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J. Biol. Chem., Vol. 278, Issue 35, 32692-32701, August 29, 2003
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From the Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
Received for publication, April 24, 2003 , and in revised form, June 2, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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How Hsp90 regulates a vastly diverse group of client proteins remains an important question. In part, this versatility can be attributed to the assistance of co-chaperone proteins that modulate the role of Hsp90 in vivo. These co-chaperones impinge on several properties of Hsp90, including its ATPase activity, physical conformations, interaction with substrates, substrate specificity, and subcellular localization (5–11). Thus, a clear understanding of how Hsp90 function requires investigation of the roles played by its co-chaperones.
In Saccharomyces cerevisiae, Hsp90 is encoded by two genes, HSP82 and HSC82, either of which is sufficient for viability (12). The largest subset of Hsp90 co-chaperones is characterized by the presence of tetratricopeptide repeat domains, which mediate binding to Hsp90. TPR-containing co-chaperones were first identified in Hsp90-steroid receptor complexes (reviewed in Ref. 13). One of these, Hop (Sti1 in S. cerevisiae) can bridge Hsp90 and another essential chaperone, Hsp70, via two sets of TPR domains (14, 15). Two classes of large TPR-containing immunophilins have also been identified in Hsp90 complexes: FK506-binding proteins FKBP51 and FKBP52, and cyclophilin CyP40, which binds cyclosporin A (CsA) (16, 17). In addition to a carboxyl-terminal TPR domain, both immunophilin groups contain peptidyl prolyl isomerase (PPIase) domains in the amino termini. Although S. cerevisiae does not possess orthologs of the large FKBPs, it contains two CyP40 relatives: Cpr6 and Cpr7 (18, 19). Another protein found in Hsp90 complexes, Cns1, is essential for viability and in this regard unique among the TPR co-chaperone family (20, 21).
Several studies have addressed the roles played by TPR co-chaperones with regard to in vivo function of Hsp90. Assays that measure the activity of heterologous Hsp90 substrates such as maturation of the glucocorticoid receptor (GR) revealed the requirement for Cpr7 and Sti1 for full Hsp90 activity (22, 23). Sti1 and Cpr7 are also required for normal growth; the functional relationship between these co-chaperones is revealed by the severe growth defect of cells containing null alleles of both genes (22).
The tetratricopeptide repeat domains in these co-chaperones consist of a
tandem array of three repeat units, each of which forms a pair of
anti-parallel 
-helices
(24). Conserved amino acids at
specific positions within the TPR units define the TPR fold, whereas other
residues line an internal groove formed by the overall structure and make
direct contacts with the EEVD sequence found at the end of the Hsp90 carboxyl
terminus (15,
25). Surprisingly, although
EEVD is conserved in all Hsp90 homologs, the deletion of this sequence does
not affect growth of S. cerevisiae cells
(26). This finding brings into
question the importance of the Hsp90 carboxyl terminus in vivo and
suggests either that there is an alternative mode of interaction between Hsp90
and TPR co-chaperones or that their physical association is not required.
The role of immunophilin PPIase domains in Hsp90 function also remains unclear. It is possible that the catalytic activity these proteins show in vitro (27, 28) is involved in protein folding and may even be important for the mechanism of Hsp90 action on some substrates. However, a mutant form of the yeast cyclophilin Cpr7 in which the PPIase domain has been deleted supports full Hsp90 activity and normal growth (29).
A functional relationship between Cpr7 and Cns1 was first revealed by the discovery that overexpression of CNS1 can suppress both the slow growth phenotype and decreased Hsp90 activity that result from deletion of CPR7 (20, 21). Although Cns1 contains a three-unit TPR domain related to those found in Hsp90-associated immunophilins, a direct interaction between Cns1 and Hsp90 has not been demonstrated prior to this study. Furthermore, Cns1 can associate with Cpr7 (20, 21), although whether this interaction is direct or whether it requires Hsp90 is unclear. In addition, the full scope of Hsp90 functions in which these co-chaperones participate remains unknown.
In this study, we have taken different approaches to investigate the function of Cns1 in general and its role in Hsp90 complexes in particular. We isolated temperature-sensitive alleles of CNS1 and assessed their effects on Hsp90 activity and Hsp90-Cns1 physical interactions. We have also more fully explored the functional relationship between Cns1 and Cpr7 by examining genetic interactions between cpr7 and cns1 mutants. Our studies reveal that in cells with compromised Cns1 function the PPIase domain of Cpr7 and the EEVD Hsp90 carboxyl terminus are essential for viability.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Geldanamycin was the gift of the Drug Synthesis & Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, NCI (National Institutes of Health). Alpha factor was the gift of L. Hicke (Northwestern University, Evanston, IL). Cyclosporin A was purchased from Affinity Bioreagents.
Strains used in this study are shown in Table I. MTY189h was generated using the gamma deletion method. cns1-1, cns1-2, and cns1-3 are MTY189h-containing TRP1-marked CEN plasmids (described later). MTY818 was generated by the integration of cns1-1 allele into the CNS1 genomic locus.
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Plasmids—His6-tagged Hsp90 for expression in
Escherichia coli was the gift of L. Pearl (University College London,
London, UK) (5). The
HSE-LacZ reporter plasmid was a gift of D. Winge (University of Utah,
Salt Lake City, UT) (31).
Plasmids expressing reporter genes for assaying the pheromone response
(FUS1-lacZ) and GR activity (p2A/GRGZ) were the gifts of S. Lindquist
(Massachusetts Institute of Technology, Cambridge, MA)
(32). pKDE3 (pGAL-Cns1), which
expresses Cns1 HA-tagged at the amino terminus under control of the
galactose-driven promoter, was the gift of J. Heitman (Duke University Medical
Center, Durham, NC) (20).
pTCA/Hsp82, used to exchange wild-type Hsp82 for
Hsp82
EEVD and, for the experiment shown below in
the Fig. 8a, was the
gift of D. Picard (Université de Genève Sciences III,
Genève, Switzerland)
(33). pYeF1 was the gift of L.
Minvielle-Sebastia (Institute de Biochimie et Génétique
Cellulaires, Bordeaux 2 Université, Bordeaux, France)
(34).
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pGAL-Cns1C (pJM165) contains a segment of the CNS1 ORF from
bp 1 to 636 in pYeF1. pJM105 was described previously
(21). pMT100 is a
TRP1-marked CEN vector expressing wild-type Cns1 3HA-tagged
at the carboxyl terminus under control of the endogenous promoter. Plasmids
encoding cns1-1 (pMT207) and cns1-2 (pMT206) were generated
by site-directed mutagenesis (Stratagene) using pMT100. A plasmid expressing
Cns13C (pMT105) was made by ligating the fragment corresponding to
the CNS1 open reading frame from the original plasmid isolated in the
screen for temperature-sensitive alleles into pMT100.
pGST-Cpr7 (pJM5) has been described previously (22). pGST-Cns1-(1–212) (pJM158), pGST-Cns1-(81–212) (pJM157), and pGST-Cns1-(213–385) (pJM159) were made by PCR amplification of the corresponding regions of CNS1 ORF and ligating into pGEX-4T3 (Amersham Biosciences). pGST-Cns1 (pJM161) was made by PCR amplification of the region of CNS1 ORF between 639 and 1155 bp and ligating into pJM158. Plasmids expressing mutant forms of GST-Cns1, i.e. pGST-Cns1G90D (pMT142), pGST-Cns1C140R (pMT140), and pGST-Cns13C (pMT138), were made by ligation of mutant cns1 from appropriate vectors into pJM161.
pURA3-GPD-His6-Hsp82EEVD
(pMT179) was made by replacing codon 705 in
pURA3-GPD-His6-Hsp82 (isolated from GPD:HSP82FP cells)
with a stop codon using site-directed mutagenesis (Stratagene). A plasmid
encoding the wild-type was replaced by pMT179 in GPD: HSP82FP to generate AS5
strain.
pSC1 was used for overexpression of Cpr7 under the control of endogenous promoter in a multicopy URA3-marked plasmid. Plasmids expressing Cpr7 and Cpr7TPR under control of the endogenous promoter have essentially been described previously (29) except that the selectable marker was replaced with LEU2, resulting in plasmids pMT160 and pMT175, respectively. A plasmid expressing GAL-driven FLAG-tagged Cpr7 (pMT194) was made by PCR amplification of the CPR7 and ligation into the pESC-LEU vector (Stratagene). For the experiment shown below in Fig. 7a, Cpr7 was expressed from pMT184, which contains a TRP1 instead of a LEU2 selectable marker. Cpr6PPI-Cpr7TPR chimera (pMT204) is expressed from a pRS425-based vector, which contains bp 1–600 of the CPR6 ORF and bp 603–1191 of CPR7 ORF under control GAL promoter. pCpr7PPI-Cpr6TPR (pMT200) was made by PCR amplification of fragments of CPR7 ORF from start to 603 bp and CPR6 ORF from 538 bp to the end and ligation into pESC-LEU vector. pCpr7R64A (pMT203) and pCpr7K67P, N68G (pMT188) were made by site-directed mutagenesis of pMT160. Normal expression levels of the wild-type and mutant forms of Cpr7 were confirmed by Western blotting.
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Screen for Temperature-sensitive Alleles of CNS1—JMY84h
cells containing pJM105 were co-transformed with 50–100 ng of linearized
pRS314 and 100–200 ng of mutagenic PCR reaction products. Conditions
used for mutagenic PCR were the following: 10 ng of pJM105 template, 100 ng of
each primer, 0.4 mM MnCl2, 0.25 mM normal
dNTPs, 0.05 mM lower dNTP, and buffer (10 mM Tris-HCl,
pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001%).
Primers were designed to amplify the entire CNS1 ORF, 600 bp of the
promoter and 
200 bp of vector sequences flanking the gene. Transformants
were selected on the medium lacking uracil and tryptophan. Colonies were grown
for 3 days and replicaplated onto medium containing 5-FOA at 23 and 37 °C.
DNA was isolated from the colonies that grew at 23 °C but not at 37
°C, transformed into Escherichia coli DH5, and
re-introduced into the original yeast strain. These plasmids were sequenced to
determine mutations responsible for the phenotype.
Screen for Multicopy Suppressors of cns1ts
Alleles—cns1-1 cells were transformed with a S. cerevisiae
genomic library prepared using multicopy URA3-marked plasmids (gift
of P. Hieter, University of British Columbia, Vancouver, Canada) and grown on
plasmid-selective medium at permissive temperature (23 °C). After 3 days,
the cells were replica-plated onto plasmid-selective medium at 23 and 37
°C and incubated for 3–5 days. Plasmid dependence of the ability to
grow at the non-permissive temperature was confirmed by streaking these cells
on 5-FOA. Plasmid DNA was isolated from cells exhibiting suppressed phenotype,
transformed into E. coli DH5, and re-introduced into
cns1-1 recipients. Genomic inserts present were identified by
sequencing.
Assays of 
-Galactosidase Activity (GRE, HSE, and
FUS1)— MTY189h or JMY84h cells containing wild-type and mutant
CNS1 on TRP1-marked plasmids were transformed with the
appropriate reporter plasmids. -Galactosidase activity was measured as
described previously (22,
35) at 23 and 37 °C.
Protein Interaction Assays—Lysis of yeast cells was performed essentially as described previously (21), except that 1 mM dithiothreitol was omitted in preparing lysates that were used for purification on Ni-NTA beads (Qiagen).
Protein Concentrations Were Determined using the Bio-Rad Protein Assay—Precipitations using GST fusion proteins were performed as described previously (21, 22). BL21-CodonPlus®(DE3)-RIL pLysS E. coli cells (Stratagene) were used in all experiments except for the one shown in Fig. 1a.
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GPD:HSP82FP or AS5 cells were used to analyze endogenous yeast Hsp90 complexes. These experiments were performed as described previously (21), except that additional washes were performed with a buffer containing 20 mM imidazole and that 200 mM imidazole was used for elution of Hsp90 complexes. Laemmli sample buffer (36) was added to the eluted fractions without precipitation with trichloroacetic acid, and samples were frozen prior to analysis by SDS-PAGE.
Recombinant His6-Hsp90 was used to test the interactions between Hsp90 and truncated forms of Cns1. In this experiment, E. coli lysate prepared as described above was bound to Ni-NTA-agarose (Qiagen) for 30 min, washed three times with phosphate-buffered saline, and subsequently bound to E. coli lysates expressing GST fusions of truncated and full-length Cns1 for 1 h at 4 °C. The agarose was washed with phosphate-buffered saline five times, and bound proteins were eluted from Ni-NTA using 150 mM imidazole as described above.
Western Blot Analysis—Protein samples were separated on 10% SDS-PAGE gels, which were either stained with Coomassie Blue or transferred onto Protran® nitrocellulose membranes (Schleicher & Schuell). Equal amounts of loaded sample and efficient transfer were confirmed by staining with Ponceau S (Sigma). ECL (Amersham Biosciences) was used according to the manufacturer's protocol. The following antisera were used for immunoblotting: GST (Amersham Biosciences), HA (12CA5, BAbCO, Inc.), FLAG (M2, Kodak), Hsp90 (gift of A. Caplan, Mount Sinai School of Medicine, New York, NY), Cpr7 (29), Cpr6 (gift of S. Lindquist, Massachusetts Institute of Technology, Cambridge, MA), Sti1 (gift of D. Toft, Mayo Graduate School, Rochester, MN), and Cns1 (this study). Cns1 antibody was raised in rabbits against the full-length recombinant protein. It was purified by passing over CNBr-activated Sepharose (Amersham Biosciences), which was bound to yeast lysates expressing the amino terminus of Cns1 and, consequently, recognizes the carboxyl terminus.
Analytical Centrifugation of Purified Cns1—Cns1 was purified
according to the following procedure. 3 liters of logarithmically growing
E. coli BL21 cells harboring pJM161 were induced to express the
fusion protein with 0.1 mM
isopropyl-1-thio--D-galactopyranoside and grown for 4 h at 30
°C. Cells were harvested and lysed as described. Lysates were cleared by
filtration on 0.2 µM membrane (Corning). Lysate was bound to the
column twice by gravity flow. Products from the overnight cleavage with
thrombin (Sigma) were eluted and concentrated using an Ultra-free filter
device, Mr cutoff limit 30,000 (Millipore). The sample was
subjected to gel filtration on a 100-ml Superdex-200 column (Amersham
Biosciences). Fractions containing Cns1 were collected and concentrated in the
same manner as described above. This sample was dialyzed with two 1.5-liter
volumes of buffer (50 mM sodium phosphate, 150 mM NaCl,
pH 7.0).
Sedimentation equilibrium experiments were conducted at 16 °Cina Beckman Optima XL-1 analytical ultracentrifuge. Six samples of purified Cns1 were prepared, resulting in A230 values of 0.15, 0.2, 0.3, 0.5, 0.8, and 1.0. Approximately 100 µl of each sample was loaded into 1.2-cm path length cells, centrifuged at 14,000 and 20,000 rpm in an An-60 Ti rotor, and allowed to reach equilibrium. Data sets were fitted individually and simultaneously using software packages that employ a non-linear least squares method: WinNonLin 1.050, Sednterp 1.01, and Beckman Optima Xl-A/XL-1 4.2. The partial specific volume was 0.7362 ml/mg, and the viscosity of the buffer (50 mM sodium phosphate, 150 mM NaCl, pH 7.0) was 1.1090 mg/ml. Two models for fitting were used: the first one assumed an ideal solution of a single species, and the second one used monomer-dimer equilibria with a fixed monomer molecular weight of Cns1 of 44,107 (determined from the amino acid sequence).
Gel Filtration—200 ml of logarithmically growing cells
expressing His6-Hsp82 or
His6-Hsp82EEVD were harvested and
lysed in the equilibration buffer (50 mM HEPES, pH 7.5, 150
mM NaCl, 1 mM EDTA, 10% glycerol) as described above.
Lysates were centrifuged twice in the TL-100 Ultracentrifuge (Beckman) at
100,000 x g, 4 °C for 30 min. A Superdex-200 column
(Amersham Biosciences) was washed in equilibration buffer. 1.5 mg of total
protein was resolved on the column, and 0.5-ml fractions were collected using
Biotech Liquid Chromatography Collector LCC-500 (Amersham Biosciences).
Collected fractions were analyzed by SDS-PAGE and Western blotting as
described above. The following molecular mass standards were used (Sigma):
thyroglobulin (669 kDa), apoferritin (443 kDa), amylase (200 kDa),
albumin (66 kDa), and carbonic anhydrase (29 kDa).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Overexpression of the Cns1 amino terminus (amino acids 1–212) was
sufficient to suppress cns1 lethality
(Fig. 1b). Although
suppression at elevated temperature (37 °C) was weaker than at lower
temperatures, the portion of Cns1 that interacts with Hsp90 is sufficient for
viability under normal growth conditions.
Generation of Temperature-sensitive Alleles of CNS1—To
investigate its in vivo functions, we performed a screen for
conditional alleles of CNS1. DNA encompassing the CNS1 open
reading frame was randomly mutagenized by PCR and cotransformed with a
linearized vector into a cns1 recipient (see
"Experimental Procedures" for details). Several
temperature-sensitive alleles of CNS1 were obtained; this study
focuses on those presented in Fig.
2a. Although growth of the three mutants is severely
impaired at 37 °C, each remains capable of very slow growth
(Fig. 2b). Even at
permissive temperatures (23–30 °C), mutants exhibit a minor slow
growth phenotype. Cns13C contains three substitutions in the
carboxyl terminus of the protein, all of which are needed for the full mutant
phenotype (data not shown). Two of the mutants contain single amino acid
substitutions in the TPR domain. Cns1G90D harbors a substitution at
a highly conserved site, normally occupied by a small neutral amino acid at
position 8 of the first TPR. In Cns1C140R, an amino acid at
position 20 of the second TPR, which typically contains a conserved small
neutral amino acid, is substituted. Interestingly, mutations at both of these
positions in tetratricopeptide repeats have been reported to impact functions
of other TPR proteins
(39–41).
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Effect of CNS1 Mutations on Hsp90 Function—Cells in which CPR7 is deleted exhibit decreased Hsp90 function (22, 35), and overexpression of Cns1 in these cells largely restores wild-type Hsp90 activity (21). To determine if Cns1 plays a basic role in Hsp90 function, the effect of geldanamycin on the growth of cns1 cells was tested. GA is a specific inhibitor of Hsp90 (42); cells lacking certain Hsp90 co-chaperones exhibit hypersensitivity to GA (20, 43, 44). In the presence of 35 µM GA, growth of cns1ts cells was severely impaired, whereas wild-type cells were unaffected (Fig. 3a).
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Maturation of glucocorticoid receptor is a commonly used assay for the ability of Hsp90 complexes to function properly in yeast cells (45). To examine the role of Cns1 in the maturation of GR, a reporter plasmid was used that expresses mammalian GR under the control of a constitutive promoter and in which glucocorticoid response elements were fused to lacZ. The activity of the reporter was measured after treatment with steroid ligand. In contrast to wild-type, the ability to support full maturation of the steroid receptor was significantly decreased in cns1ts cells (Fig. 3b). Combined with our finding that Cns1 interacts directly with Hsp90, these results reveal that Cns1 is a bona fide co-chaperone of the Hsp90 machinery.
Hsp90 plays an important role in several signal transduction pathways in
S. cerevisiae, including the pheromone response
(33) and negative regulation
of the heat shock factor (35).
The potential involvement of Cns1 in these two processes was investigated by
measuring the activity of -galactosidase expressed from reporter
plasmids containing heat shock response elements or a
FUS1-pheromone-responsive promoter. cns1ts
mutants exhibited normal regulation of both pathways (data not shown),
suggesting that Cns1 plays a significant role in only a subset of Hsp90
functions.
Physical Interactions between Hsp90 and Cns1—We first determined whether cns1ts mutations have an effect on the direct interaction between Hsp90 and Cns1. GST fusions with wild-type or mutant Cns1 proteins were expressed in E. coli, purified on glutathione-Sepharose, and incubated with recombinant yeast Hsp82. Whereas wild-type Cns1 exhibited a robust interaction with Hsp90, the interactions of Cns1G90D and Cns1C140R with Hsp90 were undetectable (Fig. 4a). Cns13C retained the ability to interact with the chaperone. Thus, mutations in the TPR domain disrupt the interaction with Hsp90, in contrast with the mutations in the carboxyl terminus of Cns1. Based on the model of TPR-Hsp90 interactions (15), the amino acids substituted in Cns1G90D and Cns1C140R are unlikely to make direct contacts with the EEVD carboxyl terminus of Hsp90. Therefore, residues previously not implicated in the binding to EEVD may also be important for the TPR-Hsp90 interaction.
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The effect of cns1ts mutations on the interaction with Hsp90 in vivo was also investigated. The presence of Cns1–3HA in Hsp90 complexes was tested using cells in which His6-Hsp82 was the sole source of Hsp90. Cells were grown at permissive temperature and either maintained at 30 °C or shifted to 37 °C and lysed, and the lysates were bound to Ni-NTA resin. Lysates prior to and after purification of Hsp90 complexes are shown in Fig. 4b. Steady-state levels of Cns13C were lower than wild-type levels at both temperatures, but the interaction of this mutant with Hsp90 was maintained to the extent proportional to the interaction of the wild-type Cns1 with Hsp90. This is consistent with our previous finding (Fig. 4a) that Cns13C interacts well with Hsp90 in vitro.
Steady-state protein levels of Cns1G90D and Cns1C140R are indistinguishable from wild-type Cns1 at both permissive and non-permissive temperatures (Fig. 4b, top two panels). In contrast to the in vitro experiments using recombinant proteins, Cns1G90D and Cns1C140R exhibited a decreased, but detectable interaction with Hsp90 at the permissive temperature. At the non-permissive temperature, the Cns1-Hsp90 association was further impaired (Fig. 4b, bottom panel). In these cells, a genomic copy of CNS1 was present, in addition to the plasmid-expressed 3HA-tagged version. To address possible competition or cooperation between epitope-tagged and endogenous Cns1, the experiment was repeated using an integrated cns1-1 allele as the sole source of Cns1 protein. The strength of interaction between Cns1G90D with Hsp90 was not affected by the presence or absence of the wild-type CNS1 allele (not shown).
Genetic Interactions between CNS1 and CPR7—Two genetic
approaches were taken to further explore the role of Cns1 in vivo. In
the first, a screen for multicopy suppressors of the temperature-sensitive
phenotype of cns1-1 was performed. Two genes were identified in the
screen: CNS1 and CPR7. Overexpression of the cyclophilin
Cpr7 from a multicopy vector strongly suppressed the temperature sensitivity
of Cns1G90D (Fig.
5a). Overexpression of Cpr7 also suppressed the
temperature sensitivity of Cns1C140R and Cns13C, albeit
to a lesser extent (Fig.
5a). Because Cns1 was originally identified by its
ability to suppress the slow growth phenotype of cpr7 cells
(20,
21), the discovery that
overexpression of Cpr7 can suppress temperature sensitivity of Cns1 mutants
provides complementary evidence for the functional relationship between these
two Hsp90 co-chaperones.
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In the second approach, genetic interaction between CNS1 and
CPR7 was explored by introducing cns1 mutants into
cpr7 cns1 cells. This experiment was performed
using the plasmid shuffle method: a wild-type copy of CNS1 was
maintained on a URA3-marked plasmid in cns1ts
cpr7 cells. The inability of these cells to grow on medium
containing 5-FOA revealed a synthetic lethal relationship between the
cns1-1 and cpr7
(Fig. 5b, top
row). A plasmid expressing Cpr7, but not vector alone, was able to
suppress this synthetic lethality (Fig.
5b, second row from top). cns1-2 and
cns1-3 also exhibited synthetic interactions with
cpr7 (data not shown). The synthetic lethality between
cns1 and cpr7 mutants, along with the reciprocal
cross-suppression of the two genes, suggests that these two Hsp90
co-chaperones share an essential function in vivo.
To identify regions of Cpr7 that are necessary for viability in a
background compromised for Cns1 function, several mutant alleles of the
cyclophilin were introduced into cpr7 cns1-1 cells.
Expression of the TPR-containing carboxyl terminus of Cpr7 was insufficient to
confer viability in these cells (Fig.
5b). This was unexpected because of our earlier finding
that the isomerase domain of Cpr7 is not needed for Hsp90-related functions
(29). Expression of the Cpr7
amino terminus, which harbors the PPIase domain, also proved insufficient for
growth of cpr7 cns1-1 cells
(Fig. 5b). To assess
specific requirements regarding the two Cpr7 domains, we took advantage of the
presence of another CyP40 homolog in yeast, Cpr6
(22). Reciprocal chimeras of
the PPIase and TPR-containing regions of Cpr6 and Cpr7 were tested for the
ability to confer viability to cpr7 cns1-1 cells.
Neither the Cpr6PPI-Cpr7TPR chimera nor the
Cpr7PPI-Cpr6TPR chimera was able to provide viability in
these cells (data not shown), suggesting a specific requirement for the TPR
and isomerase domains of Cpr7 that cannot be fulfilled by analogous domains of
a closely related cyclophilin. Overall, our data indicate that both the
isomerase and TPR domains of Cpr7 are required for viability when Cns1
function is impaired.
Role of the Isomerase Domain of Cpr7 in the CNS1-CPR7 Genetic Interaction—We used two approaches to test whether the catalytic activity of the PPIase domain of Cpr7 is required for the essential function it shares with Cns1. In the first, cells were grown on medium containing cyclosporin A, an immunosuppressant and a potent inhibitor of the isomerase activity of cyclophilins (reviewed in Ref. 46). We reasoned that, if the isomerase activity of Cpr7 were required for viability in cns1-1 cells, then these cells should be hypersensitive to CsA. Additionally, if suppression of the temperature sensitivity of cns1-1 cells depended on the catalytic activity of Cpr7, the suppression would be sensitive to CsA. However, in the presence of CsA both strains grew normally (data not shown), suggesting that the isomerase activity of Cpr7 does not play a role in the essential function that this protein shares with Cns1.
In a complementary approach, a cpr7 allele in which a conserved
amino acid within the catalytic site is altered (R64A) was tested for the
ability to confer viability to cpr7 cns1-1 cells. The
corresponding substitution in human CyP18 decreased catalytic activity to 0.1%
of wild-type (47), and an
equivalent mutation in the mammalian cyclophilin RanBP2 led to loss of
function (48). cns1-1
cells expressing Cpr7R64A grow as well as those containing
wild-type Cpr7 (Fig.
6a), providing more evidence that the Cpr7 isomerase
domain plays a role independent of its catalytic activity.
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Among the Cpr7 mutants that were tested for the ability to confer viability
in cpr7 cns1-1 cells, a Cpr7K67P,N68G
double mutant was unable to suppress lethality of these cells
(Fig. 6b).
Cpr7K67P,N68G contains amino acid substitutions at sites predicted
by the crystal structure of bovine CyP40 to reside on the surface of the
isomerase domain, removed from the PPIase catalytic site
(49). As such, these residues
may play a role in protein-protein interactions, although they do not affect
the interaction between Cpr7 and Cns1 (data not shown). Consistent with our
results shown above, these experiments reveal that the PPIase domain is
required for viability in cells with compromised Cns1 function, but in a
manner independent of the catalytic activity of the domain.
Cns1·Cpr7 Complexes—Cns1 and Cpr7 can be
found in the same complexes
(20,
21), although it is not known
whether the interaction between them is direct or indirect. Using recombinant
GST-Cpr7 and His6-Cns1 fusion proteins expressed in E.
coli, we performed GST pull-down experiments but were unable to detect
direct binding between the two co-chaperones (data not shown). Because Cns1
and Cpr7 bind directly to Hsp90, we tested the possibility that Hsp90 mediates
the interaction between the two co-chaperones.
Hsp82EEVD was used in this approach, because the
interaction between Cns1 or Cpr7 and Hsp90 is primarily mediated by the
carboxyl-terminal EEVD of Hsp90
(50). In this experiment, we
examined the ability of FLAG-Cpr7 from wild-type or
hsp82EEVD lysates to bind immobilized GST-Cns1. As
shown in Fig. 7a, the
interaction between Hsp90 and GST-Cns1 was significantly reduced in the
absence of the EEVD terminus. However, the ability of GST-Cns1 to precipitate
Cpr7 from these cells was unaffected. Therefore, the formation of
Cns1·Cpr7 complexes is independent of the ability of either
co-chaperone to interact with the EEVD region of Hsp90.
As an alternative way to examine the effect of deleting the Hsp90 EEVD
terminus on the ability of Cns1 and Cpr7 to associate, size exclusion
chromatography was performed. Extracts from wild-type or
hsp82EEVD cells were prepared and resolved on a gel
filtration column. Collected fractions were analyzed by SDS-PAGE, and Western
blotting was performed to detect the presence of the Hsp90 complex components,
including Hsp90, Cns1, Cpr7, Cpr6, and Sti1
(Fig. 7b). The elution
profile of Hsp90 did not differ between extracts from wild-type and
hsp82EEVD cells, with peaks between fractions 22 and
24 in both cases. In wild-type cells, the peaks of Cpr6 and Sti1 elution
profiles coincided with the peak of Hsp90. However, in extracts from
hsp82EEVD cells, Cpr6 eluted primarily in the
fraction 31, which corresponds to the molecular mass of 42 kDa, the
predicted size of the Cpr6 monomer. The peak of the Sti1 elution was shifted
down to fraction 27, which corresponds to about 160 kDa.
In contrast to Sti1 and Cpr6, Cns1 and Cpr7 did not co-elute with the major
peak of Hsp90 in extracts from either wild-type or
hsp82EEVD cells. Rather, the peak of Cns1 elution was
between fractions 28 and 30, and Cpr7 eluted between fractions 29 and 31.
These results indicate that, unlike Sti1 and Cpr6, Cns1 and Cpr7 are not
primarily associated with Hsp90. However, we cannot rule out the possibility
that their interaction with Hsp90 fails to withstand these particular lysis
and dilution conditions. The migration of Cpr7 is consistent with a monomeric
protein. Although the molecular masses of fractions in which Cns1 migrates
(85–110 kDa) could suggest that Cns1 homodimerizes, we believe that
this is unlikely. Using sedimentation equilibrium analysis of purified Cns1,
we determined a molecular mass of 42.500 ± 1.300 kDa, corresponding to
the monomer. Whether the interaction between Cns1 and Cpr7 is direct or
mediated by an unknown partner, it is clearly not affected by the deletion of
EEVD.
Genetic Interaction between cns1-1 and
hsp82EEVD—Since Hsp82 EEVD is apparently not needed
for the formation of Cns1·Cpr7 complexes, we investigated the in
vivo significance of this region in cells with compromised Cns1 function.
cns1-1 cells were crossed with hsp82EEVD
cells to generate double mutant recombinants. In contrast with either single
mutant, cns1-1 hsp82EEVD progeny exhibited extreme
slow growth at 23 °C and lethality at or above 30 °C
(Fig. 8a). This
phenotype was suppressed by the introduction of plasmids carrying wild-type
alleles of CNS1 or HSP82. The genetic interaction between
CNS1 and HSP82 further supports the notion that Cns1 plays a
vital role in the proper functioning of Hsp90. It demonstrates for the first
time the in vivo requirement for the EEVD region, revealing that EEVD
is critical when the function of Cns1 is compromised.
It remains unclear why there is a requirement for EEVD in cns1
mutant cells. If the interaction between TPR proteins and Hsp90 can be fully
explained by a model describing contacts between amino acids in the groove of
the TPR domain and EEVD, an Hsp90 mutant lacking the EEVD motif and a mutation
in the Cns1 TPR domain should not exhibit a synthetic interaction. To explore
the possibility that Cns1 might interact with Hsp90 even in the absence of
EEVD, Hsp90 complexes from cells expressing
His6-Hsp82EEVD as the sole source of
this protein were analyzed. As shown in
Fig. 8b, Cns1 was
capable of interacting with Hsp90 when the EEVD terminus was deleted, although
the interaction was significantly weakened. Although additional factors may
play a role in the cns1-1 hsp82EEVD synthetic
phenotype, we believe that the weak association of the two proteins in the
absence of EEVD region points to the ability of TPR co-chaperones to interact
with Hsp90 in multiple ways.
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DISCUSSION |
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We demonstrated that the TPR domain of Cns1 binds Hsp90 directly and that some mutations in CNS1 that confer temperature sensitivity alter sites within the TPR domain and disrupt this interaction. When the TPR-containing amino terminus of Cns1 is overexpressed, it is sufficient for cell viability at normal temperatures. Combined, these findings support the notion that the interaction between these two proteins is essential. Interestingly, the extent of physical interaction between Cns1 and Hsp90 corresponds with the viability of cells containing temperature-sensitive alleles of CNS1, suggesting that the inviability of cells at elevated temperatures can be attributed to the loss of this interaction. A genetic interaction between Cns1G90D and the form of Hsp90 lacking the EEVD terminus further supports the idea that a Cns1-Hsp90 interaction is essential.
The temperature-sensitive phenotype was also conferred by a mutant
harboring substitutions in the carboxyl terminus of Cns1 (cns1-3
allele). The steady-state level of Cns13C was significantly reduced
compared with wild-type. Therefore, the phenotypes associated with the
cns1-3 mutations may partly be attributed to the decrease in Cns1
protein levels. However, this mutant reveals more than simply the requirement
for normal protein levels. We have found that cns1-3 cells exhibit
both a temperature-sensitive phenotype and a decrease in Hsp90 function while
maintaining a significant degree of Cns1-Hsp90 interaction. Furthermore,
although overexpression of the amino terminus can suppress the lethality of a
cns1 null allele, it is not sufficient for normal growth at
elevated temperatures. These results underscore a requirement for the carboxyl
terminus of Cns1 in the overall function of the protein, a requirement
particularly pronounced at elevated temperatures.
Our genetic investigation of the function of Cns1 has revealed a previously
unknown involvement of the Hsp90 co-chaperone, Cpr7. Overexpression of Cpr7
can suppress the temperature sensitivity of cns1ts
mutants, and deletion of CPR7 exhibits synthetic lethality in
cns1ts cells. These results strongly support the notion
that Cns1 and Cpr7 share an essential function in vivo. It is also
apparent that their cellular functions are not simply redundant and only
partially overlap. Loss of CNS1 causes lethality, whereas loss of
CPR7 leads only to a moderate slow growth phenotype. Deletion of
CPR7 leads to the loss of negative regulation of HSF
(35), whereas mutations in
CNS1 appear to have no effect on HSF-dependent signaling (this work).
Although normal growth and full Hsp90 activity require Cpr7, they do not
require the Cpr7 isomerase domain. In contrast, an essential Cns1·Cpr7
function requires the presence of this domain. Furthermore, although
cns1 mutations lead to synthetic inviability of cells in which the
Hsp90 EEVD sequence is deleted, a similar effect is not observed in analogous
cpr7 hsp82EEVD
cells.2 The
cross-suppression of mutant cns1 and cpr7 by overexpression
of CPR7 and CNS1, respectively, and the synthetic lethality
between cns1 and cpr7 mutations demonstrate a considerable
functional overlap between the two genes and indicate that they are involved
in a common and essential cellular role.
We have discovered that the PPIase domain of Cpr7 is required for viability
in cells in which Cns1 function is compromised. This was unexpected, given
that the PPIase domain of Cpr7 is not required for normal growth or full
Hsp90-related activity (29).
Equally surprising is the evidence suggesting that, although the PPIase domain
is necessary for viability in cns1 cells, the catalytic activity of
this domain is not. First, the catalytically more active PPIase domain of Cpr6
(28) is unable to restore
viability to cpr7 cns1-1 cells, even when fused to
the TPR domain of Cpr7. Second, cns1-1 cells are not hypersensitive
to the potent cyclophilin inhibitor CsA, and CsA does not prevent suppression
of Cns1G90D temperature sensitivity by Cpr7 overexpression.
Finally, a mutation in the isomerase domain of Cpr7 predicted to drastically
reduce PPIase catalytic activity does not result in a synthetic phenotype with
cns1 mutant cells. In contrast, substitutions of two hydrophilic
residues on the predicted surface of the Cpr7 PPIase domain exhibit strong
genetic interaction with cns1-1. It is possible therefore that the
PPIase domain is involved in mediating protein-protein interactions and that
these interactions are essential for viability in cns1 mutant cells.
Such a role for an isomerase domain is not unprecedented; the isomerase domain
of another immunophilin co-chaperone of Hsp90, FKBP52, interacts with dynein
in an FK506-independent manner
(51). Our data suggest that
the isomerase domains of cyclophilins may perform similar functions. Because
mutations in the PPIase domain of Cpr7 do not affect the interaction with Cns1
(data not shown), they may disrupt interaction with a novel binding
partner.
The number of co-chaperones that Hsp90 can accommodate simultaneously has been difficult to establish. Competition studies suggested that only one TPR protein can bind to a Hsp90 dimer (52, 53). However, purified Sti1 exists in solution as a dimer and binds Hsp90 in a 1:1 molar ratio (5). Cpr6, which is monomeric, binds Hsp90 with the same molar ratio (5), suggesting that a dimer of Hsp90 could bind to two different TPR domain proteins simultaneously. Given the overall relatedness of Cpr6 and Cpr7, it is likely that Cpr7 is also a monomer in solution. In this work, we established that purified Cns1 is monomeric. Therefore, we tested the possibility that the existence of Cns1·[Hsp90]2·Cpr7 complexes might explain the physical association between Cns1 and Cpr7. Although a weak interaction between Cns1 and Hsp90 remains in the absence of the Hsp90 EEVD carboxyl terminus, this region of Hsp90 provides the major contribution to the interactions between TPR domains and Hsp90. Because deletion of EEVD had no detectable effect on Cns1·Cpr7 association, the interaction between the two co-chaperones can occur in the absence of the EEVD region and possibly even without Hsp90 itself.
Protein complexes containing Hsp90 and its co-chaperones were investigated
by gel filtration chromatography using wild-type and
hsp82EEVD cells. Although Cns1 and Cpr7 partially
co-eluted in the same fractions, they did not co-elute with the major Hsp90
peak. This finding is consistent with the observation that deletion of the
EEVD sequence does not affect the formation of Cns1·Cpr7 complexes. In
contrast, Sti1 and Cpr6 co-eluted with the Hsp90 peak and shifted to lower
molecular weight fractions in hsp82EEVD cells. Thus,
at least under these conditions, binding of Cpr6 and Sti1 to Hsp90 in
vivo appears to depend entirely on the EEVD sequence. Although all four
TPR-containing co-chaperones investigated here (Sti1, Cns1, Cpr6, and Cpr7)
presumably interact with Hsp90 primarily through highly conserved sites in the
TPR domains, there is a striking difference in the extent of their interaction
with Hsp90 in vivo. Our findings illustrate the difference between
co-chaperones that exhibit strong interactions with Hsp90, such as Cpr6 and
Sti1, and those that associate weakly, such as Cns1 and Cpr7. The detection of
Cns1·Cpr7 complexes in common fractions does not exclude their
independent binding to Hsp90 but suggests that this interaction is
transient.
The synthetic slow growth of cns1-1 hsp82EEVD
cells is the first demonstration of a functional requirement for the Hsp90
EEVD sequence in vivo and reveals that interactions with
co-chaperones are important for proper function of Hsp90. An important
question remains: why is EEVD essential only when Cns1 function is impaired?
Although several explanations are possible, the detection of a weak
association between Cns1 and Hsp82EEVD suggests
the existence of binding determinants in the Hsp90-Cns1 interaction beyond the
EEVD sequence. Multiple sites of interaction between Hsp90 and Hop have been
suggested by a study in which a substitution of EEVD for AAVA did not disrupt
the interaction with Hop (54).
Recent identification of another TPR protein that can bind to a form of Hsp90
in which the carboxyl terminus has been
truncated3 also
supports the idea that there are additional binding determinants for TPR
proteins in the Hsp90 molecule. Although binding is significantly reduced in
the EEVD mutant, the level of interaction that remains is apparently
sufficient for viability. This argument is bolstered by our finding that
Cns1-Hsp90 interaction is transient and that Cns1 mostly exists in complexes
distinct from those containing Hsp90. Overall, these results may help explain
the lack of an obvious phenotype of Hsp90EEVD
cells (26).
Our findings underscore the complexity of interactions between Hsp90 co-chaperones and demonstrate that regulation of Hsp90 can occur on multiple levels, with different co-chaperones acting both independently and in concert to ensure proper signaling through the complex.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Biochemistry, Molecular
Biology and Cell Biology, O. T. Hogan Biological Sciences Bldg., 2-100, 2205
Tech Dr., Northwestern University, Evanston, IL 60208-3500. Tel.:
847-491-5452; Fax: 847-467-1380; E-mail:
r-gaber{at}northwestern.edu.
1 The abbreviations used are: Hsp90, heat shock protein 90; 5-FOA,
5-fluoroorotic acid; CsA, cyclosporin A; FLAG, DYKDDDDK epitope; GA,
geldanamycin; GAL, galactose-inducible promoter; GR, glucocorticoid receptor;
GRE, glucocorticoid response elements; GPD,
glyceraldehyde-3-phosphate dehydrogenase promoter; GST, glutathione
S-transferase; HA, hemagglutinin epitope; HSE, heat shock
response elements; HSF, heat shock factor; His6, hexahistidine;
Ni-NTA, nickel nitrilotriacetic acid; PPIase, peptidyl prolyl isomerase; TPR,
tetratricopeptide repeat; YNB, yeast nitrogen base; ORF, open reading
frame.
2 M. Tesic and R. F. Gaber, unpublished data.
3 D. Toft and A. Chadli, personal communication.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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