors were shown to govern steroid hormone receptor function in yeast [17,33]. Here it is reported that two TPR-containing yeast proteins, Cpr7 and Cns1, are important for human Ah receptor signalling in a model yeast system.
2. Experimental procedures 2.1. Reagents Reagent grade chemicals used in this study were purchased from Fisher Scientific (Fairlawn, NJ) and Sigma (St. Louis, MO). Enzymes used for subcloning and other genetic manipulations were from New England Biolabs (Beverley, MA). Taq DNA polymerase, transcription and translation reaction mixes were from Promega (Madison, WI) and radioisotopes were purchased from New England Nuclear (Boston, MA). 2.2. Bacterial and yeast strains The DH5a strain of Escherichia coli was used for DNA manipulations and plasmid DNA amplification via routine techniques that have been described elsewhere [34]. The BL21 strain of E. coli was used to express GST fusion proteins from plasmids. W303a (MATa, ade2-1, can1-100, his3-11,15, leu2-3,112, trp1-1, ura3-1) is the parental yeast strain for all the experiments reported here. The cpr6,7- and cpr7-deficient strains have been described previously, along with the Cpr6 and Cpr7 expression plasmids used in this study [17,35,36]. The pJM88 and pAADB97 centromeric vectors were used to express Cpr6 and Cpr7, respectively. The pAADB158 vector expressed the Cpr7 derivative with three point mutations in the immunophilin-like domain and pAADB163 expressed the TPR region of Cpr7. The pJM4 and pAADB66 were used to express the Cpr7 – Gst and Cpr6– Gst fusion proteins in E. coli. The pD16 vector (a gift from Linda Hyman, Tulane University) expresses the lacZ gene under the control of a galactose-inducible promoter. Yeast was transformed with these various plasmids using a lithium acetate protocol [37]. Transformants were plated on synthetic complete medium containing 2% glucose as a carbon source that lacked the essential amino acids or nucleosides [38]. Cells were propagated in synthetic complete liquid medium that contained glucose, and galactose-containing synthetic medium was used to induce expression of genes under control of the GAL1,10 (galactose inducible) promoter. 2.3. DNA manipulations The CNS1 gene was amplified by polymerase chain reaction (PCR) using genomic DNA derived from the W303a strain as the template and the primers 50-CCGTCGACAAATACTCTGGTATTCCCCCTG and 50-GGGGATCCGCAGCATTGAATTAAAAGTGAGGTC. After an initial
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denaturation at 95 C for 2 min, the reaction was cycled twice at an annealing temperature of 42 C for 45 s, followed by 2 min extension at 72 C and denaturation at 95 C for 30 s, then 28 more cycles were performed with a higher annealing temperature of 55 C. The CNS1 PCR product was digested with BamHI and SalI restriction enzymes and subcloned between the BamHI and SalI sites of the GAL1,10 promoter of the high-copy pBEVY-GA yeast expression vector [9], to create pBEVY-GA – CNS1. The human XAP2 (ARA9) sequence was a gift from Christopher Bradfield, University of Wisconsin, and was provided in the vector pI613. The XAP2 sequence was excised using BspE1 and XmaI and inserted into the pBEVY-GA plasmid at the XmaI site to make pBEVY-GA – ARA9. To make a low-copy expression vector, the XAP2 gene expression construct in pBEVY-GA was excised with SalI and XhoI and inserted into the SalI site of the low-copy centromeric vector YCplac33 [39]. The pIGAHRC plasmid [31] was used to introduce the bidirectional Ah receptor – GAL1,10 – Arnt expression construct into chromosome III of W303a, cpr6,7-deficient and cpr7deficient strains. A two-step recombination methodology using the URA3 vector followed by negative selection on 5-fluoroorotic acid-containing plates was used for this insertion [40]. This integrated genomic construct provided for the proper expression levels of human Ah receptor and Arnt proteins when cells were grown in medium that contained galactose as the inducing carbon source. 2.4. lacZ expression assays The functional lacZ (b-galactosidase) assay to detect ligand-activated gene expression by AHRC has been described previously. A modified lacZ assay was scaled to a 96-well plate format and used to assess AHRC activation in yeast [41]. Cells from saturated cultures grown overnight in synthetic minimal glucose medium were inoculated into tubes containing 1 ml of synthetic minimal galactose medium supplemented with essential amino acids and nucleosides as required by the genotype of the yeast strain. Polystyrene culture tubes were used for exposure of yeast to the aromatic hydrocarbon b-napthoflavone. It is important to note that for some aromatic hydrocarbons other than b-napthoflavone, the type of culture tube used can determine Ah receptor ligand efficacy in yeast [31]. Dimethyl sulfoxide (DMSO) with or without the ligand b-napthoflavone (Acros Organics) was added to a final concentration of 1% for all exposures. This concentration of solvent did not affect cell growth or signalling from the reporter plasmid. All dilutions of ligands were made in DMSO rather than by serial dilution in medium to avoid loss of aromatic hydrocarbons due to the adsorption on plastic surfaces. Triplicate cultures were exposed to ligand for 18 h with shaking at 30 C. Incubations for more than 1 h during lacZ assays were avoided due to attrition of enzyme activity over time. Readings of lacZ assays and cell density were taken in the linear ranges of spectrophotometric measurement. The data
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reported here are means and standard deviations of representative experiments that have given consistent results in at least three independent trials. 2.5. In vitro interaction experiments The cDNA sequences of Ah receptor, Arnt (gifts from Drs. Christopher Bradfield, University of Wisconsin and Oliver Hankinson, UCLA, respectively) and firefly luciferase (Promega) were transcribed and translated in vitro from plasmids that contained the SP6 promoter. The TNT protocol (Promega) was used to generate 35S-radiolabelled proteins with minor modifications. Purification of the Ah receptor and Arnt plasmids by CsCl gradient centrifugation and supplementation of the TNT reaction mix with an additional 10 mM KCl and 0.25 mM MgCl2 was necessary to make sufficient amounts of 35S-labelled Ah receptor and Arnt proteins. Following synthesis, phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 mM to prevent proteolysis. The Cpr6 – GST and Cpr7 – GST expression plasmids (pAADB66 and pJM4, respectively) were expressed in BL21 cells. Isopropylthio-b-D-galatopyranoside was added to a final concentration of 0.1 mM when cultures reached an optical density of 0.6 at a wavelength of 600 nm. The cells were then cultured for an additional 3 h and harvested. Cultures were washed once in water and once in interaction buffer (20 mM Tris HCl, 100 mM NaCl, 2 mM EDTA and 0.5% Nonidet P40, pH 7.8) supplemented with PMSF (1 mM) to reduce proteinase activity. The cells were resuspended in 1 ml ice-cold interaction buffer containing 1 mM dithiothreitol (DTT) and PMSF and lysed with three pulses of 30 s each from a Vibra Cell sonicator (Sonics and Materials) with a microtip at a power setting of 10%. The lysate was clarified by centrifugation at 14,000 g for 15 min at 4 C. The supernatant (500 ml) was reacted with glutathione – sepharose beads (Sigma) to immobilize the GST fusion proteins. The beads were washed five times with interaction buffer containing 1 mM DTT to liberate unassociated proteins. The beads were blocked by a wash in interaction buffer containing 1% nonfat dry milk followed by a wash in buffer with 5% bovine serum albumin and then washed three times in interaction buffer alone. Twenty microliters packed volume of the beads was reacted with 15 ml of the 35S-labelled translated protein reaction described above in a final volume of 100 ml of interaction buffer. Protein binding reactions were incubated at room temperature with gentle mixing for 30 min and then the beads were washed five times with interaction buffer to remove unassociated proteins. Supernatants were removed from the beads and 20 ml of 2 SDS polyacrylamide gel (SDS-PAGE) sample buffer was added. Samples were heated to 70 C for 5 min and then loaded onto a 6% SDS-PAGE and resolved. The resulting gels were dried and subjected to autoradiography. The images were obtained using a phosphorimager and were compared using densitometric software (MacBAS version 2.5, Fuji).
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2.6. Western blotting and immunodetection of Ah receptor and Arnt Yeast strains expressing Ah receptor and Arnt proteins were grown to late log phase in 50-ml cultures of synthetic medium containing galactose as the inducing carbon source. The cells were sedimented, washed once in water, resuspended in disruption buffer supplemented with proteinase inhibitors and broken with glass beads. The cell lysates were clarified by centrifugation at 14,000 g for 10 min. Supernatants were mixed with an equal volume of 2 concentration of SDS-PAGE sample buffer [42], heated to 70 C for 5 min and separated on 7.5% SDS-PAGE. Gels were either fixed and stained with Coomassie Blue dye to determine protein concentrations or blotted to nitrocellulose as described previously [43]. Blots were blocked with 5% nonfat dry milk in a solution of 0.1 M NaCl and 0.1% Tween 20 detergent buffered with 25 mM Tris HCl to pH 7.8 to prevent nonspecific interaction of antibodies. Rabbit polyclonal antisera to Ah receptor and Arnt (provided by Dr. Richard Pollenz, University of South Florida) were used as the primary antibodies at a dilution of 1:100. Following antibody binding, blots were washed with five changes of the buffer above lacking nonfat dry milk. Secondary goat antirabbit peroxidase conjugated antibodies were used with an ECL detection system (Amersham) according to the manufacturer’s instructions and the resulting blot was exposed to X-ray film to visualize the Ah receptor and Arnt proteins. It was necessary to absorb primary and secondary antisera with yeast lysates bound to nitrocellulose to remove high background reactivity before detection of Ah receptor and Arnt.
3. Results 3.1. Relationship between XAP2 and yeast proteins Three groups have independently identified XAP2 as a conserved protein that interacts with Ah receptor in murine, simian and human cells (see Introduction). The relatively ubiquitous expression of XAP2 and its physical interaction with Ah receptor and Hsp90 suggest that a regulatory relationship exists between these proteins. Since Ah receptor functions in yeast, it was hypothesized that related proteins with analogous function to XAP2 might exist. A search of the Saccharomyces Database with the Blast program hwww.ncbi.nlm.nih.govi indicated that human XAP2 contains a 150 amino acid region of similarity to the yeast Cpr7 (19% identity and 39% similarity) and Cns1 (25% identity and 41% similarity) proteins. The human XAP2 sequence matched most of the carboxyl terminal half of the Cpr7 protein and a central region of the Cns1 protein to a similar extent (Fig. 1). The common region shared by these proteins contains TPR motifs. The significance of the similarity between XAP2 and Cpr7 was intriguing since
Fig. 1. Structures of TPR-containing proteins. The human XAP2 protein is compared to the yeast Cpr6, Cpr7 and Cns1 protein structures. The dotted lines denote the common multiple TPR region that is shared by the proteins. The FKBP12- and CyP18-like domains indicate sequences that resemble prolyl isomerase domains of other chaperones. Note that Cns1 has no apparent FKBP12- or CyP18-like domains and is not related to the other proteins except for the sequence similarity in the TPR region.
both are immunophilins. Furthermore, Cpr7 regulates signalling of vertebrate steroid hormone receptors expressed in yeast [17]. Another surprising result was that Cpr6, a protein that shows considerable sequence conservation ( 40% identity and 55% similarity) with Cpr7, did not match the human XAP2 sequence. Thus, the relationship among XAP2, Cpr7 and Cns1 appeared specific and spanned the regions of the proteins that included TPRs. 3.2. A specific role for Cpr7 in Ah receptor signalling A strain that contained deletions in both the CPR6 and CPR7 genes and coexpressed the human Ah receptor and ARNT genes was constructed in order to test the role of these proteins in signal transduction. An expression construct for Ah receptor and Arnt was inserted into chromosome III and a lacZ reporter plasmid, pTXRE5-Z, was transformed into the cpr6,7-deficient strain. The response to the aromatic hydrocarbon ligand, b-napthoflavone, was determined in this strain and compared to that of an isogenic strain with intact CPR6 and CPR7 genes (Fig. 2A). Ligand dependent signalling through the Ah receptor pathway was reduced by 50– 70% in the cpr6,7 strain. Expression of Cpr7 protein from a plasmid introduced into the cpr6,7 strain completely restored Ah receptor signalling to levels that were equal to that of wild-type cells. Thus, Cpr7 expression is needed for efficient signalling in yeast expressing human Ah receptor and Arnt. In contrast, expression of Cpr6 had no effect on the impaired Ah receptor signalling in the cpr6,7 strain. Note that wild-type Cpr7 strains responded well at 10 nM b-napthoflavone, whereas a high ligand concentration (1 mM) produced only modest signalling in the cpr7 strains. Thus, the Cpr7 protein modulated the apparent potency and efficacy of the Ah receptor ligand. It was surprising that the Cpr6 protein, which showed considerable conservation of primary sequence structure with Cpr7, had no apparent role in Ah receptor signalling. The effect of the cpr7 deletion on Ah receptor signalling was not due to a general (nonspecific) reduction in transcriptional or translational processes. Evidence for the specificity
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Fig. 2. Elimination of Cpr7 specifically disrupts Ah receptor signalling. Yeast strains expressing human Ah receptor and Arnt with or without disrupted CPR6 and CPR7 genes were constructed. These strains also carried a b-galactosidase reporter plasmid directed by Ah receptor – Arnt responsive DNA elements, thus permitting assessment of Ah receptor signalling. (A) The strain with no mutations that coexpresses Ah receptor and Arnt proteins is indicated as the wild-type (wt). The strain lacking both proteins is indicated as cpr6,7. Strains lacking a single protein were generated by transforming the cpr6,7 strain with a plasmid that expressed either Cpr6 (labelled cpr7) or Cpr7 proteins (labelled cpr6). Cells were treated with 0 (black bars), 10 (darker grey bars) or 1000 nM (lighter grey bars) b-napthoflavone (Ah receptor ligand) for 18 h and assessed for reporter plasmid expression. Results are reported as the mean b-galactosidase activity in units along with the standard deviation (error bars). (B) The results from wild-type and a cpr6,7-deficient strain that were transformed with a Gal4-regulated b-galactosidase expression plasmid (pD16) and assessed for enzyme activity. (C) Immunoblot analysis of Ah receptor and Arnt proteins in yeast after 18 h induction in galactose without ( ) or with (+) 1000 nM b-napthoflavone. Asterisks indicate immunoreactivity at the expected molecular weights of 96 and 82 kDa for the Ah receptor and Arnt proteins, respectively. The lane labelled ‘‘NR’’ is the parent strain of yeast that has no Ah receptor or Arnt genes, the lanes labelled ‘‘CPR7’’ express Ah receptor and Arnt in a wild-type genetic background and lanes labelled ‘‘cpr7’’ indicate expression in the mutant cells.
of the cpr7 deletion effect on Ah receptor signalling was provided by assessing the activity of b-galactosidase from a plasmid-born lacZ gene expressed under the control of a Gal4-regulated promoter (Fig. 2B). The cpr6,7 strain had no impairment of signalling from the galactose-inducible b-galactosidase expression vector pD16. Thus, the effect of the cpr7 deletion on Ah receptor signalling was not due to a general depression of transcriptional or translational processes and had no apparent effect on b-galactosidase levels, stability or activity. This result also indicates that signalling through the Gal4 transcription factor is not affected by the absence of Cpr6 and Cpr7 proteins. Expression of extra copies of either Cpr6 or Cpr7 from centromeric plasmids in the wild-type yeast strain that expressed Ah receptor and Arnt had no effect on signalling (data not shown). Thus, elevated expression of the Cpr proteins did not perturb signal transduction in this system. The possibility of Ah receptor instab-
ility in a cpr7 background was investigated by using anti-Ah receptor and anti-Arnt sera to probe Western blots. There were no changes in the levels of Ah receptor and Arnt in the cpr7 strain as compared to those in wild-type yeast in the presence or absence of ligand (Fig. 2C). Thus, Cpr7 does not appear to enhance Ah receptor signalling by stabilizing Ah receptor levels. Whether Cpr7 interacted with Ah receptor and Arnt was examined using in vitro binding assays. Evidence for a physical interaction was supported by interactions observed between a glutathione-S-transferase – Cpr7 (Gst – Cpr7) fusion protein and 35S-labelled Ah receptor that was synthesized in vitro (Fig. 3). Additionally, Gst –Cpr6 – Ah receptor complexes were formed in vitro. Measurement of the Ah receptor recovered in the complexes (data not shown) indicated that Cpr6 – Gst and Cpr7 – Gst were approximately equivalent in their ability to associate with the Ah receptor
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Fig. 3. Ah receptor interacts with Cpr6 and Cpr7 in vitro. Ah receptor and Arnt genes were transcribed and translated in vitro to generate 35S-labelled proteins. The translated proteins were reacted with Cpr6 – and Cpr7 – GST fusion proteins immobilized on glutathione – sepharose beads. The translation reactions (TRANSLATED) were run to identify the mobility of the Ah receptor (AhR, lane 1) and Arnt (Arnt, lane 2) proteins (respective arrows) in the gel, which were visualized by autoradiography. The protein (Ah receptor) that remained tightly bound to the GST fusion proteins was dissociated by SDS-PAGE buffer and is seen in the lanes labelled Cpr6 BEADS and Cpr7 BEADS.
shown). Thus, Ah receptor interacted specifically with both Cpr6 and Cpr7 fusion proteins in this in vitro assay. This interaction may direct or may consist of a ternary complex that contains Hsp90 or other proteins that were present in the reticulocyte lysate. Which region of Cpr7 is involved in the interaction with Ah receptor? The two significant features of Cpr7 are the cyclophilin-like region in the amino-terminal half of the protein that strongly resembles a prolyl isomerase domain and the multiple TPR domains that reside in the carboxylterminal half of the protein. Expression of a cpr7 derivative that contained three point mutations in the putative prolylisomerase fully restored Ah receptor signalling in a cpr7 genetic background. Additionally, a deletion derivative that expressed only the TPR-containing half of Cpr7 from a plasmid sufficed to restore full Ah receptor signalling activity in the cpr7-deficient yeast strain (Fig. 4). Thus, the carboxylterminal half of Cpr7 with its three TPR motifs played an important role in Ah receptor signalling in the yeast model system. Conversely, the immunophilin-like region in the amino-terminal half of the Cpr7 protein was fully dispensable with respect to Ah receptor signalling in yeast. 3.3. Cns1 and Ah receptor signalling
in vitro. The Gst – Cpr6 interaction with Ah receptor in vitro was surprising, considering that cells lacking Cpr6 (Fig. 2A) were competent in the Ah receptor signalling in the reporter gene assay. Additional results that demonstrated the specificity of the Cpr6 – and Cpr7 – Ah receptor interactions included the observations that Arnt and luciferase did not interact with Gst –Cpr6 and Gst – Cpr7 and that Ah receptor did not interact with GST or glutathione beads (data not
Fig. 4. Restoration of Ah receptor signalling by the TPR domain of Cpr7. Yeast expressing Ah receptor and Arnt were tested for activation of the b-galactosidase reporter plasmid under different genetic conditions. The signal from the wild-type (wt) strain is compared to cells lacking Cpr7 without (cpr7) and with an empty expression vector (cpr7 + vec.). Yeast transformed with vectors that either expressed a Cpr7 derivative with three point mutations targeted to functionally critical region of the putative prolyl isomerase domain (cpr7 + mut) or expressed the carboxyl terminal half of Cpr7 with the three TPRs (cpr7 + tpr) were capable of restoring Ah receptor signalling when transformed into the cpr7-deficient strain. Solid bars indicate the mean response from cells treated with no ligand and light grey bars indicate the response to 1-mM b-napthoflavone. Results are reported as in Fig. 2.
The sequence similarity between XAP2 and another yeast protein, Cns1, was noted in Fig. 1. Cns1 is an essential gene in S. cerevisiae and has been shown to form complexes with Hsp90, Hsp70 and Cpr7 proteins in yeast [33,44]. Cns1 is not a member of the immunophilin family, having no prolyl isomerase domain. However, Cns1 does contain three TPR motifs that are positioned in a similar manner to those in XAP2 and Cpr7. Given this similarity, Cns1 was tested for role in Ah receptor signalling. A vector designed to overexpress Cns1, pBEVY-GA – CNS1, was transformed into wild-type and cpr7-deficient yeast expressing Ah receptor
Fig. 5. Restoration of Ah receptor signalling by Cns1. Yeast expressing Ah receptor and Arnt were tested for activation of the b-galactosidase reporter in wild-type (wt) strain and in a strain lacking Cpr7 without (cpr7) or with a high-copy vector expressing the CNS1 gene (cpr7 + Cns1). Note that the chromosomal copy of the CNS1 gene is expressed in all the conditions shown, as CNS1 is an essential yeast gene. The solid black bars indicate the mean response from untreated controls and light grey bars indicate the response from cells that were treated with 1-mM b-napthoflavone.
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and Arnt and ligand-induced signalling was assessed. The Cns1 protein fully restored Ah receptor signalling in the cpr7-deficient background (Fig. 5). This result was consistent with the role of the multiple TPR regions as the relevant component needed for full activity of the Ah receptor signalling pathway.
icity as the yeast expressing this protein grew at a normal rate and its actions may be restricted to a direct effect on the Ah receptor pathway.
3.4. XAP2 and Ah receptor signalling in yeast
4.1. The role of TPR proteins in Ah receptor signalling
The restoration of full Ah receptor signalling by the TPR domain of Cpr7 and the TPR-containing Cns1 in cpr7deficient cells prompted testing of whether the human XAP2 protein could complement this signalling defect. In addition to specific effects on transcription factor signalling, strains lacking Cpr7 show a mild reduction in growth rate that is genetically complemented by the overexpression of Cns1 protein [33,44]. Expression of human XAP2 from both low- (centromere-containing) and high-copy (2 mm) vectors was assessed in cpr7-deficient and wild-type yeast strains that expressed Ah receptor and Arnt as described above. Expression of XAP2 did not genetically complement the impaired Ah receptor signalling pathway in the cpr7deficient strain and was incapable of complementing the mild growth defect as well (data not shown). In wild-type cells the expression of low XAP2 levels from the centromeric vector produced ligand-independent Ah receptor signalling and enhanced the maximal response to ligand (Fig. 6). Cells that overexpressed XAP2 from the high-copy plasmid ( 25 copies/cell) displayed enhanced ligand-independent activity that was approximately 20% of that induced by an optimal concentration of ligand (Fig. 6). Interestingly, there was an impaired ligand-dependent response in wild-type yeast expressing high amounts of XAP2. Thus, high levels of XAP2 blocked ligand-mediated Ah receptor signalling in the yeast model system, indicating that an alteration of the Ah receptor pathway had occurred. Overexpression of XAP2 had no apparent tox-
Comparison of the human XAP2 sequence with the S. cerevisiae proteome showed the existence of structural similarities with the yeast proteins Cpr7 and Cns1. The TPRcontaining Cpr7 protein was needed for efficient signalling by the human Ah receptor– Arnt complex expressed in yeast. Furthermore, the TPR-containing domain of Cpr7 sufficed in restoring full Ah receptor signalling in yeast in a cpr7 background. Cns1, another TPR-containing protein that is not an immunophilin, also restored full Ah receptor function in a cpr7-deficient background. Cpr7, Cns1 and XAP2 share the common feature of three TPR motifs (Fig. 1) and this appears to be the significant feature that influenced signalling in this model system (Figs. 4 and 5). The TPR motif is a loosely conserved 34 amino acid sequence that is involved in homotypic and heterotypic interactions among proteins [15,16]. The TPR-containing region of Cpr7, Cns1 and XAP2 physically interacts with both the MEEVD peptide sequence in the carboxyl-terminal of Hsp90 and with the ligand binding domains of receptors [26,33,44]. Furthermore, it was shown that Ah receptor signalling is enhanced when the XAP2 levels are artificially increased in cells [22,23,25]. Because Hsp90 complexes play an important role in signal transduction through the steroid and Ah receptor pathways, the TPR proteins are thought to facilitate receptor function by stimulating chaperone interactions that enhance responses to ligands. One mechanism by which XAP2 may achieve this effect is increasing receptor stability [26,27], although this effect was not apparent in the Ah receptor and Arnt immunoblotting experiments described here. Another proposed role of the receptor – chaperone complexes is facilitation of transport into the nucleus [45]. However, the chaperone – receptor shuttling hypothesis needs to be reconciled with the classical nuclear import and export processes that are directed by the localization signals within the amino terminal region of Ah receptor [46]. The association of Cpr7 with Ah receptor in vitro (Fig. 3) and the in vivo genetic effects (Figs. 2, 4 and 5) suggest that a direct regulatory interaction between these proteins occurs in the cell. However, more studies are required to verify this idea. The reticulocyte lysate used in the transcription and translation reactions contained Hsp90 chaperone complexes that may form indirect associations between TPRs and Ah receptor. Meyer and Perdew [26] have provided evidence that XAP2 can associate independently with Hsp90 and Ah receptor through different TPR domains, thus supporting the possibility of a direct interaction between a specific TPR of Cpr7 and the Ah receptor. Other possibilities that may
Fig. 6. Low XAP2 expression augments Ah receptor signalling in yeast, but high XAP2 levels block signalling. Wild-type cells expressing Ah receptor and Arnt (wt) and the same cells transformed with either a low- (low XAP2) or a high-copy vector (high XAP2) expressing XAP2 were tested for b-galactosidase expression from a reporter plasmid as described above. Solid black bars indicate the mean response from untreated cells and bars and light grey bars indicate the response to treatment with 1000 nM b-napthoflavone.
4. Discussion
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explain the results presented here include an indirect genetic effect of a Cpr7 deletion on this synthetic signal transduction pathway. The control experiment (Fig. 2B) showing equivalent b-galactosidase expression in wild-type and cpr7 mutant cells reduces the possibilities for an indirect genetic effect on Ah receptor signalling. 4.2. The relationship between Cpr6 and Cpr7 with respect to Ah receptor signalling The genetic data above indicated that Cpr7 is specifically required for full Ah receptor signalling, whereas the in vitro binding studies indicated that both Cpr6 and Cpr7 interact with Ah receptor. Cpr6 and Cpr7 are 40% identical and 55% similar in their protein sequences, are structurally related to the CyP-40 subfamily and contain the multiple TPR motifs (Fig. 1). Thus, given the in vitro binding results in Fig. 3 and the structural similarities, it is surprising that Cpr7, but not Cpr6, plays a specific role in Ah receptor signalling in yeast. In contrast, given that a Cpr7 deletion specifically impairs glucocorticoid receptor function in yeast [17], it was predicted and demonstrated that Cpr7, but not Cpr6, is involved in Ah receptor signalling in yeast (Fig. 2A). The reason why Cpr6 does not function in the synthetic Ah receptor signalling pathway is not known and is not clear from comparisons of protein sequences. However, it is interesting to note that the computer alignments matched the Cpr7 and Cns1 TPR domains to XAP2, but did not identify the TPR domain of Cpr6 as a match. Thus, some subtle difference in the TPRs or other sequences may cause Cpr7 to be active and Cpr6 to be inactive in the functional assays. This possibility could be tested using a detailed mutational analysis of the multiple TPR regions of these proteins to reveal which subportion(s) is significant for Ah receptor function. 4.3. The relationship between Cpr7 and Cns1 in Ah receptor signalling It is important to note that there was still appreciable ligand-dependent Ah receptor signalling in strains lacking Cpr7 (Fig. 2). What factor(s) could be responsible for the residual activity of Ah receptor in a cpr7-deficient strain? Given the relationships between the Cpr7 and Cns1 proteins described above, it is plausible that the residual activity is due to the basal level of Cns1 normally expressed in the yeast. Expression of Cns1 from a high-copy plasmid was capable of restoring full signalling function in a cpr7deficient strain (Fig. 5), suggesting that a sufficient amount of Cns1 can substitute for Cpr7. Thus, it may be that a low basal level of Cns1 provides for the residual Ah receptor function in the absence of Cpr7. The corollary experiment of examining Ah receptor signalling in a cns1-deficient strain cannot be readily performed because Cns1 is essential for viability. However, in future studies the generation of conditional cns1 alleles may permit this assessment to be made.
4.4. XAP2 and Ah receptor signalling in yeast Whether human XAP2 had an effect on Ah receptor signalling was examined in the model system. XAP2 failed to complement both the mild growth defect and the impaired Ah receptor signalling observed in cpr7-deficient strains. Thus, the human XAP2 may not play the same role as Cpr7 and Cns1 in the synthetic Ah receptor signalling pathway, despite the conservation of its three TPRs. Numerous reasons could explain this negative result and this issue cannot be resolved without significant experimentation. One obvious possibility for XAP2’s inability to complement the cpr7 defect is that the phylogenetic gap between organisms is too great. In support of this idea, database searches show that yeast may not contain true counterparts to the FKBP52 family of immunophilins [29]. However, XAP2 does have an effect on Ah receptor signalling in yeast. In the wild-type strain expressing Ah receptor, a relatively low level of XAP2 expression enhanced both ligand-independent and -dependent activity. This effect has been reported previously in both mammalian cells and in yeast model systems [22,23,25]. Since expression of XAP2 in yeast does not complement the cpr7 deletion’s effect on Ah receptor signalling, XAP2’s mechanism for augmenting Ah receptor signalling in the wild-type strain is unclear. It was also found that overexpression of XAP2 from a high-copy plasmid produced some ligand-independent activity in the yeast reporter assay, but eliminated the ligand-activated portion of the Ah receptor response (Fig. 6). The inhibitory effects of overexpressed XAP2 differed from a previous study that was derived using a yeast similar model system [25]. In the previous study, only augmentation of Ah receptor-directed reporter gene activation was observed and the authors concluded that XAP2 –Ah receptor complexes might be responsible for increased transactivation in the nucleus. Other interpretations of this result are possible and one explanation could reside in the differences between the experimental systems. Importantly, the Ah receptor– LexA DNA binding domain fusion protein constitutes the ligand-dependent transcription factor in the previously described study [25], whereas the Ah receptor– Arnt heterodimer constitutes the functional transcription factor in the model system presented here. In light of the finding that the PAS2/ligand binding region of the Ah receptor is a site where both XAP2 and Arnt associate with Ah receptor [26], it is possible that high XAP2 levels prevent Arnt from associating with Ah receptor and preclude the formation of a productive transcription factor (Fig. 6). This result is similar to the observation that ectopic expression of the TPR region from protein phosphatase 5 had a dominant negative function and inhibited glucocorticoid receptor signalling in mammalian cells [47]. It is important to note that XAP2 is not found in the ligand-activated Ah receptor– Arnt transcription complexes, which suggests that XAP2 must dissociate from Ah receptor to mediate gene expression under normal circumstances. Thus, high XAP2 expression levels may block signalling by preventing the formation of
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the Ah receptor –Arnt transcription factor, whereas lower XAP2 levels enhance Ah receptor activity by an unknown mechanism(s) that may include the enhancement of receptor stability [27]. In summary, the role that endogenous and human TPRcontaining proteins play in the Ah receptor– Arnt signalling pathway constructed in yeast has been investigated. Using bioinformatic, yeast genetic and biochemical approaches, Cpr7 and Cns1 were identified and shown to play a role in the Ah receptor signalling process. The genetic studies further implicate the multiple TPR regions of these proteins in Ah receptor function and provide the first evidence of impaired signalling in the absence of a TPR protein. The most likely role for these two yeast proteins appears to be the recruitment of Ah receptor to the Hsp90 chaperone complexes via their multiple TPR motifs. The yeast model system described here is useful for studying the signalling pathway of the human Ah receptor.
Acknowledgments I thank William Toscano, Sam Landry, and Marc Cox for helpful comments on this research. This publication was made possible by Grant ES 09055 from the National Institute of Environmental Health Sciences branch of the United States National Institute of Health and by the support from the Tulane-Xavier Center for Bioenvironmental Research.
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