1. Volume 27, Issue 4, Pages 675-688 (August 2007)
Multistep Disulfide Bond Formation in Yap1 Is Required for Sensing and Transduction of H2O2 Stress Signal 
Shoko Okazaki, Tsuyoshi Tachibana, Akira Naganuma, Nariyasu Mano, Shusuke Kuge 
Molecular Cell 
Volume 27, Issue 4, Pages (August 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. 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).
Molecular Cell  , DOI: ( /j.molcel )
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3. 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.
Molecular Cell  , DOI: ( /j.molcel )
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4. 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).
Molecular Cell  , DOI: ( /j.molcel )
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5. 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).
Molecular Cell  , DOI: ( /j.molcel )
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6. 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.
Molecular Cell  , DOI: ( /j.molcel )
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7. 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 (× 10−3) 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 × 103, 1.3 × 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.
Molecular Cell  , DOI: ( /j.molcel )
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8. 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.
Molecular Cell  , DOI: ( /j.molcel )
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