Multistep Disulfide Bond Formation in Yap1 Is Required for Sensing and Transduction of H2O2 Stress Signal

Molecular Cell

Article Multistep Disulfide Bond Formation in Yap1 Is Required for Sensing and Transduction of H2O2 Stress Signal Shoko Okazaki,1 Tsuyoshi Tachibana,1 Akira Naganuma,1 Nariyasu Mano,2 and Shusuke Kuge1,* 1

Laboratory of Molecular and Biochemical Toxicology Laboratory of Bioanalytical Chemistry Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.06.035 2

SUMMARY

Redox reactions involving cysteine thiol-disulfide exchange are crucial for sensing intracellular levels of H2O2. However, oxidationsensitive dithiols are also sensitive to intracellular reducing agents, and disulfide bonds are thus transient. The yeast transcription factor Yap1 is activated by disulfide-induced structural changes in the nuclear export signal in a carboxy-terminal domain. We show herein that the activation of Yap1 by H2O2 requires multistep formation of disulfide bonds. One disulfide bond forms within 15 s in an amino-terminal domain, and then disulfide bonds linking the two domains accumulate. The multiple interdomain disulfide bonds, which result in reductionresistant Yap1, are required for transduction of the H2O2 stress signal to induce the appropriate level and duration of specific transcription. Our results suggest both a mechanism wherein the H2O2 levels might be sensed by Yap1 and the way in which the NADPH levels might be maintained by altering the redox status of Yap1. INTRODUCTION Oxygen serves as an electron acceptor for the efficient production of energy. However, oxygen is converted to harmful reactive oxygen species (ROS) that include hydrogen peroxide (H2O2) and superoxide, which can damage cellular macromolecules. ROS can also act as cellular signals in response to growth factors, cytokines, and stress signals (Rhee, 2006). Thus, organisms have acquired systems for sensing levels of ROS and transducing the resultant signals to protect cells from the toxicity of ROS or determine the cell fate—either survival and proliferation or death. Superoxide generated by mitochondrial respiration and various NADPH oxidases can be converted rapidly to H2O2 by the abundant enzyme superoxide dismutase

(Halliwell and Gutteridge, 1999). H2O2 is relatively stable, and its amphiphilic properties allow it to permeate membranes. Cells control levels of ROS by exploiting the glutathione reduction-oxidation (redox) cycle and thioredoxin system, in which electrons are accepted from NADPH, with subsequent reduction of H2O2 to water. Proteins in the peroxiredoxin (Prx) family, acting in concert with a redox cycle that consists of thioredoxin, thioredoxin reductase, and NADPH (Trx/Trr/NADPH), reduce H2O2 directly (Figure 1C). Glutathione is used as the donor of electrons for the reduction of H2O2 by glutathione peroxidase. These systems play essential roles in maintaining the redox status of cells and sensing intracellular levels of ROS (Carmel-Harel and Storz, 2000). In bacteria, sensing of H2O2 involves transcription factor OxyR (Zheng et al., 1998; Aslund et al., 1999), whereas transcription factor Yap1 fulfills this role in budding yeast (Kuge and Jones, 1994; Morgan et al., 1997; Kuge et al., 1997, 1998, 2001; Delaunay et al., 2000, 2002). In response to H2O2, Yap1 and OxyR activate the transcription of genes for enzymes that mitigate oxidative stress. OxyR reacts directly with H2O2 to form sulfenic acid at Cys199, which rapidly forms a disulfide bond with Cys208, resulting in activation of the expression of the target genes. OxyR is rapidly deactivated by reduction of the disulfide bond by a glutathione-dependent mechanism (Zheng et al., 1998; Aslund et al., 1999; Tao, 1999; Lee et al., 2004). Thiol-disulfide exchange reaction is also critical to the regulation of Yap1 transcription factors in yeast. Yap1 is mainly localized in the cytoplasm under nonstress conditions. The cytoplasmic localization is determined by constitutive nuclear export that predominates over constitutive nuclear import (Kuge et al., 1997, 1998; Yan et al., 1998; Isoyama et al., 2001). In response to H2O2 stress, a disulfide bond is formed in Yap1 with resultant conformation of the nuclear export signal (NES) of Yap1, which is embedded in the carboxy-terminal cysteine-rich domain (c-CRD; Figure 1B), altered (Wood et al., 2004). This can result in inhibition of binding to the nuclear export receptor Crm1 and subsequent nuclear accumulation of Yap1 (Kuge et al., 1998; Yan et al., 1998). In contrast with the way OxyR is oxidized, formation of the disulfide bond in Yap1 is not induced by direct reaction of Yap1

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Figure 1. Establishment of a System for Oxidation of Yap1 In Vitro (A) Formation of disulfide bonds between cysteine residues in Yap1 (Yap1 [red]) is mediated by Gpx3, which has Trx-dependent peroxidase activity (Toledano et al., 2004; Delaunay et al., 2002). Cys36 of Gpx3 (also known as Orp1) is first oxidized to sulphinic acid (SOH) by H2O2, and a disulfide bond forms between Cys36 of Gpx3 and Cys598 of Yap1. A thionilate anion formed at Cys303 of Yap1 attacks the disulfide bond, and then an intramolecular disulfide bond (Cys303-Cys598) is formed in Yap1, as indicated (Yap1 [ox]). This model is based on the finding that a disulfide complex is formed between Yap1 and Gpx3 as an intermediate. (B) The six cysteine residues with the residue numbers in Yap1 in the two cysteine-rich domains (n-CRD and c-CRD) and two previously proposed disulfide bonds (bars linking two cysteine residues) in Yap1 are indicated. The regulatory domain of Yap1 (amino acid residues 122–650) is sufficient for the regulated nuclear localization of Yap1 (data not shown). We refer to this regulatory domain as ‘‘Yap1’’ in the in vitro studies described in the present report. The cysteine residues in the mutant derivatives were mutated to threonine (T) or alanine (A) as indicated. (C) A Prx (Gpx3 or Tsa1 in this study) coupled with Trx, Trr, and NADPH (Trx/Trr/NADPH system) can reduce H2O2 using high-energy electrons from NADPH. (D–F) Appearance of a high-mobility oxidized form of Yap1 was accelerated by the Gpx3-redox cycle in response to H2O2. Oxidation of Yap1 was monitored by nonreducing SDS-PAGE. Yap1 was oxidized only in the presence of H2O2 (D). The same assay was performed in the presence of Trx/Trr/NADPH (E). Yap1 was oxidized in the Trx/Trr/NADPH system in the presence of Gpx3 (F). (F) also shows reduced and oxidized forms of Gpx3 in the same reaction as that for which results for Yap1 are shown. The time (min) after addition of H2O2 is indicated. — indicates that Yap1 was included in the reaction mixture, but H2O2 was not. In this case, incubation was continued for 30 min (D) or 45 min (E and F). Reactions were stopped by the addition of IAA. Schematic representations of the reduced form of Yap1 (red) and the rapidly migrating form of Yap1 (oxII), which has a disulfide bond between n-CRD and c-CRD (Delaunay et al., 2000), are shown. The oxidized rapidly migrating form of Gpx3 (Gpx3 [ox]) and the reduced form (Gpx3 [red]) are indicated. When the rapidly migrating Yap1 was reduced by DTT, it migrated to the reduced Yap1 in a similar manner (data not shown; see below).

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cysteines to H2O2 but requires Gpx3 (glutathione peroxidase-like protein but has thioredoxin-dependent peroxidase activity; also called Orp1), which acts as a receptor for H2O2 (Delaunay et al., 2002; Figure 1A). Furthermore, OxyR exploits a single globular domain for redox sensing of H2O2, whereas Yap1 requires, for unknown reasons, formation of a disulfide bond between the two distant domains of Yap1 (c-CRD and another cysteine-rich domain in the amino-terminal region, n-CRD) for appropriate regulation in response to H2O2 stress (Coleman et al., 1999; Delaunay et al., 2002). In addition, air-oxidized Yap1 contains two disulfide bonds between n-CRD and c-CRD (Wood et al., 2003, 2004; Figure 1B), but the role of redox regulation of the cysteine residues in the two CRDs in cells that are responding to H2O2 remains to be elucidated. The rapid responses of Yap1 and OxyR to H2O2 might be mediated by kinetic mechanisms that are promoted by specificity of OxyR and Gpx3 to H2O2 (Danon, 2002; Toledano et al., 2004). However, the durations of the transcriptional active form of OxyR and Yap1 are significantly different. The transcriptional activity of OxyR in response to H2O2 is short-lived (0.5–5 min; Aslund et al. [1999]), whereas Yap1-dependent transcriptional activity persists for more than 1 hr (Delaunay et al., 2000; Kuge et al., 2001). The present study was designed to resolve the outstanding question of how the redox reactions of Yap1 affect the role of this protein in redox sensing and long-lived signal transduction. We developed a system for monitoring the oxidation of cysteine residues in Yap1 in response to H2O2 in vitro. We identified each disulfide bond according to three criteria. First, we determined interdomain disulfide bonds between n-CRD and c-CRD by mobility enhancement due to conformational constraints of the SDS-denatured polypeptide chain of Yap1. Second, intradomain disulfide bonds were detected by altered degree of mobility retardation attributed to thiol-alkylation with two maleimide derivatives. Third, mass spectrometry was used. We identified four possible oxidized forms of Yap1, which included three different active forms of Yap1 with interdomain (n-CRD and c-CRD) disulfide bonds. We also monitored the oxidation process and mutant derivatives of Yap1 in yeast. Our results suggest that the oxidation process of Yap1 and the number of interdomain disulfide bonds control the level of the active form of Yap1 in cells that are responding to H2O2.

RESULTS

Yap1 (Figure 1B): a Gpx3-induced disulfide bond between Cys303 and Cys598, which connects two distant domains (Delaunay et al., 2000), and the disulfide bond between Cys310 and Cys629, which is also formed in air (Wood et al., 2003). To study the involvement of the three cysteine residues in both n-CRD and c-CRD (Figure 1B) in the sensing of H2O2, we developed a system for monitoring the redox reaction of Yap1 in vitro. Because Gpx3-mediated disulfide bond formation in Yap1 in cells occurs under the Trx system, which can act as a reduction system for both Gpx3 and Yap1, we set up a Trx reduction system in vitro. This system consisted of Trx2, Trr1, and NADPH, in which electrons were transferred from NADPH to H2O2 by the redox reaction of Trx, as indicated in Figure 1C. We found that our Trx redox system was functional by monitoring the consumption of NADPH after the addition of H2O2 in the presence of Gpx3 and yeast Prx (Tsa1; see Figure S1 in the Supplemental Data available with this article online) as previously reported (Delaunay et al., 2002). The intramolecular disulfide bond between n-CRD (Cys303) and c-CRD (Cys598) resulted in enhanced mobility that was due to conformational constraints of the SDSdenatured polypeptide chain of Yap1 (Delaunay et al., 2000). It is possible that multiple disulfide bonds between n-CRD and c-CRD might result in the similar mobility enhancement of Yap1. We refer to such oxidized forms of Yap1 with the interdomain disulfide bonds defined by this principle as Yap1 (oxII). Yap1 (oxII) was observed time dependently when Yap1 was treated with H2O2 alone (Figure 1D). In contrast, only a small amount of Yap1 (oxII) was detected 45 min after the addition of H2O2, in the same assay, in the presence of Trx/Trr/NADPH (Figure 1E). Thus, although Yap1 was oxidized directly by H2O2, the reduction of Yap1 (oxII) by Trx was more rapid than the direct oxidation of Yap1 by H2O2. In a reaction mixture containing Trx/Trr/NADPH and Gpx3 (Figure 1F), Yap1 (oxII) appeared within 0.5 min, and almost all Yap1 was found in the Yap1 (oxII) form after 10 min in response to H2O2 (Figure 1F, upper panel), whereas most but not all of the Gpx3 was oxidized (Figure 1F, lower panel). Consistent with the results obtained in yeast cells (Delaunay et al., 2002), Gpx3 might accelerate the oxidation of Yap1 in the presence of Trx/Trr/NADPH. Thus, these results suggest that this in vitro system might represent the kinetics of the early disulfide bond formation in Yap1 in cells responding to H2O2. Note that Yap1-binding protein (Ybp1), which is essential for the formation of oxidized Yap1 in cells (Veal et al., 2003; Okazaki et al., 2005), was not required in our in vitro system.

Development of a System for Monitoring Oxidation of Yap1 In Vitro As indicated in Figure 1A, it has been proposed that the transcriptional activity of Yap1 is determined by a balance between Gpx3-mediated formation of a disulfide bond (Delaunay et al., 2002) and reduction of the disulfide bond by thioredoxin (Trx; Izawa et al. [1999]). Previous reports indicated that two disulfide bonds are formed in

Formation of a Disulfide Bond within n-CRD of Yap1 We used our kinetics-based in vitro system for further analysis of disulfide bond formation in Yap1. To elucidate the oxidation process of Yap1, it is crucial to examine the redox status of all the six-cysteine residues in Yap1. First, we examined whether the cysteine residues are in free thiol forms during the early stage of formation of Yap1 (oxII) (15 s to 15 min) by monitoring mobility retardation

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due to thiol-alkylation with 4-acetamido-40 -maleimidylstilbene-2,20 -disulphonic acid (AMS) (approximate MW, 0.5K). The mobility of reduced Yap1 (Yap1 [red]) was decreased upon treatment with AMS (compare ‘‘NEM 0 min’’ with ‘‘AMS 0 min’’ of upper panel in Figure 2A). As shown in Figure 2A, a putative oxidized form of Yap1 appeared from 15 s (0.25 min) after the addition of H2O2, in addition to the Yap1 (oxII) observed in Figure 1F. We refer to the oxidized form that was detected by an altered degree of mobility retardation by AMS (but not by the first principle) as Yap1 (oxI), in which a putative intradomain disulfide bond is likely to form (Figure 2A). The level of Yap1 (oxI) decreased as that of Yap1 (oxII) increased (5–15 min after the addition of H2O2), and no oxidized species of Yap1 was detected in the absence of Gpx3 (Figure 2B). Most of the Gpx3 was oxidized within 15 s (0.25 min), and residual reduced Gpx3 (Figure 2A, lower panel) and NADPH (see Figure 2C; 0–10 min after the addition of H2O2) decreased in a time-dependent manner. These results suggest that the repetitive redox cycling of Gpx3 might have been required for the efficient formation of Yap1 (oxI) and Yap1 (oxII). An additional form of Yap1 (oxII) with further enhanced mobility (approximately 60 kDa) was found from 5 to 15 min after the addition of H2O2 (Figure 2A upper panel; see below for further details). To characterize the covalent structure of Yap1 (oxI), we subjected lysyl endopeptidase-digested Yap1 (oxI) to MALDI-TOF mass analysis. We detected a peak that corresponded to a peptide fragment that contained Cys310 and Cys315 (peptide number 316–327) minus two protons (m/z 1573; Figure 2D). When Yap1 (oxI) was acetylated after reduction by DTT, this peak decreased markedly. A new peak (m/z 1689) appeared, which corresponded to a peptide fragment that contained acetylated Cys310 and Cys315 (Figure 2E). A peak of 1689 (m/z), but not of 1573 (m/z), was observed in the analysis of Yap1 (red) (see Figure S2). We also observed the peak of 1573 (m/z) in Yap1 (oxII). Therefore, we concluded that a disul-

fide bond between Cys310 and Cys315 (disulfide bond 1) was present in Yap1 (oxI) and Yap1 (oxII). We failed to detect peaks corresponding to a disulfide bond between the n-CRD and c-CRD peptides from Yap1 (oxII), perhaps because of the loss of peaks with the expected molecular mass due to impurities in Yap1 eluted from the acrylamide gel and/or H2O2-induced nonspecific oxidation of Yap1 polypeptides. To confirm the formation of Yap1 (oxI) and Yap1 (oxII) inside cells, we monitored Yap1 using AMS in cells treated with H2O2. We detected Yap1 (oxI) in cells treated with H2O2 for 10 s (0.17 min in Figure 2F) to 3 min. Yap1 (oxI) was then converted to Yap1 (oxII) within 5 min. These results suggest that the previously uncharacterized cysteine residues in Yap1 might also play a role in the activation process of Yap1. Multiple Oxidized Forms of Yap1 (oxII) We next examined whether the residual cysteines in Yap1 (oxI) and Yap1 (oxII) were in free thiol forms by monitoring mobility retardation due to another thiol-alkylation probe, polyethylene glycol (PEG)-linked maleimide (MW, 5 kDa). First, we reacted PEG-linked maleimide with Yap1 (six cysteine residues), Yap1CCT,CAC (four cysteine), Yap1CCC,TAT (three cysteine), Yap1CTT,CAT (two cysteine), and Yap1TTT,TAT (no cysteine) after reduction by DTT, for mobility controls of Yap1 with respective numbers of free thiol residues (Figures 2G and 2H). The mobility of Yap1 (oxI), which appeared from 15 s (0.25 min) to 1 min, was between that of Yap1, which reacted with three PEG (Yap1CCC,TAT), and that of Yap1, which reacted with four PEG (Yap1CCT,CAC; compare lanes 1 and 2 and lanes 6–8 in Figure 2G). However, after the addition of DTT to reduce disulfide bonds, the mobility of Yap1 (oxI) was equal to that of Yap1, which reacted with four PEG (Yap1CCT,CAC; compare lanes 2 and 7 in Figure 2H). Thus, we concluded that Yap1 (oxI) had four free thiols and one disulfide bond (Cys310-Cys315, designated as disulfide bond 1).

Figure 2. There Are Multiple Different Forms of Oxidized Yap1 (A) Free cysteine-SH groups were examined with the thiol-reactive probe AMS. The time-dependent oxidation (kinetics-based assay) in vitro of Yap1 in the Gpx3/Trx/Trr/NADPH redox system in response to H2O2 was monitored by using AMS. Yap1 (red), Yap1 (oxI), Yap1 (oxII), Gpx3 (red), Gpx3 (ox), Trx2 (red), and Trx2 (ox) are indicated by arrows. A thick arrow in the upper panel indicates putative Yap1 (oxII) (60K). (B) The same reaction was performed in the absence of Gpx3. (C) Levels of NADPH in the same reaction as in (A) were monitored at an absorbance of 340 nm with (+) and without () the addition of H2O2. (D and E) MALDI-TOF mass spectrometry of Yap1 (oxI). We purified Yap1 (oxI) that had been generated in the kinetics-based assay and analyzed nonreduced (D) and reduced (E) peptide fragments. (F) Yap1 was oxidized to two different forms in vivo. Yeast cells expressing an (HA)3-tagged version of full-length Yap1 were treated with H2O2 for the indicated times. Cell lysates were treated with AMS. (G) The kinetics-based assay in vitro, using PEG-maleimide to detect free thiols, showed three different oxidized forms of Yap1. As molecular weight controls, we treated recombinant Yap1CCC,TAT (lane 1), Yap1CCT,CAC (lane 2), Yap1 (lane 3), and Yap1TTT,TAT (lane 4) with PEG-maleimide after reduction by DTT. We performed the same kinetics-based assay, except that PEG-maleimide was used to alkylate free thiol groups. Bands corresponding to reduced Yap1, Yap1 (oxI), Yap1 (oxII-1), and Yap1 (oxII-2) are indicated as red, oxI, oxII-1, and oxII-2, respectively, with the corresponding diagrams. (H) Determination of the number of free thiol groups formed in Yap1 (oxII-1) and Yap1 (oxII-2) in kinetics-based assay in vitro. We used 20 mM DTT to reduce a Yap1 (oxI)-enriched fraction (lane 5; treatment with H2O2 for 30 s) and a Yap1 (oxII-1)- and Yap1 (oxII-2)-enriched fraction (lane 6; treatment with H2O2 for 30 min) and then fractioned the reaction mixtures by SDS-PAGE (lanes 7 and 8, respectively) with the molecular weight controls (see above). The numbers in parentheses indicate the number of PEG moieties (free thiol groups) that reacted with each Yap1 molecule. Diagrams of the different forms of Yap1 are also shown. The asterisks in (A), (B), (G), and (H) indicate non-Yap1 protein that copurified with Yap1 from lysates of Escherichia coli. Mobilities of molecular weight markers in (A), (B), (G), and (H) are indicated by arrowheads.

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As the level of Yap1 (oxI) decreased, two other rapidly migrating proteins, designated Yap1 (oxII-1) and Yap1 (oxII-2), were detected. Yap1 (oxII-1) appeared first (15 s to 5 min; Figure 2G, lanes 6–9). Then, it was partially converted to Yap1 (oxII-2) (5–30 min; lanes 9–11). The mobility and appearance of Yap1 (oxII-2) corresponded to that of the Yap1 (oxII), which migrated to approximately 60 kDa in Figure 2A. When Yap1 (oxII-1) was reduced by DTT, it migrated similarly to Yap1 plus two PEG moieties (compare lanes 1 and 8 in Figure 2H). The mobility of Yap1 (oxII-2) was equal to that of Yap1 without PEG moieties under reducing conditions (compare lanes 4 and 8 in Figure 2H). Thus, we concluded that Yap1 (oxII-1) had two free thiols and two disulfide bonds, whereas Yap1 (oxII-2) had no free thiols and three disulfide bonds (the disulfide bonds include at least one interdomain disulfide bond in both Yap1 [oxII-1] and Yap1 [oxII-2]). Therefore, Yap1 was converted to at least three differently oxidized forms, with different disulfide bonds, during its activation. Redox Reaction of the Yap1CCT,CAC and Yap1CTT,CAT Mutants In Vitro In contrast with our results described above, previous reports (Delaunay et al., 2000; Kuge et al., 2001) indicated that Cys315 and Cys620 are not required for the appropriate response of Yap1 to H2O2. To examine this issue, we monitored the fate of Yap1CCT,CAC, lacking Cys315 and Cys620, in our in vitro assay. As shown in Figure 3A, Yap1CCT,CAC was converted to a possible oxidized form, with altered degree of mobility retardation, in which a putative intradomain disulfide bond is likely to be formed (Yap1CCT,CAC [oxI*]) and then to oxII forms. Moreover, most of the Yap1CCT,CAC (oxI*) had no free thiol groups (designated as oxI*-2; Figures 3B and 3C), which suggested that two intradomain disulfide bonds, one in n-CRD (Cys303-Cys310) and one in c-CRD (Cys598Cys629), had formed within Yap1CCT,CAC (oxI*-2). In addition, Yap1CCT,CAC (oxII) also had no free thiol groups (Figure 3C). Thus, the two intradomain disulfide bonds in Yap1CCT,CAC (oxI*-2) were converted to two interdomain disulfide bonds, Cys303-Cys598 and Cys310-Cys629 (Figure 3D), both of which are found in air-oxidized Yap1 (Wood et al., 2003). These results suggest that the oxidation process of Yap1CCT,CAC proceeded somewhat factitiously. Thus, Cys315 and Cys620 might have a role in the oxidation of Yap1. Two Oxidized Forms of Yap1 (oxII-1) Because the extent of formation of Yap1CCT,CAC (oxII) was lower than that of Yap1 (oxII) (Figure 3A), these two forms might be qualitatively different. This possibility is supported by our observation that more of Yap1CCT,CAC (oxII) was generated when further NADPH was added to the reaction mixture (Figure 3E). Furthermore, we found that Yap1CTT,CAT (oxII), in which only one interdomain disulfide bond (Cys303-Cys598; disulfide bond 2) can be formed, appeared faster than did Yap1CCT,CAC (oxII), but slower than did Yap1 (oxII) (Figures 3A and 3F). In addition,

we observed a peptide fragment with disulfide bond 1 in Yap1 (oxII) in the MALDI-TOF mass analysis (see above). Thus, we postulated that Yap1 (oxII-1) might consist of two different forms, one with an interdomain disulfide (disulfide bond 2) with disulfide bond 1 and another with two interdomain disulfide bonds (disulfide bond 2 and Cys310-Cys629 [disulfide bond 3]). We designated these two forms Yap1 (oxII-1a) and Yap1 (oxII-1b), respectively (see Figure 4B). Thermodynamic Relationships among Different Oxidized Forms of Yap1 Our results suggest that multiple disulfide bonds between the six cysteines in Yap1 are formed at different time points. Thus, examination of qualitative differences in the different disulfide bonds might give an insight into the nature of the process. Our results suggest that the oxidation of Yap1 might involve several bidirectional redox reactions that respond to fluctuating levels of NADPH and H2O2. We postulated that the level of each disulfide bond formed in Yap1 during the redox reaction might depend on the ratio of H2O2 to NADPH. Therefore, next we compared the relative ratios of oxidized to reduced forms of Yap1 and mutant derivatives of Yap1 in our redox system with different molar ratios of H2O2 to NADPH. After Yap1 and mutant derivatives of Yap1 had been oxidized in the Gpx3/Trx2/Trr1/NADPH system with H2O2, we titrated each reaction mixture with NADPH at various levels (Figure 4A). We used the n-CRD domain of Yap1 (amino acids 122–373) to determine the ox/red ratio of disulfide bond 1. We also examined the oxII/red of Yap1CTT,CAT, Yap1CCT,CAC, and Yap1, of which the responsible disulfide bond for reduction might be disulfide bond 2, disulfide bond 3, and a disulfide bond between Cys315 and Cys620 (disulfide bond 4), respectively. As shown in Figure 4A, the ox/red ratios of these Yap1 proteins varied with the ox/red ratio of Gpx3. Although the oxII/red ratios of Yap1CTT,CAT, n-CRD, and Gpx3 were similar at a given redox state (i.e., level of NADPH), extra NADPH was required to reduce Yap1CCT,CAC and Yap1. The relative extent of formation of each disulfide bond in Yap1 in the H2O2/Gpx3/Trx/Trr/NADPH redox system with respect to the level of H2O2 and NADPH is illustrated in Figure 4B, together with the redox potential of each disulfide bond of Yap1, as estimated from the redox potential of Trx2 (see the Supplemental Data and Figure S3). Accordingly, our results indicate that the number of disulfide bonds between n-CRD and c-CRD (disulfide bonds 2, 3, and 4) influenced the level of the active (oxII) form of Yap1 at a certain ratio of H2O2 to NADPH. Redox Reactions of the Yap1CCT,CAC Mutant Protein in Cells Exposed to H2O2 To examine our hypothesis that the level of Yap1 (oxII) is determined by the number of disulfide bonds between two CRDs, we examined the oxidation of two different mutant derivatives of Yap1 in yeast cells, namely Yap1CCT,CAC and Yap1CTT,CAT, which can form disulfide

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Figure 3. Yap1CCT,CAC and Yap1CTT,CAT Were Converted to Oxidized Forms in the Kinetics-Based Assay In Vitro (A and B) Results of the kinetics-based assays of Yap1CCT,CAC in vitro in which AMS (A) and PEG-maleimide (B) were used to detect free thiol groups. Yap1CCT,CAC was converted to three different oxidized forms, namely Yap1 CCT,CAC (oxI*-1), Yap1 CCT,CAC (oxI*-2), and Yap1 CCT,CAC (oxII) (B). (C) The mobility of PEG-modified Yap1CCT,CAC (oxI*-2) was unchanged after reduction by DTT, and its mobility was the same as that of reduced Yap1CCT,CAC after reaction with IAA. (D) Possible scenario for the formation of disulfide bonds in Yap1CCT,CAC. Because most of Yap1CCT,CAC (oxI*) had no free thiol groups, and had the same mobility as Yap1CCT,CAC that did not bind PEG, it is likely that most Yap1CCT,CAC (oxI*) formed two intradomain disulfide bonds (Cys303-Cys310 and Cys598-Cys629; Yap1CCT,CAC [oxI*-2]). Yap1CCT,CAC (oxI*-2) can be converted to Yap1CCT,CAC (oxII) with Cys303-Cys598 and Cys310-Cys629 disulfide bonds. (E) Redox titration converted Yap1CCT,CAC (oxI*) to Yap1CCT,CAC (oxII). Yap1CCT,CAC was oxidized in the kinetics-based assay in vitro for 15 min (lane 1) and 60 min (lane 2). The reaction mixture (sample of 60 min) was treated with the indicated additional amounts of NADPH for 30 min until it reached equilibrium, and Yap1CCT,CAC (oxII) was analyzed. (F) Yap1CTT,CAT was converted to oxII in the kinetics-based assay in vitro. Reactions were stopped by the addition of NEM (E and F).

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Figure 4. Thermodynamic Relationships between Yap1, Yap1CCT,CAC, Yap1CTT,CAT, and Yap1 n-CRD (A) Oxidized Yap1 and mutant forms of Yap1 in the presence of the redox system were titrated with various concentrations of NADPH. (B) The relative levels of oxidized Yap1 and mutant forms of Yap1 that corresponded to each disulfide bond formed in Yap1 are shown. Yap1 with increasing numbers of disulfide bonds between n-CRD and c-CRD (disulfide bonds 2, 3, and 4) is correlated with increasing levels of NADPH and decreasing levels of H2O2, which are illustrated by the width of the blue blocks. Each disulfide bond, as well as Gpx3 and Trx2, is shown with the predicted midpoint redox potential. We calculated the midpoint redox potentials of the Trx2, Gpx3, and Yap1 proteins using the redox potential of lipoate redox buffer, Trx2, and Gpx3, respectively, as references. The redox potential of Trx2 (pH 7.0, 25 C) is similar to that recently reported by Mason et al. (2006), but there was some discrepancy in the redox potentials of Yap1 and Gpx3 (Orp1), perhaps because of differences between the systems examined (see the Supplemental Data for discussion).

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Figure 5. The H2O2-Dependent Oxidation of Yap1 and Yap1CCT,CAC In Vivo (A) A culture of yeast cells that expressed both (myc)9-tagged Yap1 (Yap1) and (HA)3-tagged Yap1CCT,CAC (CCT,CAC) was treated with H2O2. Free cysteine-SH residues in the cell lysate were allowed to react with AMS. The forms of Yap1 proteins are indicated by arrows with the names. (B) Levels of TRX2 and ACT1 mRNA were examined by northern blotting analysis after cells that expressed the full-length version of GFP-fused Yap1 (Yap1) and GFP-fused Yap1CCT,CAC (CCT,CAC), respectively, had been treated with 0.5 mM H2O2 for the indicated times. (C) Viability of the yeast cells treated with 0.2 mM H2O2 for 3 hr was examined by the microcolony assay. Averages and standard deviations (error bars) of the results for three different cultures are shown. Filled columns represent treatment with H2O2.

bonds 2 and 3 and only disulfide bond 2, respectively. To exclude the effects of differences in transcriptional activity between Yap1 and mutant forms of Yap1, we expressed only the regulatory domain of Yap1 and the mutant derivatives in YAP1D cells. First, we monitored the oxidation of Yap1CCT,CAC in vivo. As shown in Figure 5A, we showed that Yap1CCT,CAC was converted to an oxI* form within 15 s (0.25 min) after the start of treatment with H2O2. The oxII form of Yap1CCT,CAC appeared more slowly than did the oxII form of Yap1 (compare Yap1 and Yap1CCT,CAC from 15 s to 10 min in Figure 5A). We monitored Yap1dependent transcriptional activity (the level of TRX2 mRNA) in cells that expressed full-length Yap1CCT,CAC and in those that expressed full-length Yap1. As shown in Figure 5B, the initial increase in the level (20 min) of TRX2 RNA was greater in cells that expressed Yap1CCT,CAC. However, the level then decreased rapidly. Delaunay et al. (2000) reported a similar pattern of expression of TRX2 mRNA in cells that expressed Yap1CCA,CCC and in those that expressed Yap1CCC,CAC. As described above, Yap1CCT,CAC (oxI*-2) has a disulfide bond in c-CRD, which might mediate the nuclear localization of Yap1 and the activation of the specific transcription at

early times. The rapidly decreasing level of oxII in the case of Yap1CCT,CAC at late stages (Figure 5A; 120– 180 min) might be correlated with the decreasing level of TRX2 mRNA. The slightly lower viability of yeast cells that expressed full-length Yap1CCT,CAC in response to a challenge by H2O2 (Figure 5C) might be attributable to the aberrant patterns of expression of Yap1-target genes. Changes in Redox Status of Prxs and Trxs in Cells Responding to H2O2 We also monitored the redox status of Gpx3, Tsa1, and Trxs (Trx1 and Trx2) in the response of yeast cells to H2O2. Tsa1 contains 89% of the total Prxs in yeast (Ghaemmaghami et al., 2003) and has antioxidant and molecular chaperon activity under oxidative stress (Chae et al., 1993; Jang et al., 2004). As shown in Figure 6B, the increasing level of oxidized Tsa1 (formation of a dimer linked by disulfide bonds; Chae et al. [1994]) was transient (15 s to 1 min). Formation of an intramolecular disulfide bond in Gpx3 monomers was also transient (15 s to 10 min). These results suggest that the disulfide bond in Prxs formed transiently at the initial level of H2O2. Oxidation (formation of an intramolecular disulfide bond) of

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Trxs started after 15 s, and the ox/red ratio of Trxs decreased significantly at 150 min. Redox Reactions of Yap1CTT,CAT Mutant Protein that Can Form Only Disulfide Bond 2 in Cells Exposed to H2O2 Next, we monitored Yap1CTT,CAT, which can form only disulfide bond 2. As shown in Figure 6A, the oxII forms of both Yap1 and Yap1CTT,CAT appeared at 30 s (0.5 min) and remained detectable for 180 min after the addition of H2O2. A 100 kDa complex appeared concomitantly with the formation of the oxII forms of Yap1 and Yap1CTT,CAT. This complex corresponded to Yap1 linked to Gpx3 by a disulfide bond (Cys598 of Yap1 to Cys36 of Gpx3; Delaunay et al. [2002]). The ratio of oxII to red (0.5–1 min in Figure 6A) and the nuclear accumulation (1 min and 5 s to 1 min and 25 s in Figure 6C) of Yap1 were similar to those of Yap1CTT,CAT during the initial response to H2O2. However, the oxII/red ratios of Yap1CTT,CAT were lower than those of Yap1 at the later stage (5–120 min). Consistent with the lower oxII/red ratios of Yap1CTT,CAT compared with Yap1, nuclear localization of Yap1CTT,CAT in H2O2-treated cells was less efficient than in cells expressing Yap1, and the transcriptional activation of the Yap1-target gene TRX2 in cells that expressed Yap1CTT,CAT was also significantly depressed (Figure 6D). In addition, the viability of cells that expressed Yap1CTT,CAT on agar plates prepared with 0.2 mM H2O2 was significantly lower than that of cells that expressed Yap1 (Figure 6E). These results clearly showed that the steady-state oxII/red ratio of Yap1 was determined by the number of n-CRD-c-CRD disulfide bonds in yeast cells. Finally, we examined whether the level of Yap1 (oxII) might be affected by the rate of reduction and reoxidation of disulfide bonds in Yap1 (oxII). We treated cells with H2O2 for 30 min and monitored the reduction of Yap1 (oxII) after the medium had been changed to fresh medium without H2O2. As shown in Figure 6F, Yap1 (oxII) was rapidly reduced (the half life of Yap1 [oxII] was less than 3 min), which suggested that the rates of reduction and reoxidation of Yap1 in vivo might not be the determinants of the level of the oxII (active) form. Therefore, we conclude that the level of the active form of Yap1 might be determined by the number of disulfide bonds formed in equilibrium with the redox reaction. DISCUSSION In this report, we provide evidence that the six cysteine residues in Yap1 perform important roles in sensing H2O2 and transducing the signal, playing essential roles in the response of yeast cells to H2O2 stress (Figure 7). Because the rates of both Gpx3-mediated oxidation of Yap1 by H2O2 and Trx-mediated reduction of Yap1 (oxII) were rapid (Figure 6), the level of the active form of Yap1 is likely to be determined by the balance between H2O2 (level of

Gpx3-mediated oxidation of Yap1) and NADPH (level of reduced Trx) in yeast cells. We examined the early stages of the oxidation reaction using our kinetics-based oxidation system in vitro, among which the thiol-disulfide redox reactions respond to fluctuating levels of NADPH and H2O2. We found that Yap1 has four possible oxidized forms: Yap1 (oxI), Yap1 (oxII-1a), Yap1 (oxII-1b), and Yap1 (oxII-2). The findings that the appearance of Yap1 (oxII-2) was later than the other oxidized forms of Yap1 and that the redox potential of Yap1 was lower than the Yap1 mutant derivatives suggest that the three disulfide bonds formed in Yap1 (oxII-2) might be three interdomain disulfide bonds (disulfide bonds 2, 3, and 4; Figure 4B). The observations in vitro are consistent with those in vivo (Figure 2F), which suggest that Yap1 (oxI) was generated at a very early stage (10 s) of treatment of yeast cells with H2O2, whereas a further 5 min was required to convert Yap1 (oxI) to Yap1 (oxII). The fact that Yap1CTT,CAT can form oxII in cells (Figure 6A) suggests that disulfide bond 1, which is located in n-CRD, is not required for the formation of the H2O2-induced interdomain disulfide bond in Yap1 (disulfide bond 2). Disulfide bond 1 might induce a conformational strain, which triggers the formation of disulfide bond 3 to yield Yap1 (oxII-1b). Alternatively, the formation of disulfide bond 1 might be necessary to protect possible reactive cysteine residues (Cys310 and/ or Cys315) from forming inappropriate disulfide linkage to other cysteine residues prior to the formation of disulfide bond 3. These possibilities are supported by our results; an incorrect disulfide bond (Cys303-Cys315) was probably formed in n-CRD of Yap1CCT,CAC (oxI*-2) at the initial stage in yeast cells in response to H2O2, and the conversion of oxI*-2 to Yap1CCT,CAC (oxII) was delayed. The activity of Yap1, which was correlated with the amount of the oxII form generated, is probably determined by the number of disulfide bonds in Yap1 (Figure 7). The initial step in the activation of Yap1 might be initiated by disulfide bond 2, which might inhibit the function of NES (Delaunay et al., 2000). The rate of formation of disulfide bond 2 (Yap1 [oxII-1a]) was relatively low (1–5 min). A flexible structure in the Yap1 region between n-CRD and c-CRD (Wood et al., 2003) might slow the rate of interaction between these two domains. Although disulfide bond 2 was formed constitutively during the response to H2O2, it was insufficient for the maximum transcriptional activity of Yap1 (Figure 6C; CTT,CAT). The second disulfide bond between n-CRD and c-CRD, namely disulfide bond 3, might be essential for the activity of Yap1, because the activity of Yap1CCT,CAC (in which both disulfide bonds 2 and 3 formed in vitro) was similar to that of Yap1 but higher than that of Yap1CTT,CAT (in which only disulfide bond 2 was formed). The level of oxII increased significantly from 5 min after the addition of H2O2 in the case of Yap1, but not in the case of Yap1CTT,CAT (Figure 6A), which suggests that the conversion of disulfide bond 1 in Yap1 (oxII-1a) to disulfide bond 3 in Yap1 (oxII-1b) might occur at this stage (5–10 min; see Figures 6A and 7).

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Figure 6. H2O2-Induced Oxidation and Activation of Yap1 and Yap1CTT,CAT In Vivo (A and B) Time-dependent oxidation of Yap1 (Yap1) and Yap1CTT,CAT (CTT,CAT), Gpx3, Tsa1, and Trxs (Trx1 + Trx2) in cells treated with 0.5 mM H2O2 were examined. Yeast cells expressing HA-tagged Yap1 and HA-tagged Yap1CTT,CAT, respectively, were treated with H2O2. Free cysteine-SH residues in the lysate were allowed to react with NEM. Mobilities of marker proteins with molecular weight (3 103) are indicated. The forms of each protein are indicated by arrows with the names. The band of protein with a molecular weight of 100 kDa corresponds to one molecule of Gpx3 complexed with Yap1 (A). An additional unknown band of a protein of 150 kDa in Yap1-expressing cells appeared between 1 and 10 min (indicated by an asterisk in (A). This mobility corresponds to Yap1 complexed with three molecules of Gpx3 or to two molecules of Yap1 linked by a disulfide bond. Further analysis is required to characterize this complex. Both these bands disappeared on treatment with DTT (data not shown). Western blots showing Gpx3, Tsa1, and Trxs in lysates prepared from cells that expressed Yap1CTT,CAT (CTT,CAT) are also shown (B); similar results were obtained with lysates from cells that expressed Yap1 (data not shown). (C) Nuclear accumulation of GFP-fused Yap1 (Yap1) and GFP-fused Yap1CTT,CAT (CTT,CAT). (D) Northern blotting analysis of expression of TRX2 and ACT1. Cells that expressed the full-length version of GFP-fused Yap1 and GFP-fused Yap1CTT,CAT, respectively, were treated with 0.5 mM H2O2 for the indicated times. (E) Viability assay. Cells (4 3 103, 1.3 3 103, 440, 150, and 50 cells per spot) carrying pRS314 cp-GFP HA yap1CTT,CAT (CTT,CAT), pRS314 cp-GFP HA YAP1 (Yap1), and pRS314 (null) were spotted on SD dropout (TRP) agar medium (control) and on medium that contained 0.2 mM H2O2. The plates were then incubated for 50 hr at 30 C and photographed. (F) Yap1 (oxII) was rapidly reduced upon removal of H2O2 from the culture medium. Cells carrying Yap1 were treated with 0.5 mM H2O2 for 30 min, and the medium was then replaced with fresh medium without H2O2. After incubation for the indicated times (min), Yap1 was examined as described above.

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Figure 7. Multistep Disulfide Bond Formation in Yap1 Is Required for the Long-Lasting Transduction of the H2O2 Stress Signal Correlation between the levels of active forms of Yap1 (y axis) and the time after addition of H2O2 (x axis) for each disulfide bond formed in Yap1 is shown. Numbers in parentheses indicate name of the disulfide bonds (Figure 4B). Formation in the disulfide bond 1 in Yap1 (oxI) is the first step of the oxidation of Yap1 (10 s to 5 min). Then, disulfide bonds 2 linking n-CRD and c-CRD is formed (oxII-1a; 1–5 min). The third step is the formation of oxII-1b, in which disulfide bonds 2 and bond 3 are formed; disulfide bond 1 in oxII-1a might be converted to disulfide bond 3 (5 min). The diagram also includes possible formation of oxII-2, in which all six cysteine residues, including Cys315 and Cys620, form interdomain disulfide bonds. The oxidation of Yap1 is indicated by red arrows. Redox processes for Yap1CCT,CAC (CCT,CAC) and Yap1CTT,CAT (CTT,CAT) are indicated by green and blue dotted lines, respectively (see text for details). The ratio of Trx (ox) to Trx (red) and the level of Gpx3-Yap1, as estimated from Figure 6A, are illustrated by the width of the blue blocks.

Furthermore, because the level of Yap1CCT,CAC (oxII) decreased rapidly in cells at the late stage of the response, and because yeast cells carrying this mutant form of Yap1 exhibited enhanced sensitivity to H2O2 (Figure 5C), the third interdomain disulfide bond (disulfide bond 4) might generate the oxII-2 form of Yap1 in vivo to sustain the active form at the late stage of the response to H2O2 (Figure 7). Yap1 (oxII-2), which shows the lowest redox potential, might be required for nuclear localization when the level of H2O2 might become low (level of Gpx3-Yap1 depressed) and that of NADPH might become high (reduced Trx enhanced). As a result of the formation of oxII-2 in wild-type Yap1, an integral level of the transcription of the Yap1-target genes might become sufficient for the appropriate response to the oxidative stress (Figure 7). Thus, Yap1 might act as ‘‘the guardian of NADPH homeostasis’’ by increasing the number of its interdomain disulfide bonds that appeared sequentially to convert Yap1 to its reduction-resistant form (lower redox potentials; see Figure 4) in the process of recovering NADPH. Enhanced levels of oxidized forms in two Prxs, namely in dimeric Tsa1 and monomeric Gpx3, were transient in response to H2O2 (Figure 6A; 15 s to 1 min and 15 s to 10 min, respectively). Hyperoxidation of cysteine sulfhydryl groups to sulfinic acid might prevent further disulfide bond formation in Tsa1 (Biteau et al., 2003) and monomeric Gpx3. However, oxII forms of Yap1 and the mixed disulfide bond in Yap1-Gpx3 were long lived. Indirect oxidation of Yap1 via the two-component Gpx3/Yap1 system might prevent hyperoxidation of cysteine residues in

Yap1 such that disulfide bonds in Yap1 might be constitutively formed in the presence of H2O2. Our model explains how an H2O2-induced signal can be converted to an enhanced signal, as required for appropriate recovery of the cellular redox status that is determined by the NADPH and reduced Trx levels under H2O2 stress. This model might be applicable to the mechanisms that regulate other redox-regulated cysteine-containing proteins under ROS-generating physiological conditions.

EXPERIMENTAL PROCEDURES Yeast Strains and Media Yeast cells were grown in synthetic dextrose (SD) medium supplemented with amino acids (SD dropout; Dunn and Wobbe [1997]) or in YPAD medium (1% peptone, 0.5% yeast extract, 2% glucose, 0.08 mg/ml adenine sulfate). The following strains of Saccharomyces cerevisiae were used in this study: Y17202 (MATa his3D1 leu2D0 lys2D0 ura3D0 trp1::kanMX4), namely trp1D cells from a knockout library constructed using BY4742 (EUROSCARF, Frankfurt, Germany) derived from S288C, and BY4742 yap1D (MATa his3D1 leu2D0 lys2D0 ura3D0 trp1::kanMX4 yap1::URA3). The latter strain was constructed in this study from Y17202, as described by Kuge and Jones (1994). Kinetics-Based Redox Analysis of Recombinant Proteins All reagents used for redox analysis were degassed, and the experiments were performed under argon and/or nitrogen. Redox cycles of Yap1 and Yap1 mutant derivatives in the presence of Gpx3 (or Tsa1), Trx, Trr, and NADPH were examined as follows. Recombinant proteins that contained the regulatory domains of Yap1 or Yap1 mutant derivatives (amino acids 122–650) were used for all redox analyses in vitro. A reaction mixture containing proteins at a molar ratio Gpx3:Trx2:Trr1 of 14:3:1 was prepared in reaction buffer (50 mM

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Tris-HCl [pH 7.5], 1 mM EDTA, 150 mM NaCl, 0.4 mM NADPH) and incubated at room temperature for 30 min; Yap1 or a Yap1 mutant was added to this reaction mixture at the same molar ratio as that of Gpx3. Next, the oxidizing reaction was started by adding 0.4 mM H2O2. Aliquots were removed at the indicated times and mixed with iodoacetamide (IAA) or N-ethylmaleimide (NEM) at a final concentration of 10 mM, with urea at a final concentration 9 M, and one-third volume of 3 3 SDS-sample buffer (150 mM Tris-HCl [pH 6.8], 6% SDS, 30% glycerol, 0.03% bromphenol blue). For assays with other thiolalkylating reagents, the aliquots were incubated with AMS (Invitrogen, Carlsbad, CA) or methoxy PEG-maleimide (Sunbright MEMAL-50H; NOF Corporation, Tokyo, Japan) at a final concentration of 10 mM at 30 C for 30 min in the presence of urea at a final concentration of 9 M; 3 3 SDS sample buffer was added to each reaction mixture as described above. Then we fractionated the proteins by SDS-PAGE (12% polyacrylamide or a gradient of 7.5%–17.5% polyacrylamide; ratio of acrylamide to bisacrylamide, 30:0.4) in a running buffer that contained 25 mM Tris, 250 mM glycine and 0.1% SDS. Gels were stained with 0.1% Coomassie brilliant blue R-250 (Sigma-Aldrich, St. Louis, MO). Analysis of the Redox Status of Yap1 In Vivo Lysates of yeast cells that expressed the HA-tagged regulatory domain of YAP1 and/or the myc-tagged regulatory domain of YAP1 were prepared in trichloroacetic acid (TCA) essentially as described by Delaunay et al. (2000) with slight modifications, as described in the Supplemental Data. To determine the redox status of Trx1, yeast cell lysates were reduced in 5 mM DTT and then treated with AMS (25 mM). Other Methods The construction of plasmids, purification of recombinant proteins, microcolony assay, and northern blotting were performed as described in the Supplemental Data. Reproducibility All the experiments were repeated several times, and the representative results are shown. Supplemental Data Supplemental Data include Supplemental Experimental Procedures, Supplemental References, and four figures and can be found with this article online at http://www.molecule.org/cgi/content/full/27/4/ 675/DC1/. ACKNOWLEDGMENTS The authors thank Drs. K. Ohashi, G.W. Hwang, K. Kita, and T. Takahashi for helpful discussions; Ms. Y. Nagaya for skilled technical assistance; Prof. J. Goto for providing access to mass spectrometer; Drs. Y. Inoue and S. Izawa (Kyoto University) for providing Trx2-specific antiserum; and Dr. Toledano (CEA-Saclay) for expression plasmid for myc-tagged Yap1. This work was supported by Grants-in-Aid for Exploratory Research and a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (Japan), and by the ‘‘Program for Promotion of Fundamental Studies in Health Sciences’’ of the National Institute of Biomedical Innovation (NIBO). Author contributions are the following: S.O., Figures 1, 2, 3A–3E, 4, 5, and 6F and Figures S1, S2, and S3B; T.T., Figures 3F and Figure S3A; S.K., Figures 6A–6E and 7 and manuscript preparation. Received: December 1, 2006 Revised: March 7, 2007 Accepted: June 25, 2007 Published: August 16, 2007

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