Aha1 Binds to the Middle Domain of Hsp90, Contributes to Client Protein Activation, and Stimulates the ATPase Activity of the Molecular Chaperone*

Gregor P. Lotz, Hongying Lin, Anja Harst, and Wolfgang M. J. ObermannDagger

From the Protein Folding Group, Institute for Genetics, University of Bonn, Römerstrasse 164, D-53117 Bonn, Germany

Received for publication, December 16, 2002, and in revised form, February 14, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ATP-dependent molecular chaperone Hsp90 is an essential and abundant stress protein in the eukaryotic cytosol that cooperates with a cohort of cofactors/cochaperones to fulfill its cellular tasks. We have identified Aha1 (activator of Hsp90 ATPase) and its relative Hch1 (high copy Hsp90 suppressor) as binding partners of Hsp90 in Saccharomyces cerevisiae. By using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1 and Hch1. Data base searches revealed that homologues of Aha1 are conserved from yeast to man, whereas Hch1 was found to be restricted to lower eukaryotes like S. cerevisiae and Candida albicans. In experiments with purified proteins, Aha1 but not Hch1 stimulated the intrinsic ATPase activity of Hsp90 5-fold. To establish their cellular role further, we deleted the genes encoding Aha1 and Hch1 in S. cerevisiae. In vivo experiments demonstrated that Aha1 and Hch1 contributed to efficient activation of the heterologous Hsp90 client protein v-Src. Moreover, Aha1 and Hch1 became crucial for cell viability under non-optimal growth conditions when Hsp90 levels are limiting. Thus, our results identify a novel type of cofactor involved in the regulation of the molecular chaperone Hsp90.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 90-kDa heat shock protein (Hsp90)1 is a highly conserved, abundant, and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol. It is specifically involved in the folding and conformational regulation of a limited subset of proteins. Almost all natural substrates of Hsp90 are medically relevant signal transduction molecules, including the nuclear receptors for steroid hormones, several protooncogenic kinases, and disparate proteins such as nitric-oxide synthase (1-4). In order to fulfill its cellular role, Hsp90 cooperates with a cohort of cofactors such as Sti1/Hop/p60, the immunophilins FKBP51/54 and FKBP52, cyclophilin 40, cdc37/p50, Sba1/p23, and CHIP (5-8) and acts as a part of a multichaperone machine together with Hsp70 (3, 9).

Several Hsp90-associated cofactors contain tetratricopeptide repeat motifs, a degenerate 34-aa sequence, that mediate binding to the C-terminal EEVD motif of the molecular chaperone (10). Other cofactors of Hsp90, like Sba1/p23 and cdc37/p50, lack tetratricopeptide repeats and are believed to use unique sequences to associate with the molecular chaperone.

The basic mechanism of Hsp90 function is still not fully understood. Recent crystal structures of the N-terminal domain of Hsp90 (11, 12) have identified a conserved binding site for ATP. Based on this structural evidence for nucleotide binding, it was demonstrated that Hsp90 can hydrolyze ATP and that this activity of the molecular chaperone is essential in vivo (13, 14). Thus, Hsp90 was unequivocally identified as an ATP-dependent molecular chaperone.

Here we demonstrate binding of the novel cofactors Aha1 and Hch1 to the middle domain of Hsp90, currently a poorly investigated region in the molecular chaperone. The intrinsic ATPase activity of Hsp90 can be stimulated 5-fold by the cofactor Aha1. Deletion of the genes encoding Aha1 and Hch1 in Saccharomyces cerevisiae impaired activation of the heterologous Hsp90-dependent substrate protein v-Src and interfered with cell viability under non-optimal conditions.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction and Yeast Two-hybrid Experiments-- Yeast Hsp90 (yHsp90) and fragments thereof were amplified from pTGPD/P82 (a gift from S. Lindquist) and yeast Aha1 (yAha1); Hch1 and Sti1 were amplified from wild type yeast DNA. PCR products were inserted into pProExHTa expression vector to generate an N-terminal His6 sequence followed by a tobacco etch virus cleavage site. The point mutation E381K was generated using the QuickChange site-directed mutagenesis kit (Stratagene). Desired exchanges were confirmed by DNA sequencing.

Human Aha1 (hAha1) was amplified from a human brain cDNA library and cloned into pAS2-1 vector (Clontech). Human Hsp90FL (hHsp90) and hHsp90 fragments were amplified from human Hsp90alpha cDNA (StressGen, accession number X15183) (15) and cloned into pACT2 vector (Clontech). Yeast two-hybrid screening was performed as described previously (13, 16) using a human brain library based on the pACT2 vector (Clontech). Positive candidate cDNA inserts were further analyzed by analytical PCR and by nucleotide sequencing. For assaying protein-protein interactions, cotransformants were grown on SD/-Trp, -Leu plates. The interaction of the bait construct with the target construct was judged by the ability of cotransformants to grow on SD/-Trp, -Leu, -His plus 25 mM 3-amino-1,2,4-triazole selection plates.

Protein Purification-- Expression constructs in pProExHTa were transformed into Escherichia coli BL21(DE3)pLysS cells. Bacteria were grown at 18 °C in LB medium supplemented with 100 mg/liter ampicillin and 34 mg/liter chloramphenicol to A600 = 1, and protein expression was induced for 5 h with 0.25 mM isopropyl-1-thio-beta -D-galactopyranoside. After harvesting, proteins were enriched from cell pellets by Ni-NTA chromatography at pH 8.0 essentially as described (13). Proteins were further purified by ion-exchange chromatography on ResourceQ and by gel filtration on Superose 12 (Amersham Biosciences).

Protein Interaction Assays-- For gel filtration analysis, 500-µl samples containing the indicated combinations of yeast proteins yHsp90 (5 µM), yHsp90NM (5 µM), yHsp90N (10 µM), yAha1 (5 µM), Hch1 (5 µM), or Sti1 (5 µM) were incubated for 10 min at room temperature in 40 mM HEPES-KOH, pH 7.4, 100 mM KCl, 2 mM MgCl2 and then separated on a Superose 12 HR10/30 or on a Superdex 75 HR10/30 column equilibrated in the same buffer using an ÄKTA chromatography system (Amersham Biosciences). 500-µl fractions were collected for analysis beginning at 6 ml of elution volume.

For pull-down assays, yHsp90 protein fragments (10 µM) were bound to Ni-NTA resin in buffer PB (25 mM HEPES-KOH, pH 7.4, 5% glycerol, 5 mM MgCl2, 0.2% Tween 20, 100 mM KCl, 10 mM imidazole) supplemented with 2% (w/v) bovine serum albumin and 2% fish skin gelatin to minimize unspecific protein interactions. yAha1 or Hch1 proteins (10 µM), which had been cleaved off from their His6 tags by tobacco etch virus protease treatment, were added, and the mixture was agitated at 4 °C for 30 min. After two washes with buffer PB, specifically bound protein was eluted from yHsp90 fragments with 500 mM KCl in the same buffer and analyzed by SDS-PAGE.

Assay of Hsp90 ATPase Activity-- Recombinant yHsp90 protein (5 µM) or the mutant yHsp90 E381K (5 µM) were incubated at 30 °C or at temperatures indicated with or without addition of yAha1 or Hch1 in 40 mM HEPES-KOH, pH 7.4, 2 mM MgCl2 supplemented with 2 mM [alpha -32P]ATP (4 µCi/mM, Amersham Biosciences) for 15 min in a final volume of 20 µl. Aliquots of each reaction were stopped at time points by the addition of 5 mM EDTA and freezing in liquid nitrogen (13). Separation of ADP from ATP was achieved by thin layer chromatography on polyethyleneimine-cellulose sheets (Merck) in 0.5 M LiCl and 0.5 M formic acid. ATPase activity was monitored by quantitation of [alpha -32P]ADP in a liquid scintillation counter. Steady state ATPase rates were calculated from the linear range of the reactions, and ATP hydrolysis activity of 5 µM yHsp90 at 30 °C was set to 100%. For specific inhibition of the ATPase activity, geldanamycin was added to the reaction at a final concentration of 180 µM in 1% Me2SO. Control reactions received Me2SO alone.

Yeast Methods and Manipulations-- Yeast strains W303 and ILEP1 (2, 17) were used throughout this study, and standard methods for growth, transformation, and manipulation were employed. Cells were grown on YPD or on SD, SRaf, and SGal selective minimal medium (0.67% yeast nitrogen base supplemented with 2% glucose, raffinose, or galactose, respectively, and with nucleotides and amino acids depending on auxotrophy).

The HCH1 gene was amplified by PCR from yeast genomic DNA and cloned into pBluescript vector via SacI/XhoI. A disruption allele was generated by cutting a substantial part of the ORF and inserting the TRP1 gene via StyI/HincII. The disruption fragment was released from the plasmid by digestion with SacI/XhoI and transformed into haploid ILEP1 (a gift from Susan Lindquist) or haploid W303 cells. Transformants were selected on SD/-Trp plates. The AHA1::KanMX4 disruption cassette was generated by PCR amplification from genomic DNA isolated from the AHA1 knockout strain Y03573 (EUROSCARF), and transformants were selected on YPD agar plates containing 200 mg/l G418. After isolation of genomic DNA from transformants and from wild type cells, disruption of wild type alleles was analyzed by PCR using specific flanking primers. The correct replacement of the respective target gene by its deletion allele in transformed cells was verified by the appearance of PCR products of the expected size and absence of PCR products corresponding to the wild type allele as described (18).

Expression and Detection of v-Src-- Yeast cells were grown overnight in SRaf/-Ura medium, diluted to A600 = 0.3 into the same medium, and supplemented with 2% galactose for 6 h at 25 °C to induce v-Src expression. Cell viability was assessed by spotting serial dilutions onto SD/-Ura plates at 25 °C. Cultures were collected by centrifugation, resuspended for 10 min in 150 mM NaCl, 50 mM HEPES, pH 8.0, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, 1 mM Na3VO4, 5 units/µl lyticase and lysed by ultrasonification for 10 s on ice. Proteins were separated by SDS-PAGE on 7.5% gels (19). Immunoblotting was performed following the protocol of Towbin et al. (20). Antibodies 4G10 (Upstate Biotechnology, Inc.) and EC10 (Upstate Biotechnology, Inc.) were used to detect phosphotyrosine residues or v-Src protein, respectively. The ECL reagent kit (Amersham Biosciences) was used to visualize immunocomplexes.

Miscellaneous Procedures-- Protein concentrations were determined by the Bio-Rad protein assay kit.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Aha1 and Hch1 as Binding Partners of Hsp90-- HCH1 (ORF YNL281w) had been identified as a high copy Hsp90 suppressor of the temperature-sensitive Hsp90 mutant E381K in S. cerevisiae (21). We used the 153-aa sequence of the Hch1 protein (Swiss-Prot accession number P53834) as a bait to screen the EMBL and NCBI protein data bases with the Blast and Fasta3 server. As a result, we found that Hch1 shares sequence homology to a short 151-aa protein present in Candida albicans (Swiss-Prot accession number O93993) and to the N-terminal half of the 350-aa S. cerevisiae protein Aha1 (activator of Hsp90 ATPase, Swiss-Prot accession number Q12449, ORF YDR214w), which is conserved from yeast to mammals (Swiss-Prot accession numbers Q9P782 for Schizosaccharomyces pombe, 336 aa; Q93168 for Caenorhabditis elegans, 343 aa; Q9LHL7 for Arabidopsis thaliana, 360 aa; Q9V9Q4 for Drosophila melanogaster, 361 aa; Q8R3E6 for Mus musculus, 338 aa; and O95433 for Homo sapiens, 338 aa). Aha1 proteins therefore consist of a putative N-terminal domain, which is homologous to Hch1 and an additional putative C-terminal domain. Human Aha1 (hAha1) shares 23% sequence identity and 38% sequence homology to S. cerevisiae Aha1 (yAha1) (Fig. 1). Interestingly, S. cerevisiae is unique in a way that it harbors Hch1 and Aha1, whereas all other organisms investigated, with the exception of C. albicans, contain only homologues of Aha1.


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Fig. 1.   Sequence comparison aligning S. cerevisiae Hch1 (yHch1) to S. cerevisiae Aha1 (yAha1) and to human Aha1 (hAha1). Amino acids identical in all three sequences are marked by asterisks; identical residues found in two sequences are indicated by colons; gaps in the sequence are denoted by periods.

In their original study, Lindquist and co-workers (21) suspected that Hch1 might act as a cochaperone or modulator of the Hsp90 system. This idea implies that Hch1 and possibly Aha1 due to its sequence similarity could interact with Hsp90. To test this hypothesis, we cloned the cDNA for human Aha1 (hAha1) into the pAS2-1 vector and used it as the bait in a yeast two-hybrid screen against a human brain cDNA library. Screening of ~1.5 million cotransformants yielded 129 positive clones, all of them harboring hHsp90 sequences as demonstrated by analytical PCR using Hsp90-specific primers. Strikingly, besides hHsp90, no other interacting proteins were identified. Similarly, when recombinant yAha1 or hAha1 was added to S. cerevisiae or mammalian cell extracts, yHsp90 or Hsp90 could be coprecipitated (data not shown).

The Middle Domain of Hsp90 Is the Specific Binding Site for Aha1 and Hch1-- Sequencing of 16 of the 129 positive hHsp90 cDNA clones revealed 10 sequences starting within a region (aa 220-291) that links the N-terminal and the middle domain of hHsp90 (Fig. 2A). This result proposed that regions in the middle domain or in the C-terminal domain of hHsp90 were involved in binding to hAha1. The remaining cDNAs were full length or started very close to the N terminus.


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Fig. 2.   Interaction of Aha1 and of Hch1 with Hsp90 and Hsp90 fragments established by the yeast two-hybrid technology. A, schematic representation of the domain organization of human Hsp90 deduced from proteolytic data (12) and of the Hsp90 fragments used in this study; numbers refer to the sequence of human Hsp90alpha . B, hHsp90FL and fragments of hHsp90 in the pACT2 vector or empty pACT2 vector serving as a control (vertical) were cotransformed into S. cerevisiae (strain Y190) with the cDNAs encoding hAha1 and Hch1 in the pAS2-1 vector or with empty vector as a control (horizontal). To monitor protein-protein interactions, cotransformants from SD/-Trp, -Leu plates were restreaked on SD/-Trp, -Leu, -His plates containing 25 mM 3-amino-1,2,4-triazole for 8 days at 30 °C. Growth was observed only for combinations of hAha1 and Hch1 with hHsp90FL and with hHsp90M.

To delineate the binding site further, we tested hHsp90FL and the fragments hHsp90N, hHsp90M, and hHsp90C by yeast two-hybrid analysis for binding to hAha1 and to Hch1 (Fig. 2B). Although hHsp90FL and hHsp90M exhibited binding to hAha1, hHsp90N and hHsp90C did not. A similar result was obtained for Hch1 (Fig. 2B). These data indicate that the interaction with hAha1 and Hch1 is mediated by the middle domain of hHsp90 and therefore distinct from the binding of TPR-containing cofactors like Sti1/p60/Hop to the C-terminal domain of Hsp90 (10, 16) or of Sba1/p23 where regions in the N-terminal domain and in the C terminus of Hsp90 are involved (22).

Biochemical binding assays using purified yAha1 protein were performed to support and extend the outcome of our two-hybrid experiments. First, we tested yHsp90FL and the protein fragment yHsp90NM for their ability to interact with the yAha1 protein by gel filtration chromatography. yAha1, clearly migrating as a monomer, yHsp90FL, and yHsp90NM showed different elution profiles on a Superose 12 column (Fig. 3, A, B, and D). We combined yHsp90FL and yHsp90NM with yAha1 and analyzed the mixtures for protein interactions (Fig. 3, C and E). When a mixture of yHsp90FL and yAha1 was run on a Superose 12 gel filtration column, a significant amount of yAha1 coeluted with yHsp90FL (Fig. 3C, fractions 7-12), consistent with the formation of a complex between the two proteins. This binding was independent of ATP and could be disrupted by increasing the salt concentration to 500 mM KCl (not shown).


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Fig. 3.   The middle domain of Hsp90 is the specific binding site for Aha1. Purified yHsp90 and the fragments yHsp90NM and yHsp90N were incubated with yAha1 and fractionated by gel filtration chromatography on a Superose 12 column (A-E) or on a Superdex 75 column (F-H). Fractions were analyzed by SDS-PAGE. Marker proteins are shown on top (thyroglobulin 667 kDa, bovine serum albumin 67 kDa, ribonuclease A 14 kDa). A, yAha1. B, yHsp90FL. C, yAha1 incubated with yHsp90FL. D, yHsp90NM. E, yAha1 incubated with yHsp90NM. F, yAha1. G, yHsp90N. H, yAha1 incubated with yHsp90N.

When yHsp90NM and yAha1 were incubated together and run on a Superose 12 column, again a certain amount of yAha1 comigrated with this yHsp90 fragment (Fig. 3E, fractions 10-12) indicating that the C-terminal domain of the molecular chaperone is dispensable for the interaction. Binding of yAha1 to yHsp90 M is not shown in Fig. 3, and because complexes could not be resolved from either protein alone, we instead used a pull-down experiment to demonstrate this interaction (see below). However, when using a Superdex 75 column instead of a Superose 12 column, yAha1 could be separated from yHsp90N (Fig. 3, F and G). A mixture of the two proteins was analyzed by gel filtration chromatography, but no shift in the migration of yHsp90N was observed (Fig. 3H), consistent with the view that the N-terminal domain of yHsp90 is not involved in binding to yAha1.

As an independent verification of these domain mapping studies, yAha1 was assayed for interacting with purified N-terminal, middle, and C-terminal domains of yHsp90 immobilized on Ni-NTA agarose in a pull-down experiment as described under "Experimental Procedures." Mixtures of yAha1 and yHsp90 fragments were incubated to allow binding and were finally recovered by centrifugation. Protein that had specifically bound to yHsp90 fragments was eluted by 500 mM KCl and analyzed by SDS-PAGE. yAha1 bound to yHsp90M and could be specifically eluted from this protein fragment but was not retained on yHsp90N or on yHsp90C (Fig. 4).


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Fig. 4.   Aha1 binds to the middle domain of Hsp90 but not to Hsp90N or to Hsp90C. yHsp90 fragments were bound to Ni-NTA agarose via their His6 tags and incubated with non-tagged yAha1 protein as described under "Experimental Procedures." Specifically bound protein was eluted with 500 mM KCl and analyzed by SDS-PAGE. The position of marker proteins with sizes indicated in kDa is shown.

Similar results were obtained when Hch1 or the corresponding N-terminal fragment yAha1-(1-156) was used in this pull-down experiment. In contrast, the C-terminal yAha1-(157-350) fragment did not bind to yHsp90 (data not shown). Thus, the biochemical data are in full agreement with the observation of our yeast two-hybrid assays and demonstrate that the middle domain of Hsp90 is necessary and sufficient to bind Aha1 and Hch1.

Sti1 Competes with yAha1 for Binding to yHsp90-- Sti1/p60/Hop is bound in "intermediate" Hsp90 chaperone complexes to the C-terminal domain of the molecular chaperone by one of its TPR clamp domains and also contacts the N-terminal domain of Hsp90 thereby blocking access to the ATP-binding site (23). This observation prompted us to ask whether Sti1 interferes with binding of yAha1 to yHsp90, and we therefore analyzed a mixture of Sti1, yHsp90, and yAha1 for complex formation by gel filtration chromatography. Sti1 and the yHsp90-yAha1 complex showed different elution profiles on a Superose 12 column (Fig. 5, A and B). Upon addition of Sti1 to yHsp90FL and yAha1 at equimolar amounts, the yHsp90FL-yAha1 complex was apparently almost completely disrupted (Fig. 5C). This situation resembles the competition of cdc37/p50 by Sti1/p60/Hop (24, 25) and suggests a steric hindrance caused by a three-dimensional overlap of the cofactors or their binding sites on Hsp90.


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Fig. 5.   Sti1 competes with yAha1 for binding to yHsp90FL. Purified yHsp90FL was incubated with Sti1 and yAha1 and fractionated by gel filtration chromatography on a Superose 12 column. A, Sti1. B, yHsp90FL incubated with yAha1 (identical to B in Fig. 3). C, yHsp90FL incubated with yAha1 and Sti1. Marker proteins are shown on top (thyroglobulin 667 kDa, bovine serum albumin 67 kDa).

yAha1 Stimulates the Intrinsic ATPase Activity of yHsp90-- ATP hydrolysis has been shown to be essential to the in vivo function of the molecular chaperone Hsp90 (13, 14). Therefore, we were curious as to whether this capacity of yHsp90 could be affected by yAha1. We titrated increasing amounts of yAha1 into 5 µM yHsp90 (corresponding to 2.5 µM dimer) and measured ATPase activity as described under "Experimental Procedures." The ATP hydrolysis rate for various combinations of yHsp90 and yAha1 is plotted in Fig. 6A. Obviously, yAha1 was able to stimulate the intrinsic ATPase activity of yHsp90 up to 5-fold. Next we asked whether Hch1, which is homologous to the putative N-terminal domain of yAha1, would also be able to influence yHsp90 ATPase activity. Interestingly, although Hch1 bound to Hsp90 (Fig. 2B), it was not able to stimulate yHsp90 ATPase activity significantly even at 50 µM, a 10:1 molar ratio relative to yAha1 (Fig. 6B). Moreover, when yAha1 was dissected into two fragments, one comprising the putative N-terminal domain (aa 1-156) and the other one encompassing the putative C-terminal domain (aa 157-350), the intrinsic ATP hydrolysis rate of yHsp90 could not be stimulated by any of these truncated proteins (Fig. 6B). This result indicates that the entire yAha1 polypeptide is required for stimulating the ATPase activity of yHsp90.


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Fig. 6.   yAha1 stimulates the intrinsic ATPase activity of yHsp90. ATP hydrolysis rate was measured as described under "Experimental Procedures," and the ATPase activity of 5 µM yHsp90 at 30 °C without additional proteins was set to 100%. All data are corrected for unspecific background activity which could not be inhibited by addition of geldanamycin. Activities are averages of at least three independent experiments, and error bars are indicated. A, yHsp90 ATPase activity stimulated by increasing amounts of yAha1 at 30 °C. Note that yAha1 could stimulate yHsp90 up to 5-fold. B, yAha1 but neither of its fragments (aa 1-156 and aa 157-350) nor Hch1 could stimulate ATP hydrolysis of yHsp90. yHsp90 was incubated alone or with indicated amounts of yAha1, yAha1-(1-156), yAha1-(157-350), or Hch1 and assayed for ATPase activity at 30 °C. C, ATPase activity of wild type yHsp90 and of the E381K mutant at 25 and 37 °C. The ATPase activity of the E381K mutant reached nearly wild type levels.

Since Hch1 was not able to stimulate the ATP hydrolysis rate of yHsp90 but suppressed the temperature-sensitive yHsp90 mutant E381K (21), we assumed that the defect of this mutant at the non-permissive temperature is not due to a major reduction of ATPase activity. To test this assumption rigorously, we determined the rate of ATP hydrolysis for the E381K mutant at 25 and 37 °C, the non-permissive temperature, and compared it to wild type yHsp90. The shift to 37 °C did not harm the ATPase activity of the E381K mutant. Instead, the ATPase activity of the E381K mutant was raised with temperature and reached a value comparable with wild type yHsp90 (Fig. 6C). Thus, the loss of function due to the E381K mutation at the non-permissive temperature presumably results from defects in other aspects of Hsp90 biochemistry, which can be restored by the assistance of Hch1.

Aha1 and Hch1 Are Non-essential but Confer Thermotolerance to Yeast Cells When Intracellular Levels of Hsp90 Are Limiting-- Due to the relationship of both proteins (Fig. 1), Hch1 and Aha1 could serve overlapping functions and be redundant in S. cerevisiae. Therefore, we created knockouts Delta HCH1, Delta AHA1, and the Delta HCH1/Delta AHA1 double knockout in the wild type yeast strain W303 as described under "Experimental Procedures." The relative growth rate of wild type cells and of these three knockout strains was assessed using a plate assay. There was no obvious difference in the growth rate at 25, 35.5, or at 37 °C on YPD agar plates (Fig. 7). Thus a complete loss of Hch1 and Aha1 protein activity did not affect viability of W303 cells at normal and at elevated temperatures. A comparable mild effect at 37 °C has been described previously for the deletion of the gene encoding Sba1/p23 (26, 27).


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Fig. 7.   Aha1 and Hch1 are non-essential but confer thermotolerance to yeast cells when intracellular levels of Hsp90 are limiting. Viability of ILEP1 and W303 wild type (wt) or Delta HCH1, Delta AHA1, and Delta HCH/Delta AHA1 deletion strains at 25, 35.5, and 37 °C. Cells were grown overnight in YPD medium, adjusted to 1 × 108 cells per ml, and 10-fold serially diluted. 2-µl portions were spotted on YPD agar plates and incubated for 48 (at 25 °C) or 36 h (at 35.5 and 37 °C).

We next asked whether deletions of Hch1 and Aha1 would harm cells more severely if intracellular levels of Hsp90 were significantly reduced. To investigate such a situation we made use of the W303-derived strain ILEP1, a kind gift from Susan Lindquist. In ILEP1 cells expression of Hsp90 was controlled by a partially deleted promoter, which results in a reduction of Hsp90 to ~1/20 of the normal level (17). The same deletions as described above for W303 were generated in ILEP1, and cells were again assessed for viability. No difference in growth phenotype was observed at 25 and at 35.5 °C. However, when viability was tested at 37 °C, the ILEP1 Delta HCH1/Delta AHA1 double knockout cells exhibited obvious growth defects compared with the parental strain and with the single knockout strains (Fig. 7).

Deletion of Genes Encoding Aha1 and Hch1 Interferes with v-Src Activation in Vivo-- The tyrosine kinase v-Src is active in a manner that is dependent on the molecular chaperone Hsp90 and its cofactors/cochaperones. In wild type cells like W303, expression of v-Src leads to greatly increased levels of phosphotyrosine. Mutations affecting Hsp90 itself or deletions of genes encoding cofactors of the Hsp90 system resulted in a decrease of v-Src activity accompanied with lower levels of tyrosine phosphorylation (28-31) that provides a handle to estimate the activity of the Hsp90 system in vivo. Since v-Src expression is toxic in yeast cells, v-Src was expressed from a galactose-regulated plasmid.

We observed in a control experiment that most of the strains generated in this study grew equally well on glucose, galactose, and raffinose medium. As an exception, Delta HCH1/Delta AHA1 double deletion cells displayed a clearly delayed growth phenotype on galactose as the sole carbon source. When these Delta HCH1/Delta AHA1 cells were transformed with the v-Src expression plasmid and induced by transfer from SRaf/-Ura to SGal/-Ura medium, v-Src levels were strongly decreased compared with wild type or to the single deletion strains (data not shown). In contrast, addition of galactose to Delta HCH1/Delta AHA1 cells growing on raffinose medium had no negative effect on their growth rate. Therefore, W303 and ILEP1 wild type and deletion strains were transformed with the v-Src expression plasmid, grown in SRaf/-Ura medium overnight, and diluted with the same medium to an A600 = 0.3, and v-Src expression was induced at 25 °C for 6 h by addition of 2% galactose to the SRaf/-Ura medium (26) as described under "Experimental Procedures." Under these conditions v-Src protein accumulated to similar levels in wild type, Delta HCH1, Delta AHA1, and Delta HCH1/Delta AHA1 cells (Fig. 8A). However, compared with W303 cells v-Src accumulation in ILEP1 cells was reduced, likely reflecting the lower amount of Hsp90 in this strain (Fig. 8A).


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Fig. 8.   Deletion of genes encoding Aha1 and Hch1 interfere with v-Src activation in vivo. Cell lysates were prepared from normal or mutant ILEP1 and W303 cells expressing v-Src (lanes 2-5 and 7-10) or from empty vector control cells (lanes 1 and 6) as described under "Experimental Procedures." Proteins were separated by PAGE on 7.5% gels and transferred to nitrocellulose membranes. Equal protein loading was confirmed by the Bio-Rad protein assay and by Coomassie staining of a corresponding SDS gel. A, Western blot analysis of v-Src expression with antibody EC10. B, Western blot analysis of phosphotyrosine activity with antibody 4G10 in the strains corresponding to A. The position of marker proteins with sizes indicated in kilodaltons is shown. C, quantification of phosphotyrosine levels from the Western blot in B, phosphotyrosine activity of W303 wild type (wt) was set to 100%.

When protein tyrosine phosphorylation was monitored, all three mutant W303 strains showed a moderate reduction of v-Src activity to ~50% of the W303 wild type cells (Fig. 8, B and C, lanes 6-10). Next, we examined ILEP1 strains for protein tyrosine phosphorylation. Compared with W303, v-Src activity in ILEP1 was reduced as expected from the lower level of v-Src accumulation. However, deletions of the genes encoding Hch1 and Aha1 had a more pronounced effect, which resulted in a decrease of v-Src activity in ILEP1 Delta HCH1/Delta AHA1 as low as ~10% compared with the parental ILEP1 cells (Fig. 8, B and C, lanes 1-5). From this result we conclude that Hch1 and Aha1 became important for client protein activation under non-optimal conditions at higher temperatures when Hsp90 levels are limiting as observed in ILEP1 cells.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

An increasing number of cochaperones/cofactors are now known to regulate the Hsp90/Hsp70 chaperone machine for fulfilling different tasks in the cell. A defined group of cofactors like Sti1/p60/Hop, the immunophilins FKBP51/54 and FKBP 52, cyclophilin 40, protein phosphatase PP5, and CHIP bind to the C-terminal EEVD motif of the Hsp90 component of this system by virtue of their modular TPR clamp domains (10). In the case of p23, regions in the N-terminal ATPase domain and induced dimerization of Hsp90 are required for binding (22).

The results presented here demonstrate that Aha1 and Hch1, in remarkable contrast, associate with the middle domain of Hsp90, whereas C- and N-terminal domains of the molecular chaperone are dispensable. As a consequence of this interaction, the cofactor Aha1 stimulates the inherent ATPase rate of Hsp90 5-fold, comparable with the 7-fold stimulation of Hsp70, the other major chaperone of the eukaryotic cytosol, by the J-domain protein Hsp40 (32). In association with our results, a stimulating effect of Aha1 on the ATPase activity of Hsp90 has also been reported by the recent work of Panaretou and co-workers (44). When we used Hsp90 and Aha1 at a 1:1 molar ratio, Hsp90 ATPase was almost fully stimulated, whereas Hch1 and the N-terminal Aha1 fragment displayed no stimulating effect under the same conditions (Fig. 6B). When the Hch1:Hsp90 ratio was increased from 1:1 to 10:1, a slight stimulation of ATP hydrolysis could be observed (Fig. 6B). Panaretou and co-workers (44) raised this molecular excess further and could thereby stimulate Hsp90 ATPase by Hch1 and by the N-terminal Aha1 fragment, however, only to ~30% that achieved by full-length Aha1. These in vitro data suggest that Aha1 is by far a more potent stimulator of Hsp90 ATPase activity than Hch1. Another point questions a function of Hch1 as an authentic stimulator of Hsp90 ATPase. Remarkably, Hch1 suppressed the growth defect of the Hsp90 mutant T22I in S. cerevisiae (21). Since the toxicity of this mutant was attributed to its 6-fold increased ATPase activity (43), a pivotal role of Hch1 as a stimulator of Hsp90 ATPase seems to be virtually unlikely.

Like Sba1/p23 (26, 27), Aha1 and Hch1 are not essential in S. cerevisiae for general chaperone-mediated protein folding under normal conditions. However, Aha1 and Hch1 turned out to be crucial when growth temperatures were raised to 37 °C and Hsp90 levels were limiting (Fig. 7). Under conditions where Hsp90 becomes limiting, both cofactors contributed to the activation of the substrate protein v-Src by Hsp90 (Fig. 8); thus our study and the work of Panaretou and co-workers (44) identify Aha1 and Hch1 as cofactors crucial for the activation of a client protein in yeast. Compared with wild type and single deletion strains, Delta HCH1/Delta AHA1 cells accumulated slight levels of v-Src protein when expression was induced by transfer from raffinose to galactose medium as mentioned under "Results" and reported by Panaretou and co-workers (44). This may be due to rapid degradation (44) or by lower synthesis of the v-Src protein under those conditions. However, we realized that Delta HCH1/Delta AHA1 cells were sensitive to galactose as the sole carbon source and that this stress situation might interfere with the analysis of v-Src activation. Therefore, we induced v-Src expression by addition of galactose to cells growing on raffinose medium, rather then by transfer of cells from raffinose to galactose medium. Under those conditions v-Src protein reached comparable levels in wild type, Delta HCH1, Delta AHA1, and in Delta HCH1/Delta AHA1 strains (Fig. 8).

At higher temperatures, Hsp90 is thought to prevent unfolding of substrates and allow recovery of sensitive client proteins which had been partially unfolded (33, 34). Remarkably, Aha1 and to a lower extent Hch1 is up-regulated by heat stress to the same degree as Sti1 and Hsp82, the inducible form of Hsp90 in S. cerevisiae (data from expression profiling analysis may be obtained from the Saccharomyces Genome Data base at genome-www4.stanford.edu). Aha1 and Hch1 cannot prevent aggregation of unfolded proteins (data not shown) and are therefore no chaperones on their own. Instead, they add to the efficiency of the Hsp90 system, which becomes crucial under non-optimal conditions. Whether the stimulating effect of Aha1 on the activation of client proteins like v-Src is a direct consequence of its ability to stimulate Hsp90 ATPase has yet to be demonstrated. For this purpose, rather than a wholesale disruption of Aha1, mutants of Aha1 would be helpful that bind indifferently to Hsp90 but fail to stimulate its ATPase activity. However, such mutants are not readily available.

It is generally believed that unfolded substrate proteins associate first with the Hsp70 system and are then targeted to Hsp90 via Sti1/p60/Hop that binds both Hsp70 and Hsp90 (1, 3, 35-38). This interaction leads to the formation of an "intermediate complex" in which Sti/p60/Hop inhibits the ATPase activity of Hsp90 and locks the molecular chaperone in a conformation allowing substrate protein to bind (23). Further remodeling and dissociation of Sti1/p60/Hop, Hsp70 and its cochaperones then leads to the "mature complex," the state in which Hsp90 consumes ATP (23) and actively contributes to the activation of client proteins. Our data suggest that binding of Aha1 and Sti1 to Hsp90 is mutually exclusive (Fig. 5C), probably due to steric hindrance. In contrast, Panaretou and co-workers (44) proposed in their recent work that Aha1 and Sti1 can coexist in Hsp90 complexes. To resolve this potential inconsistency it is important to point out that this group used the C-terminal fragment cSti1 (aa 237-589) in their experiments, whereas in our studies natural full-length Sti1 was used. It is plausible that the N-terminal truncation prevents a three-dimensional overlap of cSti1 with Aha1, and therefore both cofactors can bind simultaneously to Hsp90, whereas full-length Sti1 competes with Aha1 for Hsp90 binding. The affinity of Aha1 for Hsp90 seems to be too weak to replace Sti1 directly from Hsp90 complexes at physiological salt conditions (Fig. 5C), but it might be increased by modifications or by the presence of a client protein, although there is no experimental evidence for such a regulation. On the other hand, Panaretou and co-workers (44) have shown that Aha1 can coexist in Hsp90 complexes with cochaperones of the "mature" type like full-length Cpr6. Thus, displacement of Sti1 by Cpr6 during the transition from the intermediate to the mature Hsp90 chaperone complex allows Aha1 to enter the Hsp90 complex at this stage. Hence, the cell can keep Aha1 at moderate levels as Aha1 does not have to replace Sti1 directly from Hsp90. Therefore, it is conceivable that Aha1 binds to the middle domain of Hsp90 and contributes to protein folding at this stage of the mature complex once Sti1 has been displaced by the mechanism described. Interestingly, it has been suggested recently that in addition to the N- and C-terminal domains (39, 40), the middle domain of Hsp90 can interact with client proteins and serves as a molecular scaffold for their activation (41). Aha1 and Hch1 bind to the same domain that would enable them to act on this process.

The more complex the organism from which the Hsp90 protein is derived, the less efficient the ATP hydrolysis rate, which predicts that the molecular chaperone would require cofactors to regulate ATPase activity or binding of substrate especially in higher organisms (42). Compared with more complex organisms, Hsp90 from S. cerevisiae has a relatively high ATPase activity, and consequently further stimulation would not be urgently required. This may explain the relative redundancy of Aha1 and Hch1 in S. cerevisiae. On the other hand, in higher organisms tuning of Hsp90 ATPase activity by Aha1 according to the demands of the client protein might become important to trigger substrate activation and account for the absence of an Hch1 homologue. However, it has still to be demonstrated experimentally that Hsp90 systems of humans and other higher organisms can indeed be stimulated by their respective Aha1 cofactors similar to yeast Hsp90 by yeast Aha1.

The mechanism of ATPase stimulation by Aha1 is elusive. A direct interaction of Aha1 with the catalytic N-domain of Hsp90 could not be demonstrated in this study (Figs. 3 and 4); however, a transient interaction could contribute. Alternatively, Aha1 might give rise to a conformational transition within the Hsp90 dimer and thereby induce contacts of the N-terminal domains required for significant ATPase activity (43).

    ACKNOWLEDGEMENT

We thank Dr. S. Lindquist for supplying yeast strain ILEP1 and the vector pTGPD/P82.

    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 284/project Z3 and by European Commission Grant QLK3-CT2000-00720.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 49-228-734445; Fax: 49-228-734263; E-mail: obermann@uni-bonn.de.

Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212761200

    ABBREVIATIONS

The abbreviations used are: Hsp90, 90-kDa heat shock protein; hHsp90, hAha1, yHsp90, yAha1, human or yeast Hsp90 and Aha1; Hsp90FL, Hsp90N, Hsp90M, Hsp90C, Hsp90 full-length, N-terminal, middle, and C-terminal domain; aa, amino acid; ORF, open reading frame; Ni-NTA, nickel-nitrilotriacetic acid; SD, synthetic dropout medium; TPR, tetratricopeptide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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