Yap1p and Yap8p control As(III)-induced expression of different subsets of defense genes. (A) Northern blot analysis of total RNA extracted from wild-type, yap1Δ, yap8Δ and yap1Δ yap8Δ cells in the absence (lane 1) or presence of 1 mM As(III) for 60 min (lane 2) and 300 min (lane 3). The filter was hybridized to ³²P-labeled DNA fragments recognizing the indicated genes. (B) Metalloid-dependent induction of the ACR3-lacZ reporter in wild-type, yap1Δ, yap8Δ and yap1Δ yap8Δ cells. Cells exposed to metalloids were assayed for β-galactosidase activity as described in MATERIALS AND METHODS. The results are the average of three independent experiments. The error bars represent the SD.

Phenotypes of S. cerevisiae cells lacking AP-1 proteins and proteins involved in metalloid detoxification. (A) Metalloid sensitivity. Cells of W303-1A were grown in liquid medium, and 10-fold serial dilutions of the cultures were spotted on agar plates with or without metalloid salts. Growth was monitored after 3 d at 30°C. (B) Sensitivity of yap8Δ is limited to metalloids, whereas yap1Δ is sensitive to several oxidizing agents. Cells were cultivated as described above, and growth was monitored after 2–3 d at 25°C.

Cellular localization of Yap1p and Yap8p. (A) Plasmids carrying GFP fusions of Yap1p and Yap8p were transformed into yap1Δ and yap8Δ cells, respectively. Transformants were cultivated in selective medium with either 2% glucose (GFP-Yap1p) or 2% raffinose +0.5% galactose followed by induction with 2% galactose for 1–2 h (GFP-Yap8p). Living cells were analyzed by fluorescence microscope for Yap1p and Yap8p localization (GFP) before and 10 min after exposure to As(III) (1 mM), Sb(III) (10 mM), and H₂O₂ (0.5 mM). (B) Localization of GFP-Yap8p in yap8Δ cells transsformed with a plasmid expressing the fusion protein from the endogenous YAP8 promoter before and after 10 min of As(III) exposure. (C) Cells with a genomic copy of Yap8p fused to the Myc-tag were grown in YPD, either untreated or exposed to 1.0 mM As(III) for 3 h, and nuclear extracts and cytoplasmic fractions were prepared and probed by anti-myc antibodies. Cells lacking Yap8p-Myc was used as a control.

Functional analysis of Yap1p and Yap8p mutants bearing substitutions of conserved CRD cysteines. (A) Domain organization of Yap1p and Yap8p and position of conserved cysteine residues involved in redox regulation and metalloid tolerance. NES, nuclear export sequence. (B) Subcellular localization of Yap8p cysteine mutants. Plasmids carrying GFP fusions of Yap8p and the Yap8p-C132A, Yap8p-C274A, and Yap8p-C132A C274A mutants were transformed into yap8Δ cells, and the proteins were visualized by fluorescence microscopy. For simplicity, the GFP-Yap8p mutants are labeled in abbreviated form. (C) Yap8p cysteines C132 and C274 are both required for Yap8p function. yap8Δ cells were transformed with plasmids expressing either native GFP-Yap8p or the indicated GFP-Yap8p mutants. The yap8Δ mutant was also transformed with an empty control plasmid. Transformed cells were grown in liquid medium containing 2% raffinose and 0.5% galactose followed by induction with 2% galactose for 1–2 h. Serial 10-fold dilutions of the cultures were spotted on agar plates (with glucose as a carbon source) with or without As(III). (D) GFP-Yap8p cysteine mutants are unable to induce ACR3-lacZ expression. yap8Δ cells were transformed with empty control plasmid and with plasmids expressing either native GFP-Yap8p or the indicated GFP-Yap8p mutants. β-Galactosidase activity was measured as described in Figure 3. (E) Yap1p cysteines are important for As(III) and oxidative stress tolerance. yap1Δ cells were transformed with plasmids expressing either native GFP-Yap1p or the indicated GFP-Yap1p mutants. Wild-type and yap1Δ cells were also transformed with an empty control plasmid. Ten-fold serial dilutions of the cultures were spotted on agar plates and incubated at 30°C (As(III)) or 25°C (t-BOOH and diamide) for 2–3 d. For simplicity, the GFP-Yap1p mutants are labeled in abbreviated form. (F) Subcellular localization of Yap1p CRD mutants. Plasmids carrying GFP fusions of Yap1p and the indicated Yap1p mutants were transformed into yap1Δ cells, and the proteins were visualized before and 10 min after exposure to As(III) (1.0 mM) or H₂O₂ (0.5 mM) by fluorescence microscopy.

ACR3 induction requires the TTAATAA promoter element. (A) Promoters of ACR2, ACR3, and YAP8 contain putative AP-1 binding sites with the sequence TTAATAA. (B) Presence of the TTAATAA sequence in the ACR3 promoter is required for Acr3p-mediated As(III) tolerance. Cells were grown in liquid medium and 10-fold serial dilutions of the cultures were spotted on agar plates with or without As(III). (C) Induction of ACR3-lacZ expression is dependent on the TTAATAA sequence. β-Galactosidase activity was measured as described in Figure 3.

Binding of FLAG-Yap8p to the ACR3 promoter as detected by ChIP. PCR was performed on chromatin fragments isolated after immunoprecipitation (Anti-FLAG-IP) in cells expressing FLAG-Yap8p or cells without tagged protein (no tag) with primers that specifically amplify the ACR3 and YAP8 promoters. Cells were either untreated or exposed to 1 mM As(III) for 3 h. PCR was also performed on extracts that were not immunoprecipitated (input).

Model for As(III)-activation of Yap1p and Yap8p. (A) As(III) triggers nuclear accumulation of Yap1p and transcriptional activation of its target genes, including TRX2 (thioredoxin). Mutations within the CRDs that affect Yap1p function under As(III) exposure (Yap1p-TAT and Yap1p-3Cys C620A) are indicated with an asterisk (*). Whether As(III) activates Yap1p through oxidative modification of critical cysteines or through another mechanism is presently unknown. (B) Yap8p is bound to the promoter of ACR3 (arsenic efflux protein) in an apparently inactive form in the absence of stress, whereas As(III) exposure activates Yap8p through an as yet unknown mechanism. Yap8p activation requires specific cysteine residues. Cysteine mutations (C132A or C274A) that affect Yap8p function are marked with an *.