Redox Control of Hsp70-Co-chaperone Interaction Revealed by Expression of a Thioredoxin-like Arabidopsis Protein*

By using a yeast functional complementation assay, we have identified AtTDX, a new Arabidopsis thaliana gene, encoding a two-domain 42-kDa protein. The amino-terminal domain of AtTDX is closely related to the co-chaperone Hsp70-interacting protein HIP, whereas its carboxyl-terminal part contains a thioredoxin domain. Both in vivo and in vitro assays showed that AtTDX is a protein-disulfide reductase. We next found that the HIP domain of AtTDX is capable of interacting with the ATPase domain of Ssb2, a yeast heat-shock protein 70 chaperone. Strikingly, the AtTDX-Ssb2 interaction can be released under oxidative stress, a redox-dependent regulation involving the thioredoxin activity of AtTDX. A mutation inactivating the cysteine 20 of the ATPase domain of Ssb2 was found to stabilize the AtTDX-Ssb2 interaction that becomes redox-insensitive. As cysteine 20 is conserved in virtually all the Hsp70 chaperones, our results suggest that this residue might be more generally the target of redox regulations of chaperone binding activity.

Thioredoxin (TRX) 1 is a small 12-kDa ubiquitous protein containing two redox-active half-cystine residues in an active center with conserved amino acid sequence Cys-X-X-Cys (where X indicates various amino acids) that functions as a protein-disulfide reductase. The two cysteine residues in the active site provide the sulfhydryl groups involved in the thioredoxin-dependent reducing activity. Under an oxidized form, the TRX-S 2 protein contains a disulfide bridge within the active site that is reduced to a TRX-(SH 2 ) dithiol by NADPH and the flavoprotein TRX reductase (for review, see Ref. 1). Under this reduced form, thioredoxin becomes a very powerful reductant of disulfide bridges in target proteins.
Thioredoxin was initially characterized from Escherichia coli extracts as a hydrogen donor for ribonucleotide reductase (2). Thioredoxins were later shown to act as hydrogen donors to peroxiredoxins that reduce hydrogen peroxide (3) and to methionine-sulfoxide reductases that reactivate proteins damaged by stresses that generate reactive oxygen species (ROS) (4). They are necessary for a number of other metabolic enzymes that form a disulfide as part of their catalytic cycle (5). In addition, thioredoxins induce conformational changes of the targeted proteins by disulfide bond reduction and assist in the protein folding pathway (6). Thioredoxins directly or indirectly interact with several nuclear factors, such as the mammalian transcription factor NFB and Ref-1 (see Ref. 7 and references therein). Thioredoxins play important roles in cell cycle and division and embryogenesis (8) and inhibit spontaneous apoptosis in tumoral cells (9). Mammalian thioredoxin has also been shown to associate directly with nuclear receptors such as the glucocorticoid receptor GR (10), whose regulation requires cooperation between heat-shock proteins such as Hsp70 and Hsp90 (11).
Hsp70 and Hsp90 are molecular chaperones that are involved in many important biological processes by appropriately folding nascent polypeptides on ribosomes as well as by assembling multisubunit protein complexes (12). Chaperones also play a pivotal role by renaturing proteins after exposure to various stresses, driving protein translocation across membranes, and disassembling protein complexes prior to protein degradation. Molecular chaperones often function together, and in this respect, members of the Hsp70 family are of prime importance. They are highly conserved from bacteria to human and exhibit a well defined structure as follows: the 44-kDa amino-terminal part of the protein binds nucleotides, whereas the 28-kDa carboxyl-terminal domain interacts with misfolded or partially unfolded polypeptides (13).
The chaperone activity of Hsp70 is regulated by cofactors that catalyze the interconversion between the ATP and ADP states (14). In bacteria, cycling of the Hsp70 homologue DnaK between different nucleotide states is regulated by the chaperone cofactors DnaJ and GrpE (15). DnaJ stimulates the Hsp70 ATPase activity, and the conversion of Hsp70 into the ADPbound state allows it to interact with polypeptide substrates (16). In contrast, GrpE binds to the ATPase domain of DnaK and triggers the release of ADP, hence accelerating substrate dissociation upon ATP re-binding (17). In eukaryotes, the yeast DnaJ homologue Hsp40/Zuo1 (18) and the mammalian Hsp40/ Hdj (19) have also been shown to associate with Hsp70. However, structural homologues of GrpE are limited to compartments of prokaryotic origin, i.e. mitochondria and chloroplasts (20). Mammals contain other classes of Hsp70 co-chaperones, such as the p48/Hip (Hsc70-interacting protein), BAG-1/Hap (Hsp70-activating protein), and CHIP (carboxyl terminus of Hsc70-interacting protein) (21-23) cofactors. BAG-1 and Hop accelerate ADP release and stimulate the re-binding of ATP. CHIP decreases net ATPase activity and reduces chaperone efficiency, thus negatively regulating the Hsp-substrate binding cycle. In contrast, Hip stabilizes the ADP-bound form of Hsp70, extending the time window during which Hsc70 interacts stably with a polypeptide substrate. All these co-chaperones have a spatial distribution that is critical to the coordination of Hsp70 functions, because they ensure that substrates are both bound and released at an appropriate place and time by regulating the ATP/ADP cycle (24).
In this report, we describe the molecular cloning and characterization of a novel bipartite protein from Arabidopsis thaliana that may be a new component of the Hsp70 chaperone system. This protein exhibits a unique domain structure with a carboxyl-terminal thioredoxin domain, the amino-terminal domain containing three tetratricopeptide repeats similar to that of the rat and human Hip (21). We termed this new protein AtTDX for Tetratricopeptide domain-containing thioredoxin. We present evidence that AtTDX displays a disulfide reductase activity both in vitro and in vivo due to its thioredoxin domain, whereas its amino terminus interacts specifically with the yeast Hsp70 Ssb2 protein. We show that the interaction between AtTDX and Ssb2 is sensitive to the redox status, and we demonstrate that the thioredoxin domain of AtTDX acts as a redox switch that turns the complex with Ssb2 on and off. We also show that a conserved cysteine in the ATPase domain of Ssb2 is required for disruption of the complex with AtTDX. The work we present here provides the first example of a redox-dependent interaction of a thioredoxin-like protein with a member of the Hsp70 family.

EXPERIMENTAL PROCEDURES
Recombinant DNA and DNA Analyses-The A. thaliana two-leaf stage cDNA library used for the yeast complementation assays was constructed in a pFL61 vector (25). PCR amplifications were done using the Expand Long Template kit (Roche Molecular Biochemicals). Directed mutagenesis was done by primer-mediated mutagenesis using QuikChange TM Site-directed Mutagenesis Kit (Stratagene). All the constructs and PCR products were checked by sequencing. Details concerning plasmids created in this study are available upon request. The program Darwin used to create the phylogenic tree is at cbrg. inf.ethz.ch. Swiss-PDB-Viewer and Rasmol programs used to analyze the Ssb2 sequence are at www.expasy.ch/spdbv/mainpage.htm and klaatu.oit.umass.edu:80/microbio/rasmol, respectively.
Protein Analyses and Antibodies-Protein concentrations were determined with a protein assay kit (Bio-Rad). Western blots were developed by enhanced chemiluminescence. Anti-HA monoclonal 12CA5 and anti-GST antibodies were purchased from Roche Molecular Biochemicals and Amersham Biosciences, respectively. Polyclonal antibodies against His-AtTDX were raised in rabbit (Eurogentec).
For H 2 O 2 sensitivity assays, transformed cells were first grown on histidine-containing minimal medium to a density of 10 7 cells per ml and then serial diluted in histidine-less minimal medium in the presence of increasing concentrations of H 2 O 2 . Plates were then incubated at 30°C for 3 days.
Expression of Recombinant Proteins-A pET16b vector (Novagen) was used for the expression of His-tagged version of AtTDX and its derivatives in E. coli, allowing the amino-terminal introduction of a His 6 tag. The coding region of AtTDX and AtTDX⌬-(1-269) were subcloned as NdeI-BamHI fragments into corresponding sites of pET16b. The coding region of ySsb2 was cloned as a BamHI-BamHI fragment into the pGEX-4T1 vector (Amersham Biosciences) for the expression of a amino-terminal GST-tagged version of the protein. BL21 (DE3) cells harboring the appropriate plasmids were cultured at 37°C in LB medium with ampicillin. Expression of the recombinant proteins was induced at A 600 of 0.2 by addition of isopropyl-1-thio-␤-D-galactopyranoside to 1 mM, and cells were further grown for 3 h. Cells were then harvested, and total proteins were extracted from bacteria using a hydraulic press (Carver, model 3968) according to Verdoucq et al. (30). Cells expressing His-AtTDX and derivatives were resuspended in a 1ϫ imidazole binding buffer (Novagen), and His-tagged proteins were pu-rified on Ni 2ϩ His.Bind resin according to the manufacturer's instructions. Cells expressing ySsb2 were resuspended in a 1ϫ phosphatebuffered saline buffer (Amersham Biosciences), and GST-ySsb2 was purified on glutathione-Sepharose 4B column according to the manufacturer's instructions.
His-tagged AtTDX and AtTDX⌬-(1-269) proteins used for determination of thioredoxin activity were tested using the insulin-disulfide reduction assay as described previously (31). All assays were monitored by addition of 1 mM dithiothreitol, and measurements were performed at A 650 for 45 min on a spectrophotometer (model DU7400, Beckman Instruments).
Two-hybrid Experiments-All the experiments were performed in the yeast reporter strain YRG2 using the Gal4 Two-hybrid Phagemid Vector kits (Stratagene). The vector pBDGal4 was used to clone AtTDX and its derivatives as the baits, and pADGal4 was used to clone the target protein Ssb2 and its derivatives as the targets. Two two-hybrid cDNA libraries were screened: an A. thaliana cell suspensions cDNA library cloned into a pADGal4 vector (gift of B. Lescure), and a yeast cDNA library cloned into a pGAD-GH vector (32). Library screening and control/test experiments were as described by Stratagene using 20 mM 3-aminotriazole. Double transformants were grown on selective medium and tested by histidine prototrophy and ␤-galactosidase activity.
All the fragments for both the bait and the target cDNAs, as well as truncations, were obtained by PCR amplification, using restriction sitecontaining primers on yeast genomic DNA extracted with standard procedures. The pBD.AtTDX⌬-(112-213) and pAD.Ssb2⌬-(387-549) constructs were obtained by a two-step PCR. The clone pAD.AtHsc70-1 was obtained by PCR amplification on clone G11F4T7 (NCBI stock center).
Co-immunoprecipitation Assays-In vivo interaction between HA-AtTDX and GST-Ssb2-tagged proteins was assayed by co-immunoprecipitation experiments. Briefly, the full-length AtTDX sequence was cloned into a pFL39 vector, whereas the full-length Ssb2 sequence was cloned into a pEG.KT vector. Cultures of yeast cells, protein extractions, and immunoprecipitation experiments were performed as described previously (29). Precipitates were separated on 10% SDS-PAGE gels and analyzed by Western blotting.
Reconstitution of AtTDX-ySSB2 Complex Using Affinity Chromatography-Recombinant His-AtTDX and GST-ySSB2 were incubated in a 20 mM imidazole-containing buffer (Buffer A) for 15 min at 30°C as described previously (21). Using a batch method, 100 l of Ni 2ϩ His-.Bind resin (Novagen) equilibrated with 1ϫ binding buffer was added, and the samples were further incubated for 30 min at 4°C. After washing with Buffer A, bound proteins were eluted with 3 volumes of Buffer A, 150 mM imidazole. Proteins were then separated on 10% SDS-PAGE gels and analyzed by Western blotting.

RESULTS
Cloning of a Novel TPR-containing Thioredoxin-like Protein from A. thaliana-To isolate new Arabidopsis genes encoding thioredoxins or thioredoxin-like proteins, we screened an A. thaliana cDNA library by functional complementation of trx1⌬, trx2⌬ double disrupted yeast cells. In S. cerevisiae, the simultaneous disruption of the two thioredoxin encoding genes, TRX1 and TRX2, leads to several growth defects, including organic sulfur auxotrophy and sensibility to oxygen peroxide (33). trx1⌬, trx2⌬ mutant cells were transformed with a library of 2-day-old seedlings cDNA that are expressed from the strong PGK1 promoter on vector pFL61 (25), and uracil prototroph clones able to grow without methionine were selected. From a screen of 5⅐10 6 independent transformants, 20 methionine prototroph colonies were recovered. Further analyses demonstrated that these clones correspond to the expression of two different A. thaliana cDNAs from the PGK1 promoter. The first one encodes AtTRX3 protein, a thioredoxin shown previously to complement the EMY63 mutant for sensibility to oxygen peroxide but not for sulfate assimilation when expressed from a centromeric low copy plasmid (34). In the present study, At-TRX3 is expressed from a strong promoter on a high copy plasmid, and therefore, methionine auxotrophy complementation is believed to result from high AtTRX3 synthesis. The second cDNA encodes a novel thioredoxin-containing protein.
This full-length cDNA is 1365 nucleotides in length and con-tains an open reading frame with the potential of coding a 380-residue polypeptide (Fig. 1A). This protein displays a bipartite structure with an amino-terminal part that comprises three 34-amino acid tetratricopeptide domains (TPR, residues 112-213) and a carboxyl-terminal domain (residues 270 -380) that is highly similar to thioredoxin proteins. The two domains are separated by a central region rich in charged residues. We named this novel A. thaliana protein, AtTDX, for Tetratricopeptide domain-containing thioredoxin.
Occurrence of TDX in Plant Genomes--To confirm the occurrence of the AtTDX gene within the A. thaliana genome as well as to determine the structure of this gene, we amplified by PCR genomic fragments using two oligonucleotides flanking the AtTDX cDNA coding sequence (Fig. 1A). The chromosomal copy of AtTDX contains 9 introns that are all located upstream from the thioredoxin domain, the 9th being found at the beginning of the thioredoxin domain (Fig. 1B). Southern blot experiments demonstrated that AtTDX is unique within the A. thaliana genome (not shown), data confirmed by the recent completion of the sequence of this plant genome. Northern assays showed that AtTDX is ubiquitously expressed but at low levels in A. thaliana tissues (data not shown). To get more insight on the occurrence of TDX-encoding genes in plants, we also sequenced a cDNA from tobacco encoding an AtTDX homologue (NtTDX, gift of N. Chaubet). The AtTDX and NtTDX proteins share more than 65% of identical residues (Fig. 1C). These observations confirmed that AtTDX is a real plant protein and does not arise from the fusion of two unrelated sequences during the cDNA library construction. AtTDX is located on chromosome 3 (BAC MEB5, accession number AB019230) but is erroneously annotated, each domain being considered as an independent gene.
The amino-terminal part of AtTDX (from amino acids 1-256), which contains the three TPR repeats, is also highly similar to the A. thaliana HIP co-chaperone (Fig. 1C). Moreover, the TPR repeats found within the AtTDX protein exhibit typical features of most of the TPR-containing proteins. A phylogenic analysis of some TPR-containing protein families from A. thaliana clearly shows that these proteins are grouped according to their cellular function and that the TDX family is closely related to the HIP family (Fig. 1D). Interestingly, AtHIP is positioned within the TDX group. On a separate Darwin tree, thioredoxin domains of AtTDX and NtTDX form a new family, closely related to the one containing the cytosolic thioredoxin h (data not shown).
The Thioredoxin Domain of AtTDX Is a Functional Proteindisulfide Reductase--The functional complementation of the yeast trx1⌬, trx2⌬ double mutant strongly suggested that At-TDX exhibits thioredoxin activity in vivo. Indeed, AtTDX expression not only relieved the methionine auxotrophy of trx1⌬, trx2⌬ cells but also allowed them to reduce methionine sulfoxide ( Fig. 2A). To assess further the possibility that AtTDX was indeed a disulfide reductase, the AtTDX protein was expressed in E. coli as a fusion to a polyhistidine tag, and the recombinant protein was purified by Ni 2ϩ affinity chromatography. Recombinant AtTDX was assayed in vitro for thioredoxin activity by measuring its ability to catalyze the reduction of insulin disulfide bridges (31). Moreover, to determine whether thioredoxin activity could be restricted to the AtTDX carboxyl-terminal domain, we purified a truncated form of AtTDX containing only the last 110 amino acids (AtTDX⌬-(1-269)). As shown in Fig.  2B, a rapid induction of insulin reduction is observed in the presence of 5 M recombinant AtTDX. The in vitro insulin reduction activity displayed by AtTDX is equivalent to the activity displayed by AtTRX3, a thioredoxin previously shown to exhibit high TRX activity (35). Moreover, the assays revealed that the truncated derivative AtTDX⌬-(1-269) was also able to reduce insulin. Therefore, this experiment demonstrated that AtTDX possesses disulfide reductase activity in vitro due to the thioredoxin domain found at its carboxyl-terminal part.
AtTDX Interacts with the Yeast Ssb2 Hsp70 Chaperone --To gain more insights on the function of the AtTDX protein, we next tried to understand what could be the role of its aminoterminal TPR-containing domain. Most TPR-containing proteins have been shown to be associated with large protein complexes through their TPR motifs (36,37). We thus tested whether proteins capable of interacting with the amino-termi-nal domain of AtTDX could be identified in a two-hybrid screen. Indeed, heterologous functional assays in S. cerevisiae have already been successfully used to characterize the functional specificity of thioredoxins from plants (34). Moreover, the success in the functional complementation of the trx1⌬, trx2⌬ mutant cells by AtTDX cDNA (leading to its own characterization, this work) suggested that AtTDX interacts with yeast proteins. We therefore reasoned that a similar two-hybrid approach using a yeast cDNA library could be applied for deciphering the function of AtTDX, to determine whether yeast proteins capable of specifically interacting with AtTDX could be identified. A first experiment was first performed using as the bait the first 269 amino acids of AtTDX (AtTDX⌬-(270 -380)) fused to the Gal4 DNA-binding domain, against a yeast cDNA library as a heterologous two-hybrid screening assay. From a screen of 1 ϫ 10 6 independent transformants, 14 histidine, aminotriazole-resistant prototroph colonies were selected. Plasmid DNAs were extracted from the 14 selected colonies and used to retransform the reporter strain. Only one positive clone displaying a strong positive Hisϩ phenotype was recovered, and the corresponding plasmid DNA was subsequently analyzed. The sequencing of this plasmid revealed an open reading frame of 573 amino acids fused in-frame with the Gal4 activation domain. Data base searching showed that the cloned DNA fragment encodes the yeast Ssb2, a member of the heat-shock protein 70 family protein (38). Because the isolated DNA lacked the last 40 amino acids of Ssb2, we amplified the full-length SSB2 nucleotide sequence by PCR with yeast genomic DNA, and we tested the interaction of the entire Ssb2 with both AtTDX and AtTDX⌬-(270 -380). As shown in Fig. 3, strong interactions were visualized between the full-length Ssb2 and either AtTDX or AtTDX⌬-(270 -380).
The interaction between AtTDX⌬-(270 -380) and Ssb2 was next assayed by co-immunoprecipitation experiments. Wildtype yeast cells (W303-1A) were co-transformed with plasmids expressing a hemagglutinin (HA) epitope-tagged AtTDX and a glutathione S-transferase (GST)-tagged Ssb2 (Fig. 4A). Proteins of the resulting transformants were extracted and immunoprecipitated with either anti-GST (Fig. 4B) or anti-HA (Fig.  4C) antibodies. Immunoprecipitated proteins were next analyzed by immunoblotting with either anti-GST or anti-HA antibodies. In both cases, co-immunoprecipitation assays revealed a specific and strong interaction between AtTDX and Ssb2. Identical results were obtained when the truncated AtTDX⌬-(270 -380) protein was used (data not shown).
We next tested the direct physical interaction between At-TDX and Ssb2, using an in vitro affinity procedure. For this purpose, both His-tagged AtTDX and GST-tagged Ssb2 proteins were produced in E. coli BL21 cells and purified, and formation of a protein complex between the two recombinant FIG. 2. AtTDX possesses disulfide reductase activity. A, AtTDX is able to complement the trx1⌬,trx2⌬ mutant. Cells expressing AtTDX and AtTRX3 from the pGK1 promoter were grown to a density of 10 7 cells per ml and plated on galactose agar-containing medium at A 600 ϭ 0.2 in the presence or absence of methionine (ϩMet, ϪMet) or in the presence of methionine sulfoxide (ϩMetSO) as sole source of sulfur. Plates were incubated 6 days at 30°C. B, both AtTDX and AtTDX⌬-(1-269) are capable of in vitro disulfide reduction of bovine insulin. The thioredoxin activity was measured as turbidity at 650 nm due to insulin precipitation by the addition of the proteins (5 M each) at 25°C. Thioredoxin AtTRX3 was used in this assay as a positive control, whereas bovine serum albumin and dithiothreitol alone are negative controls.
FIG. 3. AtTDX interacts with the yeast Hsp70 Ssb2 in the yeast two-hybrid system. Left panel represents minimal medium containing histidine (ϩHis) such that interaction is not required for growth (control plate), and right panel is His-free but contains 20 mM 3-aminotriazole (3AT), such that interaction is required for YRG2 cell growth (test plate). Four independent transformed cells were tested. proteins was assayed by monitoring their retention on a Ni 2ϩ column. Following incubation of the two proteins, Ssb2 was specifically retained on the column (Fig. 4D), confirming the results of the two-hybrid screen.
AtTDX Interacts with the ATPase Domain of Ssb2 via Its TPR Domain--To determine which region of AtTDX mediates its binding to Ssb2, we constructed a series of deleted derivatives in the pBDGal4 vector, and the resulting constructions were tested for interaction with Ssb2. As reported above, the thioredoxin domain of AtTDX is not required for its interaction with Ssb2. Further dissection of the remaining protein indicated that the TPR domain located between residues 112 and 213 (Fig. 5A) is necessary for the interaction between AtTDX and Ssb2.
Likewise, to characterize which domains of Ssb2 were necessary for its interaction with AtTDX, we next generated deleted derivatives of SSB2 that were expressed as fusions to the Gal4 activation domain. Two-hybrid assays revealed that the deletion of either the central peptide binding domain (Ssb2⌬-(387-613)) or the 10-kDa carboxyl-terminal domain (Ssb2⌬-(550 -613)) of Ssb2 did not affect the Ssb2-AtTDX interaction (Fig. 5B). In contrast, a derivative of Ssb2 lacking the ATPase domain (Ssb2⌬-(1-386)) was no longer capable of interacting with AtTDX.
The Interaction between AtTDX and Ssb2 Is Sensitive to Oxidative Stress-Eukaryotic cells continuously produce ROS from multiple sources. Both thioredoxin and Hsp protein families are among the cellular enzymes that protect cells against damage induced by ROS. Because AtTDX and Ssb2 each belong to one of these families, we wondered whether the AtTDX-Ssb2 interaction that we observed might be regulated in response to an oxidative stress. To test such an hypothesis, we first decided to analyze the effect of oxygen peroxide (H 2 O 2 ) that is known to induce an oxidative stress in yeast cells (39). Second, we decided to perform again a two-hybrid assay to test the effect of H 2 O 2 , because the yeast two-hybrid system involving Gal4 has been shown to be adapted to the study of protein interactions under oxidative stress (40).
Serial dilutions of cells co-expressing Ssb2 fused to the Gal4 activation domain and AtTDX fused to the Gal4 DNA-binding domain were plated on minimal medium containing increasing amounts of H 2 O 2 , in the presence and absence of histidine. In the presence of histidine, the addition of H 2 O 2 up to 1.5 mM on histidine-containing plates impaired cell growth only very weakly (Fig. 6A, upper panels). In the absence of histidine, cells were able to grow in the presence of H 2 O 2 at concentrations as low as 0.5 mM, confirming that Ssb2 and AtTDX interact each other. However, in a striking contrast, the addition of higher concentrations of H 2 O 2 severely prevented cell growth on plates lacking histidine, no growth being observed as soon as 1 mM H 2 O 2 was added to the medium (Fig. 6A, lower panels). A similar result was obtained when the thiol-oxidizing agent diethyl maleate was used (data not shown). These results therefore suggested that in the presence of oxidizing compounds, the Ssb2-AtTDX interaction is abolished.
We next tried to decipher whether the thioredoxin domain of AtTDX might be involved in the above reported regulation of Ssb2-AtTDX interaction. We therefore used the AtTDX derivative lacking the thioredoxin domain (AtTDX⌬-(270 -380)) in place of the full-length AtTDX in the two-hybrid assays, and we tested the transformed cells for their sensitivity to H 2 O 2 . Results of the experiments showed that, in this case, the addition of H 2 O 2 only weakly impaired the growth of the cells, in both the presence and absence of histidine (Fig. 6B). Thus, the thioredoxin domain of AtTDX appears to be involved in the bind to the target protein (41). A second mutant was generated by replacing the second cysteine (Cys-307) of the thioredoxin domain active site by a serine residue (AtTDX Ser307 ). In this case, the first step of the reduction of the target by thioredoxin occurs while the second step is abolished, therefore leading to a covalent link between thioredoxin and its target protein, with thioredoxin being unable to be released from the intermediate complex (42). Both AtTDX mutant alleles were cloned in-frame downstream from the Gal4 DNA-binding domain and tested by two-hybrid assays for their interaction with Ssb2 under H 2 O 2 regulation. The results shown in Fig. 7A clearly demonstrated that, unlike what was observed with the wild-type AtTDX, the addition of 1 mM H 2 O 2 had no effect on the cells expressing either AtTDX Ser302 or AtTDX Ser307 fused to the Gal4 DNAbinding domain and grown in the absence of histidine. Thus, in two-hybrid assays, each of the two mutations mimics the behavior of the large deletion ⌬-(270 -380) that removed the entire thioredoxin domain from AtTDX. Taken together, these results demonstrated that the thioredoxin active site of AtTDX is directly responsible for the H 2 O 2 sensitivity of the Ssb2-AtTDX interaction and suggest that, in response to oxidative stress, the complex formed between AtTDX and Ssb2 is disrupted via the reducing power of the thioredoxin domain of AtTDX.
The Conserved Cys-20 in Ssb2 ATPase Domain Is Involved in the Release of AtTDX/Ssb2 Interactants--The disulfide reductase activity of the thioredoxin domain of AtTDX and the involvement of the thioredoxin active site in the H 2 O 2 -mediated regulation of AtTDX-Ssb2 interaction prompted us to investigate whether a cysteine residue within the Ssb2 protein would be targeted by the thioredoxin active site under oxidative stress. The Ssb2 protein contains only two cysteines, one located in the ATPase domain at position 20 (Cys-20) and the second in the peptide-binding domain at position 455. We noticed that only Cys-20 of the ATPase domain is highly conserved among Ssb2 homologues from eukaryotes. To test whether the H 2 O 2 regulation of the Ssb2-AtTDX interaction might rely on this residue, we constructed a mutated version of Ssb2 in which Cys-20 was replaced by a serine residue (Ssb2 Ser20 ). This new Ssb2 allele was cloned in-frame downstream from the activation domain of Gal4, and the resulting construction was used in two-hybrid experiments performed as described above. We first checked that the mutation did not affect the interaction between Ssb2 Ser20 and AtTDX in the absence of H 2 O 2 (data not shown). Next we observed that the Ssb2 Ser20 -AtTDX interaction was resistant to the presence of H 2 O 2 (Fig. 7B), in contrast to the interaction between the wild-type Ssb2 and AtTDX. These results therefore indicate that the oxidation of Cys-20 of Ssb2 is necessary to release the Ssb2-AtTDX interaction under oxidative stress. DISCUSSION During the last 10 years, an increasing number of genes encoding members of the thioredoxin family have been reported in all prokaryotic and eukaryotic organisms. In plants, the complexity of the thioredoxin reductase/thioredoxin systems was first shown by the sequencing of A. thaliana expressed sequence tags that demonstrated the presence of at least five thioredoxin h-encoding genes in this organism (35). Today, more than 18 thioredoxin and thioredoxin-like sequences have been found in A. thaliana (43). Here, we report the cloning and the characterization of AtTDX, a novel and striking member of the thioredoxin family from A. thaliana. AtTDX is the first member of the thioredoxin family described to date that possesses an extra domain with tetratricopeptide repeats. This domain interacts strongly with the ATPase domain of Ssb2, a member of the Hsp70 family. Moreover, the thioredoxin active site of AtTDX and a conserved cysteine residue within the ATPase domain of Ssb2 were shown to mediate the release of the AtTDX-Ssb2 interaction under oxidative stress conditions.
AtTDX, a New Thioredoxin-like TPR-containing Protein-By using yeast mutant cells that do not express endogenous thioredoxin, we have isolated by functional complementation At-TDX, a novel thioredoxin containing protein from A. thaliana. The AtTDX protein displays an interesting bipartite structure encompassing both a thioredoxin domain and a TPR repeat containing domain is highly similar to the co-chaperone Hip protein. Both in vitro and in vivo assays demonstrated that AtTDX has disulfide reductase activity and therefore that At-TDX could be assigned to the thioredoxin superfamily. In both prokaryotic and eukaryotic cells, several proteins containing a thioredoxin catalytic domain fused to an unrelated extra domain have been described (43). However, the function of the associated domain has been established in two instances only, the human cytosolic PICOT, a 37-kDa protein kinase C-interacting protein with amino-terminal thioredoxin domain (44), and the plant APS reductases, three 50-kDa exhibiting a carboxyl-terminal thioredoxin domain (45). The functional characterization of such proteins was of particular interest because it demonstrated coordinated functions between each domain.
Among the members of the thioredoxin family, AtTDX is the first described as comprising TPR repeats associated to the thioredoxin domain. TPRs are 34-amino acid motifs that were originally identified in yeast (46,47) and then found in a large number of both prokaryotic and eukaryotic proteins (36). TPR repeats mediate protein-protein interactions. TPR-containing proteins play diverse roles in many cellular processes like cell cycle regulation, transcriptional repression, heat shock response, protein kinase inhibition, and peroxisomal protein transport (36). It is noteworthy that the TPR domain of AtTDX is more particularly related to TPR proteins known to interact with members of the heat-shock protein family. In particular, we found that the TPR repeats of AtTDX are highly related to those of Hip and Sti1p (the yeast Hop counterpart), with Hip being a co-chaperone that specifically binds to Hsp70 and Hop providing a physical link between Hsp70 and Hsp90 (48). In addition, AtTDX displays a significant degree of similarity with TPR domains of protein phosphatase 5, cyclophilin CyP40, and FKBP52, all factors known to interact with Hsp90 chaperones (11).
AtTDX Is Capable of Interacting with a Yeast Hsp70 Chaperone-The homology of AtTDX with HIP suggested that At-TDX could interact with heat-shock proteins through its TPR repeats. By using a heterologous two-hybrid strategy, we indeed isolated one yeast protein capable of interacting with AtTDX, the Ssb2 protein, a member of the yeast Hsp70 family (38).
Interaction domain mapping experiments favor the hypothesis that the AtTDX-Ssb2 interaction is specific and does not result from the targeting of AtTDX by Ssb2 in response to overproduction and/or misfolding. Indeed, we show here that the ATPase domain of Ssb2 mediates its interaction with At-TDX, whereas it is well established that misfolded proteins are selectively recognized by the peptide-binding domain of Hsp70 chaperones (13). Furthermore, we show that a single cysteine replacement within the thioredoxin active site of AtTDX strongly enhances the AtTDX-Ssb2 interaction, whereas it was shown that such a mutation has a very limited impact on thioredoxin structure (49,50). Finally, we searched for Hsp70 genes in A. thaliana data bases and found that the AtHsc70-1 gene (51) encodes the closest A. thaliana homologue of Ssb2. The two proteins Ssb2 and AtHsc70-1 share 53% identical residues (68% similarity). We cloned the AtHsc70-1 gene into the pADGal4 vector and succeeded in obtaining a two-hybrid interaction with AtTDX, even weaker than that involving Ssb2 (not shown). These results, considered as a whole, are strong evidence that AtTDX is a new Hsp70 interactant.
Surprisingly, we did not isolate any clone encoding Hsp70 proteins from the Ssa family in our two-hybrid screens. Ssa and Ssb share 65% identity (more than 72% of similarity) in their ATPase domain, which is precisely the region of Ssb2 that interacts with AtTDX. In our screens, only positive clones strongly interacting with AtTDX were selected, and one can imagine that proteins interacting with AtTDX with a low specificity were not retained. We also did not isolate clones encoding members of the Hsp90 proteins, because of the high degree of similarity between the TPR repeats of AtTDX and the Hop/ Sti1 proteins. The absence of such proteins among the positives clones suggests that AtTDX does not act as a physical link between Hsp70 and Hsp90 proteins, as Hop does (48).
The fact that AtTDX interacts with Ssb2 and with an A. thaliana Ssb homologue raises the question as to how the AtTDX function correlates with Hsp70 chaperone activity. Ssb proteins are ribosome-associated chaperones that interact with nascent chain and likely play an important role in early protein folding events (38,52). Several partner proteins that regulate Hsp70 function as a molecular chaperone have already been identified, including positive (Hsp40 and HIP) and negative (CHIP and Bag-1) regulators (18,(21)(22)(23). HIP and Bag-1, like AtTDX, interact with the Hsp70 ATPase domain (21,54) and functionally compete in regulating the in vivo chaperone activity of Hsp70 (55), within the cycle between the ADP-and ATP-bound states. Interestingly, AtTDX and AtHIP are highly homologous in their amino-terminal interacting TPR region. Because both are expressed in A. thaliana, one could imagine that they sequentially interact with some common Hsp70 at different times and/or in different circumstances during the plant life. The next step in understanding AtTDX function will be to investigate in which of the ATP/ADP states Ssb2 interacts with AtTDX and whether AtTDX binds to Ssb2 by displacing Hsp70 cofactors or by synergy with other co-chaperones. This could bring new elements to determine whether AtTDX as a new Hsp70 co-chaperone.
A Redox Switch Dissociates the AtTDX-Hsp70 Complex-One major result from our two-hybrid experiments indicated that the AtTDX-Ssb2 interaction is specifically released upon oxidative stress. Both the removal of the thioredoxin domain from AtTDX and the replacement of one cysteine from the thioredoxin active site by a serine residue renders the AtTDX-Ssb2 interaction insensitive to oxidative stress. This is the demonstration that the thioredoxin domain of AtTDX is directly involved in the oxidative process that dissociates AtTDX from Ssb2. Furthermore, we showed that Cys-20 located in the ATPase domain of Ssb2 is not necessary for AtTDX-Ssb2 complex formation but is mandatory for its dissociation. Taken altogether, our results demonstrate that a redox switch governs the association/dissociation of AtTDX-Ssb2 complex.
Oxidizing conditions like exposure to H 2 O 2 are known to cause disulfide bonds to form and chaperone function to be turned on. Proteins from distinct Hsp families were also shown to be affected in their activity and/or conformation by the redox status. The murine small Hsp25 that carries a single Cys residue equilibrates between reduced protein and protein dimer, depending on the oxidoreduction conditions (56). The E. coli Hsp33 has been described recently (57) as a chaperone with an on-off mode of activity that uses reactive disulfide bonds as molecular switches. Exposure to hydrogen peroxide causes zinc to be released from Hsp33 conserved cysteines and disulfide bond formation and the chaperone function to be turned on. Another example is given by Tsai et al. (58) that showed that the human protein-disulfide isomerase is capable of redox-driven chaperone activity. All these examples are consistent with our results that show an in vivo redox regulation of a member of the Hsp70 family by a thioredoxin.
Recently, Hsp70 chaperoning activity was shown to be increased by environments that mimics oxidative stress (59). Because association with peptides under oxidative conditions is not reversible by reducing agents, it has been proposed that other chaperone-associated factors are required for substrate release. AtTDX may be one of these chaperone-associated factors, acting as an oxidative stress detector through its disulfide reductase activity. In the case of AtTDX/Ssb2 dissociation, involvement of AtTDX disulfide reductase activity and oxidation of Ssb2 cysteine 20 are two mandatory events demonstrated by the present work. At present, the exact function of Ssb2 Cys-20 under normal conditions and oxidative stress remains to be demonstrated. A survey of all Hsp70 of bacteria, fungi, animals, and plants shows the presence of a conserved cysteine in the position homologue to that of Ssb2 Cys-20. This suggests that the mechanism of a redox-dependent release is conserved. Moreover, analysis of the Ssb2 sequence with Swiss-PDB-Viewer and Rasmol programs proposed that Cys-20 is located in a small convex cavity near the protein surface and is thus easily accessible for disulfide bond formation/oxidation. These findings suggest that Ssb2 Cys-20 may be engaged in a disulfide bond with a third partner, which could be another Hsp70 molecule in a self-association (53) or another protein with conserved cysteine. How thioredoxin activity can affect, directly or indirectly, the redox state of Ssb2 Cys-20 under oxidative stress remains unsolved. Several hypotheses exist that require further experiments, including fine analysis of determinants and other partners involved in the interaction.