H2O2 sensing through oxidation of the Yap1 transcription factor

Abstract

The yeast transcription factor Yap1 activates expression of antioxidant genes in response to oxidative stress. Yap1 regulation involves nuclear accumulation, but the mechanism sensing the oxidative stress signal remains unknown. We provide biochemical and genetic evidence that upon H2O2 treatment, Yap1 is activated by oxidation and deactivated by enzymatic reduction with Yap1-controlled thioredoxins, thus providing a mechanism for autoregulation. Two cysteines essential for Yap1 oxidation are also essential for its activation by H2O2. The data are consistent with a model in which oxidation of Yap1 leads to disulfide bond formation with the resulting change of conformation masking recognition of the nuclear export signal by Crm1/Xpo1, thereby promoting nuclear accumulation of the protein. In sharp contrast to H2O2, diamide does not lead to the same Yap1 oxidized form and still activates mutants lacking cysteines essential for H2O2 activation, providing a molecular basis for differential activation of Yap1 by these oxidants. This is the first example of an H2O2-sensing mechanism in a eukaryote that exploits the oxidation of cysteines in order to respond rapidly to stress conditions.

Fig. 1. Kinetics of Yap1 activation. (A) Expression of TRX2. Total RNA was isolated from a wild-type strain, grown to an OD₆₀₀ of 0.3 in selective minimal medium, before and 10, 20, 30, 45 and 60 min after treatment with 300 µM H₂O₂, and TRX2 and ACT1 (control gene) mRNAs were quantified by RT–PCR. An autoradiogram of the separated ³²P-labelled PCR products is shown in the upper panel and a plot of the TRX2/ACT1 signal ratio quantified on a PhosphorImager in the lower panel. (B) Nuclear redistribution of Yap1. A Δyap1 strain expressing GFP-Yap1 at an OD₆₀₀ of 0.3, treated with 300 µM H₂O₂ for the time indicated and analysed for GFP staining. (C) Yap1 phosphorylation. Extracts of a Δyap1 strain expressing Myc-tagged Yap1 (yMyc-Yap1), before and 5, 10, 15, 30, 45 and 60 min after treatment with 300 µM H₂O₂ were immunoblotted with the anti-Myc monoclonal antibody after SDS–PAGE. (D) Yap1 phosphorylation correlates with Yap1 nuclear localization. Δyap1 strain cultures expressing Myc-Yap1, Yap1^L619S,L623S or Yap1^{C598T,C620A,C629T}, before and 20 min after H₂O₂ treatment, were processed as in (C).

Fig. 2. In vivo oxidation of Yap1. (A) yMyc-Yap1 cultures (grown to an OD₆₀₀ of 0.4) before and after 2.5 min treatment with 400 µM H₂O₂ (lanes 1 and 2) or 1 mM t-butyl hydroperoxide (lanes 5 and 6) were lysed using TCA, treated with iodoacetamide and immunoblotted after non-reducing SDS–PAGE. Lanes 3 and 4 are the same as lanes 1 and 2 except that the TCA-precipitated protein pellet was dissolved in the presence of 200 mM DTT, before adding a 3.2 M excess of iodoacetamide. (B) Time course of Yap1 oxidation by H₂O₂. A yMyc-Yap1 culture before and 2.5, 5, 15, 30, 45 and 60 min after treatment with 400 µM H₂O₂, processed as in (A) (lanes 1 and 2), except that Yap1 was dephosphorylated and separated under non-reducing or reducing conditions as indicated. (C) The minimum H₂O₂ concentration required to oxidize Yap1 in vivo. A yMyc-Yap1 culture treated for 5 min with 25, 50, 100, 150, 200, 300 and 800 µM H₂O₂ and processed as in (A) (lanes 1 and 2). (D) Time-course analysis of Yap1 redox status in response to diamide. A yMyc-Yap1 culture before and 5, 15, 30, 45 and 60 min after treatment with 2 mM diamide, processed as in (A) (lanes 1 and 2) and separated under non-reducing or reducing conditions as indicated.

Fig. 3. Identification of Yap1 redox-active cysteines. (A) Schematics of Yap1. (B) Both N- and C-terminal cysteines are required to oxidize Yap1. Δyap1 strain cultures expressing Myc-Yap1, Yap1^{C303A,C310A,C315A} or Yap1^{C598T,C620A,C629T} before and 5 min after treatment with 400 µM H₂O₂ were processed as in Figure 2A (lanes 1 and 2) and immunoblotted after non-reducing SDS–PAGE. (C) Cysteines C303 and C598 are the redox-active Yap1 cysteines. Δyap1 strain cultures expressing Myc-Yap1, Yap1^C303A, Yap1^C310A, Yap1^C315A, Yap1^C598A, Yap1^C620A or Yap1^C629A, and Yap1^{C310T,C315A,C620A,C629T} before and 5 min after H₂O₂ treatment, as in Figure 2B and immunoblotted after non-reducing or reducing SDS–PAGE, as indicated.

Fig. 4. Activity of Yap1 cysteine substitution mutants. (A) Yap1 cysteine residues important for tolerance to H₂O₂. The Δyap1 strains not expressing or expressing Myc-Yap1, Yap1^C303A, Yap1^C310A, Yap1^C315A, Yap1^C598A, Yap1^C620A or Yap1^C629A were tested for their tolerance to increasing concentrations of H₂O₂ by the patch assay on solid medium. (B) The same strains as in (A) were used to measure the amount of TRX2 and ACT1 mRNAs by RT–PCR as in Figure 1A. Cells were treated with 300 µM H₂O₂ for the time indicated. (C) The TRX2/ACT1 signal ratios quantified from the RT–PCR shown in (B) on a PhosphorImager were calculated and plotted as a function of time of exposure to H₂O₂.

Fig. 5. Essential role of C303 and C598 for H₂O₂-induced Yap1 nuclear redistribution. (A) Analysis of the cellular distribution of GFP-tagged Yap1^C303A, Yap1^C310A, Yap1^C598A and Yap1^C629A. Cells taken before or 12 min after treatment with 300 µM H₂O₂ were analysed for GFP or DAPI staining or under visible light. (B) Phosphorylation of Yap1 cysteine mutants as an indicator of their subcellular localization. Δyap1 cultures expressing Myc-Yap1, Yap1^C303A, Yap1^C310A, Yap1^C315A, Yap1^C598A, Yap1^C620A or Yap1^C629A were taken before and after treatment with 300 µM H₂O₂ and processed as in Figure 1C.

Fig. 6. The critical role of C303 and C598 is at the level of Crm1–Yap1 interaction. β-galactosidase production from a lacZ reporter gene was assayed in strain EGY48 carrying pKW442 (Crm1–lexA^BD) or pEG202 (empty vector) and one of the following: pLDB439 (YAP1-B42^AD or pYAP1^C303A-B42^AD), pYAP1^C598A-B42^AD or pJG4-5 (empty vector). (A) Assay performed on solid medium in the absence or presence of H₂O₂ at the concentration indicated. (B) Assay performed on liquid medium in the absence (–) or presence (+) of 500 µM H₂O₂. The results are the averages of four independent experiments.

Fig. 7. Deactivation (reduction) of Yap1 by the thioredoxin system. (A and B) Expression of TRX2 (A) and TRR1 (B) in wild-type, Δtrr1 (A), Δtrx1Δtrx2 (B), Δgsh1Δglr1pro2-1 and Δtsa1 strains. Cells were treated with 300 µM H₂O₂ for the indicated time and processed for RT–PCR as in Figure 1A. The ACT1 autoradiograms are not shown. (C) Yap1 is oxidized in mutants of the thioredoxin system. yMyc-Yap1, Δtrx1Δtrx2 and Δtrr1 expressing Myc-Yap1 (grown to an OD₆₀₀ of 0.3) were processed as in Figure 2A and extracts were analysed by immunoblotting after non-reducing PAGE. (D) Reduction of oxidized Yap1 by the thioredoxin system. yMyc-Yap1 (grown to an OD₆₀₀ of 0.4), treated with 400 µM H₂O₂, lysed with glass beads in 100 mM Tris–HCl pH 8, 0.1% SDS, 1 mM EDTA, complete protease inhibitors and PMSF. Twenty-five micrograms of extracts were incubated with 1, 10 or 20 µM thioredoxin (from Spirulina sp.), 1.3 µM E.coli thioredoxin reductase and 1 mM NADPH (Sigma) for 30 min at 37°C. Iodoacetamide (75 mM) was added and the samples were analysed by immunoblotting after non-reducing PAGE.