Yap1p activates gene transcription in an oxidant-specific fashion

Abstract

Positive regulation of gene expression by the yeast Saccharomyces cerevisiae transcription factor Yap1p is required for normal tolerance of oxidative stress elicited by the redox-active agents diamide and H(2)O(2). Several groups have provided evidence that a cluster of cysteine residues in the extreme C terminus of the factor are required for normal modulation of Yap1p by oxidant challenge. Deletion of this C-terminal cysteine-rich domain (c-CRD) produces a protein that is highly active under both stressed and nonstressed conditions and is constitutively located in the nucleus. We have found that a variety of different c-CRD mutant proteins are hyperactive in terms of their ability to confer diamide tolerance to cells but fail to provide even normal levels of H(2)O(2) resistance. Although the c-CRD mutant forms of Yap1p activate an artificial Yap1p-responsive gene to the same high level in the presence of either diamide or H(2)O(2), these mutant factors confer hyperresistance to diamide but hypersensitivity to H(2)O(2). To address this discrepancy, we have examined the ability of c-CRD mutant forms of Yap1p to activate expression of an authentic target gene required for H(2)O(2) tolerance, TRX2. When assayed in the presence of c-CRD mutant forms of Yap1p, a TRX2-lacZ fusion gene fails to induce in response to H(2)O(2). We have also identified a second cysteine-rich domain, in the N terminus (n-CRD), that is required for H(2)O(2) but not diamide resistance and influences the localization of the protein. These data are consistent with the idea that the function of Yap1p is different at promoters of loci involved in H(2)O(2) tolerance from promoters of genes involved in diamide resistance.

FIG. 1

Transactivation of ARE-TRP5-lacZ expression by alanine scanning mutations in CSE629. Strain YSC6 (yap1-Δ2::hisG ARE-TRP5-lacZ) was transformed with low-copy-number plasmids expressing the indicated forms of Yap1p. Transformants were grown on SC medium (20) to an A₆₀₀ of 0.6 and then split into three equal aliquots, which were subjected to diamide- or H₂O₂-induced oxidative stress or left untreated (No Stress) for 1.5 h. Cells were then processed and β-galactosidase activity was measured as described previously (6). The locations of the basic-region leucine zipper DNA binding domain and the two separable transactivation domains in Yap1p are indicated on the left-hand side of the figure. The drawing represents the Yap1p protein chain, and the numbers indicate the position along the factor.

FIG. 2

Phenotype and expression of alanine-scanning mutations in CSE629. (A) Cells lacking the YAP1 gene (Δyap1) were transformed with low-copy-number plasmids expressing the indicated forms of Yap1p or the vector only (pRS316). Transformants were grown to an A₆₀₀ of 1, and spots of 1,000 cells were placed on YPD containing diamide or H₂O₂. Each oxidant was present in a concentration gradient, as indicated by the bar at the top of the figure. (B) Δyap1 cells expressing the indicated forms of Yap1p were grown in the absence of oxidants (U) or challenged for 1.5 h with diamide (D) or H₂O₂ (H). Protein extracts were prepared, and either 50 μg (wild-type Yap1p) or 100 μg (mutants) of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The gel was then transferred to nitrocellulose and probed with a rabbit anti-Yap1p antiserum (24). Bound antibody was detected by using goat anti-rabbit antibody and chemiluminescence (Pierce).

FIG. 3

ARE-TRP5-lacZ expression supported by diamide-hyperresistant mutants with mutations in the c-CRD region. (A) The sequence of the C-terminal 53 amino acids of Yap1p is shown in the single-letter amino acid code. The numbers refer to the position of each amino acid along the 650-residue length of Yap1p, and the locations of the three CSE repeats are indicated by underlining. The highlighted region corresponds to a putative nuclear export signal as suggested previously (29). The position and sequence of each mutant analyzed here are indicated above the amino acid sequence. (B) The ability of each mutant to regulate gene expression was assayed by introducing low-copy-number plasmids expressing the indicated forms of Yap1p into a Δyap1 strain containing the ARE-TRP5-lacZ reporter gene, as described for Fig. 1. Levels of ARE-dependent β-galactosidase activity were determined in the unstressed cells (No Stress) and in cells subjected to diamide- or H₂O₂-induced oxidative stress.

FIG. 4

Oxidative stress phenotypes and expression level of random mutants with mutations in the c-CRD. (A) Δyap1 cells were transformed with low-copy-number plasmids expressing the indicated forms of Yap1p. Transformants were assayed for their ability to tolerate diamide- or H₂O₂-induced stress by a spot test assay as described in the legend to Fig. 2. (B) Steady-state protein levels of the indicated Yap1p derivatives were determined by Western blotting with the rabbit anti-Yap1p antiserum as described in the legend to Fig. 2.

FIG. 5

The n-CRD is required for normal redox regulation. The extent of the Yap1p sequence deleted in each construct is indicated by the gap and by the numbers on the left-hand side of the drawing. The other symbols and labeling are as in Fig. 1. ARE-dependent β-galactosidase activity was determined for each construct as described in the legend to Fig. 1.

FIG. 6

Oxidant resistance and expression profiles of n-CRD mutant proteins. Δyap1 cells were transformed with low-copy-number plasmids expressing the indicated forms of Yap1p. Oxidative stress phenotypes (A) and steady-state protein levels (B) were assayed as described in the legend to Fig. 2.

FIG. 7

The n- and c-CRD regions contribute regulatory information to the redox response of Yap1p. (A) All Yap1p derivatives were introduced on low-copy-number plasmids into the Δyap1 ARE-TRP5-lacZ reporter strain and assayed for ARE-dependent β-galactosidase activity as described in the legend to Fig. 1. (B) Oxidative stress resistance phenotypes of Δyap1 cells carrying low-copy-number plasmids expressing the indicated forms of Yap1p were assayed as described in the legend to Fig. 2. (C) Steady-state protein levels of the double and single CRD mutants were analyzed by Western blotting. This blot was probed with affinity-purified anti-Yap1p antiserum that was depleted for antibodies that recognize the nonspecific protein species. The double CRD mutant was grown in the absence of stress (U) or subjected to oxidative stress by exposure to diamide (D) or H₂O₂ (H) prior to preparation of protein extracts.

FIG. 8

Oxidant-specific subcellular localization of Yap1p. Low-copy-number plasmids expressing the indicated alleles of YAP1 as GFP fusion proteins were introduced into Δyap1 cells. Transformants were grown under nonstressed conditions (Uninduced) or subjected to oxidative stress elicited by diamide or H₂O₂. Living cells were then stained with DAPI and analyzed by microscopy for Yap1p localization (GFP) and DAPI fluorescence.

FIG. 9

A TRX2 YRE is capable of responding to Yap1p C629A when assayed in a heterologous promoter context. An oligonucleotide corresponding to the YRE present at position −181 in the TRX2 promoter (YRE_TRX2) was inserted into a CYC1-lacZ fusion plasmid in place of the normal CYC1 upstream sequences. This YRE_TRX2-CYC1-lacZ fusion gene was carried on a low-copy-number plasmid and introduced into a Δyap1 strain along with a second low-copy-number plasmid containing the indicated forms of Yap1p. Transformants were grown and assayed for β-galactosidase activity by using a chemiluminescent substrate as described in Materials and Methods.

FIG. 10

An intact Yap1p c-CRD region is required for normal induction of the TRX2 promoter during H₂O₂ challenge. Cells lacking the YAP1 structural gene were transformed with a low-copy-number plasmid vector (Δyap1) or the same vector expressing the wild-type or C629A form of Yap1p. Along with these three alleles of YAP1, various fusion genes carrying the indicated segments of the TRX2 promoter region placed upstream of a CYC1-lacZ fusion gene were introduced. The numbers are relative to the start of the TRX2 coding sequence. The solid vertical bars indicate the positions of the two YREs, and the single open box denotes the location of the Skn7p binding site. β-Galactosidase activities were determined by using a chemiluminescent substrate as above.

FIG. 11

A hyperactive YAP1 allele cannot bypass the Skn7p requirement of H₂O₂ activation of the TRX2 promoter. A low-copy-number plasmid expressing the indicated forms of YAP1 was introduced into Δyap1 skn7 or Δyap1 cells along with a TRX2-lacZ gene fusion. TRX2-dependent β-galactosidase activity was determined under nonstressed (No Stress) or H₂O₂-challenged conditions. (B) Aliquots of 1,000 cells of each of the transformants described in panel A were placed on YPD containing a gradient of H₂O₂ increasing from left to right.