INTRODUCTION

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To grow in the presence of oxygen, cells must be able to deal with reactive oxygen species (ROS) that are produced during metabolism. Aerobes have the ability both to detoxify ROS and to repair macromolecules that are damaged by these highly reactive compounds (26). Owing to the potentially lethal action of ROS, cells maintain constant surveillance of intracellular ROS levels and rapidly activate the expression of loci involved in oxidative stress tolerance (7).

The yeast Saccharomyces cerevisiae has been a useful model for studies of the eukaryotic response to oxidant challenge (15). S. cerevisiae produces a variety of enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, and small molecules and peptides (glutathione and thioredoxins) that detoxify ROS (reviewed in reference 10). Recent data have provided insight into the regulation of the biosynthesis of these enzymatic activities.

One of the key regulators of oxidative stress tolerance in S. cerevisiae is the Yap1p transcription factor. The YAP1 gene is required for normal tolerance to a wide variety of oxidants and is essential for normal synthesis of a variety of antioxidant activities, including glutathione and glutathione reductase (4, 5, 19, 27). Later studies established that Yap1p-dependent transactivation was markedly enhanced when cells were challenged with oxidants including diamide and H₂O₂ (13). Western blotting experiments demonstrated that the increased activity of Yap1p was probably due to a posttranslational modification of the factor (14, 24). Mutational analyses have implicated a set of three cysteine residues contained in the C-terminal cysteine-rich domain (c-CRD) of the factor as being required for the normal elevation of Yap1p transactivation upon oxidative stress (14, 24). Cysteine residues have been implicated as likely sensors of the reducing environment of cells in other redox-regulated transcription factors (reviewed in reference 22). Recent studies have implicated the c-CRD in maintaining Yap1p in the cytoplasm until oxidant challenge drives the protein into the nucleus (14, 29). Deletion of the c-CRD produced a mutant Yap1p that appeared to be constitutively localized in the nucleus and conferred a hyperresistance phenotype to diamide (14). These data led to the model that regulation of Yap1p by the redox state of the cell was via oxidant-regulated nuclear localization regulated by the c-CRD of the protein (14, 29).

In this work, we demonstrate that the regulatory role of the Yap1p c-CRD cannot be solely to modulate nuclear localization of the factor. Loss of the c-CRD results in a Yap1p derivative that confers hyperresistance to diamide but hypersensitivity to H₂O₂. This phenotypic pattern is reproduced by a number of different mutant forms of Yap1p lacking normal c-CRD regions. A second CRD, in the amino terminus of Yap1p (n-CRD), is also required for normal oxidative stress regulation of the protein. Finally, we show that while Yap1p c-CRD mutants activate the expression of a synthetic reporter gene to high levels, these mutant derivatives fail to normally activate the promoter of a gene required for H₂O₂ tolerance. These data argue that mutants lacking wild-type CRD regions are defective in an additional regulatory function required for activation of transcription at promoters involved in H₂O₂ resistance.

	MATERIALS AND METHODS

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Yeast methods. The yeast strains used in this study were SEY6210 (MAT leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801 suc2-9 Mel), SM12 (MAT leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801 suc2-9 Mel yap1-1::HIS3 ARE-TRP5-lacZ), SM13 (MAT leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801 suc2-9 Mel yap1-2::hisG), YSC6 (MAT leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801 suc2-9 Mel- yap1- 2::hisG ARE-TRP5-lacZ), and YSC18 (MAT leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801 suc2-9 Mel yap1-2::hisG TRX2-lacZ). YSC6 was constructed by cutting pTEP9 (ARE-TRP5-lacZ) with KpnI (to direct recombination to LEU2) and transforming SM13 to Leu2⁺. YSC18 was generated by cutting pSC99 containing TRX2-lacZ with NheI and integrating this linear plasmid in the HIS3 gene of SM13. YSC6 and YSC18 were both checked by Southern blotting to confirm proper chromosomal structure of the recombinants. YSC25 was constructed by transforming SEY6210 with SacI-cleaved pGC61 (skn7::TRP1). YSC26 (skn7 yap1) was produced by transforming YSC25 with a Asp718-SacI fragment containing yap1-2::hisG-URA3-hisG. Ura3⁺ transformants were treated with 5-fluoroorotic acid to remove the URA3 gene. Yeast cells were grown either in rich, nonselective medium (yeast extract-peptone-dextrose medium [YPD]), minimal medium (synthetic dextrose [SD]) with required supplements, or SD supplemented with Casamino Acids (20). Transformation was performed by the lithium acetate technique of Ito et al. (9). Assays for -galactosidase activity were carried out on permeabilized cells as described previously (6) or with a luminescent substrate for detection of low levels of -galactosidase activity as described by the manufacturer (Clontech). Diamide and H₂O₂ resistance assays were carried out by spot tests (28).

Plasmids. The integrating ARE-TRP5-lacZ construct pTEP9 has been described previously (25). Low-copy-number plasmids carrying the wild-type or CSE629AAA forms of YAP1 (pSM58wt and pSMS37, respectively) were constructed previously (24). The YAP1 deletion mutant plasmids pJAW1 and pJAW15 were described previously (25). All PCR products were sequenced in their entirety to ensure that no errors had occurred during amplification.

Construction of a YAP1 mutant library and selection of hyperactive YAP1 alleles. The c-CRD-encoding region of YAP1 was subcloned as an Asp718-HindIII fragment into pBluescript KSII+ to form pSMS26. A 20-µg portion of this plasmid was then mutagenized with 100 µl of 45% formic acid for 10 min. DNA was recovered by ethanol precipitation, and the carboxy terminus of YAP1 was amplified by PCR with flanking T3 and T7 primers. The PCR product was cleaved with Asp718-HindIII and ligated into the context of wild-type YAP1. The resulting library was amplified in bacteria, and plasmid DNA was generated.

Western blotting analysis. Cells were grown in 100 ml of minimal medium to an absorbance at 600 nm (A₆₀₀) of 0.6, drug was added, and cells were incubated for an additional 1.5 h. Diamide or hydrogen peroxide was added to give final concentrations of 1.5 mM and 1.0 mM, respectively. Cells were harvested, washed, and broken by glass bead lysis in buffer containing 300 mM sorbitol, 100 mM NaCl, 5 mM MgCl₂, 10 mM Tris (pH 7.4), and complete protease inhibitors (Boehringer Mannheim). Cell lysates were cleared, and the Bradford protein assay (Bio-Rad) was used to determine the protein concentration of the supernatant. Portions (50 or 100 µg) of protein from each sample were run on an 8% polyacrylamide gel. The proteins were transferred to nitrocellulose, blocked with 2.5% nonfat dry milk in phosphate-buffered saline, and probed with the anti-Yap1p polyclonal antiserum. Horseradish peroxidase-conjugated secondary antibody and the ECL kit (Pierce) were used to visualize immunoreactive protein. The affinity-purified Yap1p antiserum was prepared by passing serum over columns of protein extract prepared from a yap1 strain or bacterially purified Yap1p linked to Sepharose beads as specified by the manufacturer (Pierce Amino-link).

Fluorescence microscopy analysis. Transformants expressing GFP-Yap1p fusions of interest were grown and subjected to oxidative stress as described above. After the desired stress regimen had been imposed, the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) to visualize DNA. Cells were then viewed with an Olympus BX60 fluorescence microscope. Images were captured with a Hammamatsu ORCA charge-coupled device camera and processed with Adobe Photoshop 5.0 software.

	RESULTS

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Alanine-scanning mutagenesis of the CSE629 repeat. Previous studies from our laboratory (24) and others (14, 29) have implicated cysteine residues in the c-CRD as being critical for the normal response of Yap1p to oxidant challenge. These cysteines in the Yap1p c-CRD are present as three CSE repeat clusters, and our data suggested that the most C-terminal of these clusters (CSE629) played a major role in regulation of Yap1p function (24). However, these mutagenesis experiments involved replacing all three amino acids of the CSE repeats with alanine residues and precluded an individual assessment of the regulatory contribution of each individual amino acid. To explore the necessity of each position in the CSE629 repeat for normal control of Yap1p function, each amino acid was individually replaced with an alanine residue. The resulting single-amino-acid substitution mutants were then assayed for their ability to transactivate a Yap1p-dependent lacZ reporter gene, oxidative stress phenotypes, and steady-state protein levels.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Transactivation of ARE-TRP5-lacZ expression by alanine scanning mutations in CSE629. Strain YSC6 (yap1-2::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 -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.

View larger version (52K): [in this window] [in a new window]   FIG. 2.   Phenotype and expression of alanine-scanning mutations in CSE629. (A) Cells lacking the YAP1 gene (yap1) 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) yap1 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).

Random mutagenesis of the Yap1p C terminus. To gain additional insight into the amino acids required for normal function of the Yap1p C terminus, we generated a collection of random mutations in a restriction fragment encoding this region of the factor (see Materials and Methods for details). This restriction fragment was then reintroduced into an otherwise unaltered YAP1 gene carried on a low-copy-number plasmid. This mutant plasmid pool was transformed into a yap1 strain carrying the ARE-TRP5-lacZ gene, and transformants that were hyperresistant to diamide were selected. Cells that exhibited enhanced diamide tolerance were then assayed for their levels of ARE-directed -galactosidase activity. Plasmids were recovered from colonies that expressed elevated -galactosidase levels and tested for their ability to retransform both the diamide hyperresistance and the high-level ARE-TRP5-lacZ expression on a fresh version of the original yap1 strain. Plasmids that were able to retransform these traits were then subjected to DNA sequence analysis to identify the altered position in the C terminus.

View larger version (20K): [in this window] [in a new window]   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 yap1 strain containing the ARE-TRP5-lacZ reporter gene, as described for Fig. 1. Levels of ARE-dependent -galactosidase activity were determined in the unstressed cells (No Stress) and in cells subjected to diamide- or H2O2-induced oxidative stress.

View larger version (54K): [in this window] [in a new window]   FIG. 4.   Oxidative stress phenotypes and expression level of random mutants with mutations in the c-CRD. (A) yap1 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.

An N-terminal cysteine-rich domain regulates Yap1p function. Analysis of an internal-deletion derivative of Yap1p that lacked residues 220 to 335 (Yap1p 220-335) indicated that this segment of the protein was required for normal regulation (24, 25). Loss of the region from residues 220 to 335 resulted in a Yap1p derivative that conferred hyperresistance to diamide but failed to provide normal H₂O₂ resistance. A striking feature of the region of Yap1p from residues 220 to 335 was the presence of the only other three cysteine residues in the protein. Since the cysteine residues in the c-CRD play important roles in regulation of Yap1p, we prepared a series of mutations to examine in more detail the possible role of these N-terminal cysteines in control of Yap1p function. Each mutant factor was expressed from a low-copy-number plasmid and assayed for the ability to complement both the ARE-TRP5-lacZ expression levels and oxidative stress phenotypes of a yap1 strain (Fig. 5 and 6).

View larger version (16K): [in this window] [in a new window]   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 -galactosidase activity was determined for each construct as described in the legend to Fig. 1.

View larger version (42K): [in this window] [in a new window]   FIG. 6.   Oxidant resistance and expression profiles of n-CRD mutant proteins. yap1 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.

Genetic interaction of two negative regulatory domains in Yap1p. The above data strongly suggested that both the n-CRD and c-CRD are crucial for the normal response to oxidants. To evaluate if these two spatially separate regulatory domains control Yap1p activity through a common regulatory step, we constructed a double-mutant factor lacking the region from residues 220 to 335 and carrying the CSE629AAA allele. This double-mutant factor (Yap1p 220-335/CSE629AAA) was introduced into yap1 strains carrying the ARE-TRP5-lacZ reporter gene. Plasmids expressing the 220-335 or CSE629AAA forms of Yap1p alone were assayed as controls.

View larger version (43K): [in this window] [in a new window]   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 yap1 ARE-TRP5-lacZ reporter strain and assayed for ARE-dependent -galactosidase activity as described in the legend to Fig. 1. (B) Oxidative stress resistance phenotypes of yap1 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.

Trafficking information for Yap1p is contained in both the n- and c-CRD regions. Previous studies have demonstrated that Yap1p subcellular localization changes from primarily cytoplasmic in the absence of stress to nuclear when cells are challenged with either diamide or diethylmaleate (14). We wanted to examine the localization of Yap1p in response to mutations in the n-CRD and H₂O₂ challenge, two issues that have not been previously explored. To facilitate evaluation of the Yap1p subcellular location, we used PCR to insert the GFP after the ATG codon of the wild-type YAP1 gene. The resulting GFP-Yap1p chimera was carried on a low-copy-number plasmid. Mutant coding sequences were introduced into this fusion gene in vitro and then transformed into a yap1 strain carrying the ARE-TRP5-lacZ reporter gene.

View larger version (95K): [in this window] [in a new window]   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 yap1 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.

Differential activation of a TRX2-lacZ reporter gene in response to oxidant exposure. One complicating feature of the behavior of several mutant Yap1p derivatives characterized in this work and previously (24) was the ability of some mutants (such as Yap1p K626X [Fig. 4]) to strongly activate ARE-TRP5-lacZ expression in the presence of H₂O₂ but their inability to confer even normal H₂O₂ resistance on a yap1 strain. One hypothesis to explain this discrepancy was that a unique action of the Yap1p C terminus was required at promoters involved in H₂O₂ tolerance but not at promoters of genes playing a role in diamide resistance. To directly test this idea, we analyzed the ability of several different mutant derivatives of Yap1p to stimulate the expression of a gene involved in H₂O₂ resistance, TRX2.

TRX2-lacZ yap1

The TRX2 YRE is capable of responding to Yap1p C629A. To examine if the failure of a c-CRD mutant form of Yap1p to elicit overproduction of TRX2 was due to some property of the YRE in TRX2, we examined the ability of a YRE in the TRX2 promoter to serve as a Yap1p-dependent upstream activator sequence of a heterologous promoter. An oligonucleotide that corresponds to the TRX2 YRE at position 181 in the promoter of this gene was synthesized. This oligonucleotide (YRE_TRX2) was placed upstream of a CYC1-lacZ promoter in place of the normal CYC1 upstream activator sequence elements. The resulting construct was then introduced into yap1 cells along with a low-copy-number plasmid expressing either the wild-type or C629A forms of Yap1p. The vector plasmid alone was included as a control. Transformants were grown in selective media and YRE_TRX2-dependent -galactosidase activity determined as described above.

View larger version (21K): [in this window] [in a new window]   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 (YRETRX2) was inserted into a CYC1-lacZ fusion plasmid in place of the normal CYC1 upstream sequences. This YRETRX2-CYC1-lacZ fusion gene was carried on a low-copy-number plasmid and introduced into a yap1 strain along with a second low-copy-number plasmid containing the indicated forms of Yap1p. Transformants were grown and assayed for -galactosidase activity by using a chemiluminescent substrate as described in Materials and Methods.

The 141 to 61 region of the TRX2 promoter blocks H₂O₂ induction in the absence of a normal c-CRD region of Yap1p. To map the region of the TRX2 promoter that requires the function of the Yap1p c-CRD for induction, we fused various segments of the 5'-flanking DNA of TRX2 to a CYC1-lacZ fusion gene lacking its normal upstream activator sequences. These TRX2-CYC1-lacZ fusion genes were introduced into a yap1 strain along with a low-copy-number plasmid vector expressing wild-type or C629A mutant forms of Yap1p. -Galactosidase activities under unstressed and H₂O₂-challenged conditions were then determined (Fig. 10).

View larger version (20K): [in this window] [in a new window]   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 (yap1) 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. -Galactosidase activities were determined by using a chemiluminescent substrate as above.

View larger version (34K): [in this window] [in a new window]   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 yap1 skn7 or yap1 cells along with a TRX2-lacZ gene fusion. TRX2-dependent -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.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Control of the activity of the positive transcriptional regulatory protein Yap1p is one of the key steps modulating the response of S. cerevisiae to oxidative stress. While the action of Yap1p as a positive regulator of gene expression is required for normal tolerance to the oxidants diamide and H₂O₂, the responses of Yap1p to these oxidants exhibit important differences. These differential responses are most clearly seen in the analysis of mutant forms of Yap1p. A number of lesions that either perturb (C629A) or remove (K626X) the c-CRD in Yap1p produce derivatives that confer hyperresistance to diamide yet fail to provide normal H₂O₂ tolerance to a yap1 strain. These findings strongly suggest that the role of the Yap1p c-CRD is different in the face of the distinct oxidative challenge elicited by either diamide or H₂O₂. Diamide is believed to shift the pool of glutathione into a predominantly oxidized state (12), while H₂O₂ is a promiscuous oxidizing agent that acts to damage many different macromolecules in the cell (2). Since the modes of oxidative damage induced by these two agents are not identical, the cellular responses to these stresses are likely to be different. At least part of this selective cellular response to these oxidants is programmed into the oxidant-specific behavior of Yap1p.

The oxidant-specific behavior of the Yap1p c-CRD has important implications for the physiological action of this domain of the protein. Previous work has shown that the c-CRD is involved in the control of Yap1p nuclear localization (14), probably at the level of nuclear export (29). These studies led to the proposal that the Yap1p c-CRD acted to enhance nuclear export in the absence of oxidants but that Yap1p would accumulate in the nucleus upon oxidant challenge (14, 29). Our findings indicate that while c-CRD-regulated localization of Yap1p may be a role of this domain of the factor, it clearly is not the sole activity of this region.

In addition to the already recognized role of the c-CRD in regulation of Yap1p activity, we provide evidence that the amino terminus of Yap1p is also required for the appropriate redox response of the factor. Importantly, the n-CRD also contains information that is required for normal trafficking of Yap1p. Loss of small segments of Yap1p (residues 220 to 243 or 220 to 307) or a single-amino-acid substitution in the n-CRD leads to constitutive nuclear localization of the resulting mutant protein. However, a larger deletion mutant (Yap1p 220-335) is normally excluded from the nucleus in the absence of oxidative challenge. These data suggest that the n-CRD contains nuclear targeting information that is normally masked in the absence of oxidative stress. Loss of the sequences between positions 220 and 243 or the C303A mutation unmasks this targeting determinant, and the mutant proteins are found in the nucleus. A larger deletion mutant lacking residues 220 to 335 is found in the nucleus only upon oxidant challenge. This derivative (Yap1p 220-335) may lack the targeting information that was unmasked by the two more amino-terminal mutations.

Along with this newly identified role in redox-dependent trafficking of Yap1p, the n-CRD is a key contributor to the ability of the protein to activate transcription of a subset of downstream target genes. A wild-type n-CRD is essential for normal H₂O₂ tolerance but dispensable for diamide resistance. This finding suggests that genes involved in H₂O₂ resistance require both CRD regions to be normally activated by Yap1p but that genes required for diamide resistance do not. Previous mutagenesis experiments have identified other substitution mutations in both CRD regions that confer an H₂O₂ sensitive phenotype on cells and prevent normal activation of TRX2 expression upon H₂O₂ challenge (23). Interestingly, loss of the n-CRD leads to a H₂O₂-specific defect in the ability of the resulting mutant Yap1p to activate both the ARE-TRP5-lacZ and the TRX2-lacZ reporter genes whereas c-CRD mutations prevent induction only of TRX2 transcription in the presence of H₂O₂. These data suggest that Yap1p-mediated transactivation requires normal n-CRD function at all promoters but that c-CRD function is specifically needed to induce expression of genes involved in H₂O₂ tolerance.

To determine the possible regulatory relationship of these two domains of Yap1p, we constructed a mutant derivative that lacked both the n-CRD (Yap1p 220-335) and c-CRD (Yap1p CSE629AAA). This was done to determine if the n-CRD could still influence Yap1p function in the absence of c-CRD function. One possible model explaining the roles of the two CRD regions would be that the n-CRD acts simply to modulate c-CRD function. In the absence of a functional c-CRD, n-CRD mutations would not be expected to influence function of the resulting Yap1p derivative. Contrary to this prediction, our results suggest that both CRD regions play critical roles in Yap1p function and regulation. The mutant lacking both the n-CRD and c-CRD regions (Yap1p 220-335/CSE629AAA) is more active than either single mutant. Both of the CRD regions act to repress the factor during conditions of diamide stress, and both are required for Yap1p to normally mediate resistance to H₂O₂ stress.

The analysis of the c-CRD was complicated by the observation that most c-CRD mutants behaved as constitutive activators of the ARE-TRP5-lacZ reporter gene but failed to normally complement the H₂O₂ hypersensitivity of the yap1 mutant. Our finding that the same c-CRD mutants that drive greater expression of the ARE-TRP5-lacZ reporter than wild-type Yap1p fail to properly regulate transcription of the TRX2 gene clarifies this apparent paradox. The ARE-TRP5-lacZ reporter serves as a model promoter for the response of isolated YREs to Yap1p. This synthetic promoter accurately predicts the diamide resistance phenotype of all Yap1p mutant derivatives that we have analyzed. Yap1p mutants that drive high-level expression of ARE-TRP5-lacZ also confer a strong diamide resistance phenotype on yap1 cells. However, this artificial reporter fails to predict the corresponding H₂O₂ resistance phenotype of the same mutants. When c-CRD mutants are analyzed for their ability to induce transcription of TRX2 in response to H₂O₂, a marked defect is observed (Table 1).

We believe that these observations suggest that the mode of transactivation of Yap1p at promoters involved in diamide tolerance is different from that at promoters involved in H₂O₂ resistance. Transcriptional activation of genes involved in diamide tolerance seems to be able to be reduced to the behavior of an isolated YRE. Up-regulation of genes required for H₂O₂ resistance is more complex and must be examined in the context of the native promoter. This is illustrated by the analysis of the H₂O₂ induction of the TRX2 promoter by Yap1p. A mutant Yap1p lacking an intact c-CRD region cannot induce TRX2 in the presence of H₂O₂ unless a region of the TRX2 promoter is removed. The presence of this inhibitory region of TRX2 also prevents the otherwise hyperactive C629A form of Yap1p from driving high-level constitutive expression of TRX2. These data suggest the presence of a binding site for a factor that interacts with the Yap1p c-CRD before activation of TRX2 can occur. This behavior is a unique feature of TRX2, since other Yap1p target genes exhibit dramatic increases in expression if the c-CRD is defective (1, 23). Current work focuses on identification of the factor binding this TRX2 inhibitory site.

Finally, it is important to contrast the physiological significance of tolerance to either diamide or H₂O₂. While both of these oxidants are useful tools to elicit oxidative stress in the laboratory, H₂O₂ is arguably the more relevant phenotype to S. cerevisiae cells in particular and fungi in general. Neutrophils, a major host defense against fungal infections, eliminate invading fungal cells through oxidative killing (reviewed in reference 17). While there are several different redox-active chemicals that can be produced by neutrophils, a major oxidant is H₂O₂ (11). Understanding how fungal cells tolerate H₂O₂ toxicity will have important implications in the treatment of pathogenic fungal infections.