The Saccharomyces cerevisiae AP-1 protein discriminates between oxidative stress elicited by the oxidants H2O2 and diamide.

The Saccharomyces cerevisiae AP-1 protein (yAP-1) is a key mediator of oxidative stress tolerance. Transcriptional activation by yAP-1 has been shown to be inducible by exposure of cells to H2O2 and diamide, among other oxidative stress eliciting compounds. Here we define the segments of the yAP-1 protein that are required to respond to this environmental challenge. Western blotting analyses indicated that levels of yAP-1 do not change during oxidative stress. Deletion mutagenesis and gene fusion experiments indicate that two different segments of yAP-1 are required for oxidative stress inducibility. These two domains function differentially depending on the type of oxidant used to generate oxidative stress. Three repeated cysteine-serine-glutamate sequences located in the carboxyl terminus are required for normal regulation of yAP-1 function during oxidative stress. Replacement of these cysteine-serine-glutamate repeats by alanine residues does not similarly affect H2O2 and diamide regulation of yAP-1 function. While yAP-1 transactivation is enhanced by exposure to either H2O2 or diamide, the protein responds to the oxidative stress produced by these compounds in nonidentical ways.

a key mediator of oxidative stress tolerance. Transcriptional activation by yAP-1 has been shown to be inducible by exposure of cells to H 2 O 2 and diamide, among other oxidative stress eliciting compounds. Here we define the segments of the yAP-1 protein that are required to respond to this environmental challenge. Western blotting analyses indicated that levels of yAP-1 do not change during oxidative stress. Deletion mutagenesis and gene fusion experiments indicate that two different segments of yAP-1 are required for oxidative stress inducibility. These two domains function differentially depending on the type of oxidant used to generate oxidative stress. Three repeated cysteine-serine-glutamate sequences located in the carboxyl terminus are required for normal regulation of yAP-1 function during oxidative stress. Replacement of these cysteine-serine-glutamate repeats by alanine residues does not similarly affect H 2 O 2 and diamide regulation of yAP-1 function. While yAP-1 transactivation is enhanced by exposure to either H 2 O 2 or diamide, the protein responds to the oxidative stress produced by these compounds in nonidentical ways.
Oxidative stress is a challenge faced by all cells that grow in an aerobic environment. Cellular damage resulting from oxidative stress has been implicated in a wide variety of pathological conditions including Down's Syndrome (1), familial amyotrophic lateral sclerosis (2), and cancer (3) as well as normal cellular processes like aging (4) and apoptosis (5). Not surprisingly then, cells possess a robust ability to detoxify the reactive oxygen species that mediate cellular damage by oxidative stress. Regulation of the production of this defense network has been the focus of much recent work. Studies in animal cells have shown that the activity of the transcription factors c-Jun and NF-B is modulated in response to the intracellular redox environment (6 -8). These regulatory proteins are likely to act to link oxidative stress to a downstream response to this challenge.
The yeast Saccharomyces cerevisiae serves as an invaluable model system for fundamental eukaryotic processes such as oxidative stress tolerance. As first shown by Schnell et al. (9), the YAP1 locus is a key determinant of oxidative stress tolerance. YAP1 encodes a basic region-leucine zipper (bZip) transcription factor of 650 amino acids that is a positive regulator of gene expression (9 -12). Domain mapping experiments indicate the presence of two separable transactivation domains in the protein, located between amino acids 220 -379 and 430 -537 (13). We have found that transcriptional control of GSH1 and production of normal levels of glutathione require the action of yAP-1 (14). Kuge and Jones (15) demonstrated that both an artificial yAP-1-responsive reporter and TRX2 gene expression were inducible by a variety of oxidative stress agents in a yAP-1-dependent fashion. The induction of yAP-1-dependent transactivation correlated with an increase in yAP-1 DNA binding activity.
In this work, we have explored the yAP-1 protein sequences required for oxidative stress induction of yAP-1 transactivation. Through the use of deletion mutagenesis and lexA fusion proteins, two different regions of yAP-1 have been identified that mediate the response of this protein to H 2 O 2 and diamideinduced oxidative stress. Intriguingly, while both of these two regions affect the response of yAP-1 to H 2 O 2 and diamide, the consequences of mutating either region varies with the type of oxidant used to generate stress. These data strongly suggest that oxidative stress conditions generated by H 2 O 2 and diamide are differentially sensed by yAP-1.
Plasmids-The production of the carboxyl-terminal and internal deletion series of YAP1 derivatives has been described before (13). The yap1-⌬2::hisG allele was generated by insertion of a BamHI/SalI fragment, containing hisG-URA3-hisG, from the plasmid pNKY51, into BamHI/SalI-cleaved pJAW4 (13). The resulting construct, pSM107, contains a yap1 allele lacking amino acids 64 -469. This new yap1 allele was constructed to replace the previous yap1-⌬1::HIS3 allele (20). This * This work was supported in part by National Institutes of Health Grant GM49825 and a grant-in-aid from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  was done to prevent introduction of the yeast promoters associated with each end of the BamHI fragment carrying the HIS3 gene (21) which could lead to production of truncated but still immunoreactive yAP-1 protein species. Construction of the chimeric yAP-1-VP16 fusion protein was accomplished via insertion of the VP16 transactivation domain into pSMM1 (13) that had been treated with SalI, end repaired with the Klenow fragment, and then digested with HindIII. The VP16 transactivation domain was obtained from the pCRF3 plasmid (22) as a Klenow-repaired BglII/HindIII segment. This plasmid was named pSMS17 and expressed the 219 NH 2 -terminal residues of yAP-1 fused to the VP16 transactivation domain. To express yAP-1 chimeras lacking the YAP1 promoter and NH 2 terminus, a plasmid was constructed to provide a yeast promoter and convenient cloning sites. This expression plasmid, pAK1, was generated by inserting an ADH1 promoter segment as a Klenow-repaired EcoRI/BamHI fragment into SmaI/BamHI-cleaved pRS315 (23). ADH1 transcription is directed from BamHI to SmaI. The lexA-VP16 fusion plasmid (pSMS54) was constructed by inserting a BglII/BamHI fragment from pCRF2 into BamHI-cleaved pEG202, a lexA expression plasmid (24). The lexA-VP16 fusion gene was then transferred into pAK1 as a HindIII/SalI fragment. Similarly, the lexA-yAP-1 fusion was constructed by inserting a HpaI-BamHI linker/SalI fragment from YAP1 into BamHI/SalI-cut pEG202. This lexA-YAP1 fusion protein consisted of the lexA DNA-binding domain fused to residues 156 -650 of yAP-1. This fusion gene was then transferred into pAK1 as a HindIII fragment to generate pAK2.
The alanine scanning mutations (25) were generated via polymerase chain reaction using a two-step procedure as described (26). The mutagenic primers were: Site 1, GGC TCT TTA CTA AGG GCC GCG GCA ATT TGG GAT AGA ATA ACA; Site 2, GAT GTC GAT GGT TTA GCC GCG GCA CTA ATG GCA AAG GCA AAA; Site 3, ATG GCA AAG GCA AAA GCC GCG GCA AGA GGG GTT GTC ATC AAT. Note that each alanine scanning mutation introduces a new SacII restriction site into the YAP1 coding sequence. All mutant forms of YAP1 were sequenced in their entirety to avoid introduction of spurious mutations along with the desired alanine scanning lesion. Each alanine scanning mutation was cloned as a Asp718/HindIII fragment into pJAW15 (13), replacing a wild-type version of this fragment. Presence of the desired mutation(s) was confirmed by cleavage with SacII.
Production of Anti-yAP-1 Rabbit Polyclonal Serum-Production of yAP-1 was carried out in bacteria using a histidine-tagged form of yAP-1. A BamHI/SalI fragment encoding yAP-1 residues 63-650 was inserted into the plasmid pET28bϩ (Novagen). The resulting fusion protein contained six histidine residues fused in-frame with the large yAP-1 carboxyl-terminal fragment. This fusion was then transferred to the pL promoter-containing plasmid pOTSNco12 (27) as an NcoI/XhoI fragment. This new chimera (pJAW140) contained the polyhistidine-yAP-1 fusion gene under control of the heat-inducible pL promoter. pJAW140 was transformed into an Escherichia coli strain (AR68) containing a lysogen carrying the cI857 temperature-sensitive repressor allele. Protein extracts were prepared as described (10) from heatinduced AR68 transformants carrying pJAW140. Purification of the polyhistidine-yAP-1 fusion protein was accomplished through nickel chelate chromatography as described by the manufacturer (Novagen). Purified fusion protein was then used to immunize New Zealand White rabbits using standard techniques (28). The crude antiserum was then directly used for Western blot analysis.
Western Blotting Analysis-For Western blotting, cells were grown in 50 ml of minimal media to an A 600 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 or 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 MgCl 2 , 10 mM Tris, pH 7.4, and protease inhibitors. Cell lysate was cleared and the Bradford protein assay was used to determine the protein concentration of the supernatant. 100 g of protein of each sample was run on a 5-15% polyacrylamide gradient gel. Proteins were transferred to nitrocellulose, blocked with 2.5% nonfat dry milk in phosphate-buffered saline, and probed with the anti-yAP-1 polyclonal antiserum or a mouse monoclonal antibody directed against carboxypeptidase Y (Molecular Probes, Eugene, OR). Horseradish peroxidase-conjugated secondary antibody and the ECL kit (Pierce) were used to visualize immunoreactive protein.

RESULTS
Oxidative Stress Stimulation of Wild-type yAP-1-As a reporter system for yAP-1-dependent transactivation, we em-ployed the yeast strain SM12. This strain contains a deletion allele of the chromosomal YAP1 locus and an integrated copy of a yAP-1-dependent gene fusion (ARE-TRP5-lacZ). A low-copy vector plasmid either carrying or lacking the YAP1 structural gene was introduced into the SM12 background. Appropriate transformants were then assayed for yAP-1-dependent transactivation of the ARE-TRP5-lacZ fusion gene.
Exposure of SM12 transformants carrying the wild-type YAP1 gene to the oxidative stress agents diamide, H 2 O 2 , or diethylmaleate led to a strong induction in ARE-dependent ␤-galactosidase activity (Fig. 1). The presence of a carboxylterminal deletion mutant of yAP1 that has been previously shown to be transcriptionally inactive (13)  The locations of the three cysteine-serine-glutamic (CSE) acid repeats are indicated by CSE with the number following denoting the amino acid position of the cysteine residue. B, SM12 cells (relevant genotype: yap1-⌬1::HIS3, ARE-TRP5-lacZ, ura3-52) were transformed with lowcopy plasmids expressing the amino-terminal 219 residues of yAP-1 (yAP-1-⌬219) or the wild-type form of the protein. Appropriate transformants were grown with selection for the URA3-containing plasmid to an A 600 of 0.6, induced with the indicated oxidative stress agent, and then assayed for yAP-1-dependent ␤-galactosidase activity as described (18). DEM, diethylmaleate. C, protein extracts were prepared by glass bead lysis of SM13 (relevant genotype: yap1-⌬2::hisG, ura3-52) transformants carrying either a low-copy plasmid expressing wild-type yAP-1 (wild-type) or the empty vector (vector), pRS316 (23). Transformants were grown and subjected to oxidative stress as described above. 100 g of each protein extract was electrophoresed through SDS-PAGE and processed for Western blot analysis using the anti-yAP-1 rabbit polyclonal antiserum (upper panel) or a control antibody directed against carboxypeptidase Y (CPY) (lower panel). NS denotes the presence of a protein species that cross-reacts with the anti-yAP-1 antiserum.
stress-inducible lacZ expression. These results establish that the ␤-galactosidase activity produced by the ARE-TRP5-lacZ fusion gene faithfully reproduces the previously described elevation of yAP-1-dependent transcription elicited by oxidative stress (15) and that the COOH terminus of the protein is required for this response.
Western blot analysis was next carried out to directly examine yAP-1 protein levels during oxidative stress. Protein extracts were prepared from control and oxidatively-stressed cells, electrophoresed on SDS-PAGE, 2 and transferred to nitrocellulose. The location of immunoreactive yAP-1 was detected through use of the rabbit polyclonal anti-yAP-1 antiserum. In response to oxidative stress elicited by diamide, H 2 O 2 , or diethylmaleate, steady-state levels of yAP-1 did not change (Fig.  1). Probing the same membrane with a monoclonal antibody against carboxypeptidase Y demonstrated that approximately equal amounts of protein were loaded in each sample. This observation provides definitive evidence that the elevation in yAP-1-dependent transactivation does not involve an increase in the level of yAP-1 protein. A previous examination of this issue employed Northern blotting as the assay for yAP-1 synthesis and did not directly examine protein levels of the factor (15). Additionally, pulse-chase experiments indicate that the stability of the yAP-1 protein did not change in response to oxidative stress. 3 These data establish that oxidative stress modulation of yAP-1 function occurs through post-translational modification of the factor.
A lexA-YAP1 Gene Fusion Is Responsive to Oxidative Stress-To identify the regions of yAP-1 that are required for oxidative stress induction of yAP-1 transactivation, we employed a set of carboxyl-terminal truncation mutations of this protein (13). This analysis was not informative as even a mutant form of YAP1 consisting of amino acids 1-627 was unable to significantly activate ARE-TRP5-lacZ expression, failed to complement the H 2 O 2 or diamide hypersensitivity of a ⌬yap1 strain, and was not detectable by Western blotting (data not shown). We concluded from this analysis that the carboxyl terminus of yAP-1 was indispensable for normal steady-state levels of the protein and possibly for function. To analyze and compare the role of the yAP-1 amino-and carboxyl-terminal segments in oxidative stress induction, we prepared chimeras between these yAP-1 protein domains and appropriate heterologous domains from other transcription factors. These chimeras were then tested for their effect on oxidative stress induction of appropriate reporter genes.
A carboxyl-terminal fragment of yAP-1, extending from amino acid 156 to 650, was fused to the bacterial lexA protein (29). This lexA-yAP-1 chimera was then expressed in an S. cerevisiae strain carrying a GAL1-lacZ fusion with multiple lexA operator (lexAop) sites in place of the normal GAL1 UAS (29,30). As a control for the specific oxidative stress inducibility of the lexA-yAP-1 fusion protein, a lexA-VP16 fusion protein was also expressed in these cells. ␤-Galactosidase expression from the lexAop-GAL1-lacZ fusion gene was then assayed under non-stressed and H 2 O 2 -or diamide-stressed conditions (Table I). The lexA-yAP-1 chimeric protein was able to respond to diamide induction but not to exposure to H 2 O 2 . ␤-Galactosidase activity produced in response to the presence of the lexA-VP16 fusion protein was not responsive to either oxidative stress agent. Western blotting experiments using the anti-yAP-1 antiserum indicated that the lexA-yAP-1 fusion protein was produced at equivalent levels under non-stressed and stressed conditions (data not shown). This result supports the view that the carboxyl terminus of yAP-1 is necessary and sufficient for response to diamide-induced oxidative stress. However, this same segment of yAP-1 is required but is not sufficient for the response to H 2 O 2 .
A second yAP-1 chimera was produced in which a yAP-1 amino-terminal region (residues 1-219) was fused to the strong transactivation domain from VP16 (22). We have previously demonstrated that normal yAP-1 DNA binding activity can be provided by a polypeptide extending from position 63 to 156 (10). This yAP-1-VP16 chimera was unable to induce expression of the ARE-TRP5-lacZ fusion gene after exposure to either diamide or H 2 O 2 (Table II). These data are consistent with the notion that the carboxyl-terminal segment of yAP-1 provides the major site for regulation of this factor by diamide-induced oxidative stress but neither the carboxyl or amino terminus of the protein alone is sufficient for the response to H 2 O 2 .
Mutants Lacking Internal Segments of yAP-1 Are Still Inducible by Oxidative Stress-To refine the localization of the domain(s) important in oxidative stress regulation of yAP-1, the function of a collection of internal deletion mutant proteins was analyzed. The nomenclature of this set of mutant proteins is yAP-1⌬X-Y, where X-Y refers to the range of amino acids deleted from the yAP-1 protein. Each of these internal deletion mutant proteins was assessed for the ability to activate expression of the ARE-TRP5-lacZ gene and confer oxidative stress resistance to diamide and H 2 O 2 . Additionally, the steady-state level of these polypeptides were measured by Western blot analysis under both normal or oxidative stress conditions.
Loss of the region from residues 220 to 335 produced a yAP-1 derivative with increased responsiveness to diamide induction (Fig. 2). Upon exposure to diamide, yAP-1⌬220 -335 produced 443 units/OD of ARE-dependent enzyme activity compared with 246 unit/OD produced by the wild-type protein. This result suggests the presence of a site for negative regulation of yAP-1 during diamide-induced oxidative stress. Further deletion of yAP-1 sequences from 220 to 430 caused a reduction in non-stressed levels of ␤-galactosidase to 4% of wild-type but maintained 24% of normal diamide stressed enzyme activity. The internal deletion derivative lacking the most yAP-1 sequence information (yAP-1⌬220 -469) was unable to stimulate ARE-TRP5-lacZ expression under nonstressed conditions but was still responsive to diamide. The remaining internal deletion mutants were all inducible by diamide exposure and varied in their relative ability to transactivate due to the different extents of the yAP-1 transactivation domains remaining, as seen before (13).
The activity of several internal deletion mutants varied depending on the use of H 2 O 2 or diamide as the oxidant. The yAP-1⌬220 -335 protein was not capable of significantly elevating expression of the ARE-TRP5-lacZ fusion gene in response to H 2 O 2 . A yAP-1 derivative lacking amino acids 220 -430 produced 13 units/OD of ␤-galactosidase upon H 2 O 2 challenge. 2 The abbreviation used is: PAGE, polyacrylamide gel electrophoresis. 3 S. Steggerda, unpublished data.  : leu2-3,-112, lexAop-GAL1-lacZ) was transformed with plasmids expressing the indicated fusion proteins. Appropriate transformants were grown under non-stressed conditions (no Stress) or subjected to diamide-induced (diamide) or H 2 O 2 -induced (H 2 O 2 ) oxidative stress. lexAop-dependent ␤-galactosidase activity was then determined.
b The indicated lexA fusion proteins were all expressed from the low-copy plasmid pAK1.
The level of H 2 O 2 -dependent ␤-galactosidase activity produced by this mutant protein represented 6% of wild-type H 2 O 2 -dependent expression and only 22% of the activity this same mutant elicited in the presence of diamide. A yAP-1 derivative lacking amino acids between 220 and 469 was not able to respond to H 2 O 2 -produced oxidative stress. These data strongly suggest that the 220 -335 region serves to inhibit the ability of yAP-1 to transactivate in response to diamide but is required for normal transactivation during H 2 O 2 -elicited stress. This assertion is supported by the observation that none of this group of three internal deletion mutations was able to normally complement the H 2 O 2 hypersensitivity of a ⌬yap1 mutant strain (see below).
Each internal deletion mutant allele was also tested for the ability to complement the diamide and H 2 O 2 hypersensitivity of a strain lacking the YAP1 locus (Fig. 3). As predicted from the relative degree of activation of the ARE-TRP5-lacZ fusion gene, the yAP-1⌬220 -335 protein elevated diamide tolerance relative to the wild-type factor. In agreement with its defect in H 2 O 2 induction of reporter gene expression, the yAP-1⌬220 -335 protein was not able to normally complement the H 2 O 2 hypersensitivity caused by lack of the YAP1 gene. The other two internal deletion mutant proteins lacking the 220 -335 region (yAP-1⌬220 -430 and yAP-1⌬220 -469) were both able to grow at least weakly on diamide plates but were unable to complement the H 2 O 2 hypersensitive phenotype of the ⌬yap1 strain. The remaining internal deletion mutants were able to correct both the diamide and H 2 O 2 hypersensitive phenotypes of the ⌬yap1 strain.
Western blotting analysis was also carried out on each internal deletion mutant protein to assess the relative steady-state levels of each factor (Fig. 4). Protein extracts were prepared from selected ⌬yap1 transformants expressing each mutant factor under normal and diamide-or H 2 O 2 -stressed conditions. Equal amounts of protein were resolved on SDS-PAGE and the relative levels of immunoreactive yAP-1 assessed by probing with the anti-yAP-1 antiserum.
All internal deletion mutant proteins were produced at comparable levels and migrated at the expected molecular mass, irrespective of the presence of diamide as a stress agent (Fig.  4). The same analysis was performed for selected mutant proteins using H 2 O 2 as the stress agent. The levels of the mutant proteins did not change in response to the different oxidative stress agents. We conclude that the observed differences in transactivation and inducibility of the various yAP-1 derivatives are due to alterations in the function of each mutant protein rather than the level of production.
A Repeated Amino Acid Triplet Important in Activation of yAP-1-While the above analysis of yAP-1 localized a major oxidative stress responsive segment of the polypeptide, no information is available on specific residues that might contribute to regulation of yAP-1. In other oxidative stress-responsive factors, including c-Jun (31), SoxR (32), and OxyR (33), cysteine residues have been implicated in oxidative stress modulation of function. The carboxyl-terminal 52 amino acids of yAP-1 possesses three cysteine residues, present as cysteine-serine-glutamic acid (CSE) triplet repeats. The three CSE repeats begin with cysteine at positions 598, 620, and 629 and are referred to as sites 1, 2, and 3, respectively. To assess the role of each CSE repeat in regulation of yAP-1, we prepared alanine scanning mutations in each of these CSE repeats and analyzed the function of the resulting yAP-1 derivative. Three single CSE and three double CSE alanine scanning mutations were generated.
The site 1 and site 2 mutant proteins were both defective in their ability to be regulated by oxidative stress (Table III). The site 1 mutant had a pronounced defect in the ability to activate the ARE-TRP5-lacZ fusion under normal conditions but was still able to be induced by diamide or H 2 O 2 exposure, although FIG. 2. Internal deletion derivatives of yAP-1 maintain inducibility by oxidative stress. A set of deletion derivatives of yAP-1 lacking various internal segments of the coding sequence was introduced into the SM12 strain. Transformants were grown in minimal medium with appropriate supplements and either untreated (No Stress) or subjected to oxidative stress with the addition of diamide or H 2 O 2 . After 1.5 h of stress the cells were harvested and ARE-TRP5-lacZ-dependent ␤-galactosidase activities determined (18). For each internal deletion mutant, the numbers following the ⌬ symbol denote the amino acid region deleted in each factor. The symbols depicting the locations of the previously mapped functional domains in yAP-1 are as in Fig. 1. ND, not determined. a SM12 (relevant genotype: yap1-⌬1::HIS3, ARE-TRP5-lacZ, ura3-52) cells were transformed with URA3-containing plasmids containing the indicated fusion gene. Appropriate transformants were grown in SD medium plus necessary supplements and either not treated (no stress) or subjected to oxidative stress with the addition of diamide (diamide) or hydrogen peroxide (H 2 O 2 ) as described above. ARE-dependent ␤-galactosidase activities are presented. Pdr3p-VP16 served as an unrelated DNA-binding domain-VP16 gene fusion (44).
b Each type of transcription factor listed below was introduced on a plasmid. All YAP1 derivatives were carried on a low-copy plasmid while the PDR3-VP16 gene was cloned into the 2-m vector pRS426. Vector refers to the presence of the low-copy vector pRS316 lacking any yeast transcription factor gene. the induction by H 2 O 2 was reduced to only 14% of wild-type. Loss of the CSE repeat at site 2 produced a factor that had higher than normal transactivation function under nonstressed conditions but was not significantly inducible upon oxidative stress. The site 3 mutant protein had a dramatic defect in regulation and produced 1325 units/A 600 under nonstressed conditions. The loss of the site 3 CSE led to a mutant yAP-1 protein that produced high level ARE-dependent ␤-galactosidase activity irrespective of the presence of oxidant. Combining each of the three single alanine scanning mutants into the three possible double mutants demonstrated that the effect of the site 3 lesion predominated in each of the two double mutants containing this mutation. The site 1,2 double mutant exhibited a phenotype intermediate in function between each single mutant.
Each alanine scanning mutant protein was also assessed for the ability to confer oxidative stress resistance to a ⌬yap1 strain when introduced on a low-copy plasmid vector. As expected from the behavior of the ARE-TRP5-lacZ fusion gene, all mutants lacking the site 3 CSE element exhibited a striking elevation in diamide tolerance (Fig. 5). The site 3 mutants were all more resistant to diamide than the wild-type protein. Mutant yAP-1 derivatives lacking only site 1, site 2, or both of these sites produced diamide resistance that was similar to that of the wild-type factor. Both the site 1 and site 3 single mutant factors were defective in their ability to restore H 2 O 2 resistance to a cell lacking a chromosomal YAP1 locus while a mutant protein lacking site 2 fully restored H 2 O 2 resistance. The defect in the site 1 mutant protein to confer H 2 O 2 resistance correlates well with the reduction in H 2 O 2 -induced ARE-TRP5-lacZ activity of this mutant. The inability of the site 3 mutant to correct the defect in H 2 O 2 tolerance in the ⌬yap1 strain was unexpected since we found that this mutant protein was capable of high-level transactivation of the ARE-TRP5-lacZ in the presence of H 2 O 2 . We believe this discrepancy is caused by the high levels of ␤-galactosidase elicited in the absence of oxidative stress and the marked stability of this enzyme in yeast (34). This and other possible reasons for the differential H 2 O 2 behavior of the site 3 mutant protein are discussed below. All of the three double mutant proteins were defective in their ability to complement H 2 O 2 hypersensitivity.
The steady-state level of each single CSE mutant protein was next analyzed (Fig. 6). These altered yAP-1 molecules were produced under control or diamide or H 2 O 2 -stressed conditions and subjected to Western blot analysis as described above. Mutants lacking the wild-type site 3 CSE element produced markedly reduced levels of immunoreactive yAP-1. The steadystate level of each double mutant protein was also assessed (data not shown). Alteration of sites 1 and 2, alone or in com- FIG. 3. Complementation of oxidative stress hypersensitivities of a ⌬yap1 strain by internal deletion mutants of yAP-1. SM13 cells were transformed with low-copy plasmids expressing each of the indicated forms of YAP1. Transformants of interest were grown to an A 600 of 1, diluted, and 1000 cells were placed on YPD plates containing the indicated concentrations of oxidative stress agents. Wild-type denotes the presence of a low-copy plasmid carrying the wild-type YAP1 gene, while vector represents a transformant containing the vector plasmid lacking YAP1 sequences.
FIG. 4. Steady-state levels of internal deletion derivatives of yAP-1. Western blot analysis was performed to assess the steady-state levels of the various internal deletion mutant forms of yAP-1. SM13 cells were transformed with low-copy plasmids expressing the various forms of yAP-1, subjected to oxidative stress with the addition of diamide or H 2 O 2 or untreated, and protein extracts prepared. 100 g of each protein sample was electrophoresed on SDS-PAGE and processed for Western blotting using the rabbit anti-yAP-1 antiserum. NS, nonspecific cross-reacting protein. A, assessment of the steady-state levels of mutant yAP-1 derivatives in response to diamide-induced oxidative stress. Ϫ and ϩ refer to the absence or presence of 1.5 mM diamide. A carboxyl-terminal deletion derivative of yAP-1 expressing only the amino-terminal 219 amino acids (yAP-1⌬219) was included as a control. The numbers on the left-hand side of the figure indicate molecular mass markers in kilodaltons. B, comparison of steady-state levels of selected yAP-1 derivatives in response to H 2 O 2 -induced oxidative stress. This assay was performed as described in part A with the exception that 1 mM H 2 O 2 was used to induce oxidative stress. A SM13 transformant containing the pRS316 vector lacking YAP1 sequences (pRS316) was analyzed as a negative control for yAP-1 production. bination, did not affect the level of protein produced. When double mutants were made by combining either a site 1 or 2 mutant with a site 3 lesion, the resulting mutant protein behaved indistinguishably from a protein lacking only site 3.

DISCUSSION
The transcriptional response to oxidative stress can be broken down into two phases: early or primary responses and later or secondary responses. Primary response genes are likely to act to provide rapidly acting buffers of oxidants. The function of yAP-1 in the response of S. cerevisiae cells to oxidative stress is most consistent with this factor playing a role as a primary response regulator. Transactivation by yAP-1 is strongly induced over a short time period and this induction does not require an increase in translation of yAP-1. Additionally, two known yAP-1 target genes, GSH1 (14) and TRX2 (15), both encode functions that can serve to buffer increases in the oxidizing potential inside the cell. Increased GSH1 transcription leads to elevated levels of glutathione while enhanced TRX2 synthesis produces more thioredoxin. Both of these molecules are important intracellular reductants that serve to maintain the normal reducing environment of the cell (35,36).
Detailed studies in animal cells have provided evidence that NF-B and AP-1 are likely to serve as a pair of primary and secondary response factors during oxidative stress. NF-B DNA binding activity is rapidly and strongly induced within 60 min of exposure to H 2 O 2 but is repressed by antioxidants (6). Interestingly, AP-1 DNA binding activity is weakly induced by H 2 O 2 but is highly responsive to antioxidants such as pyrrolidine dithiocarbamate (6) or thioredoxin (7). The role of yAP-1 in S. cerevisiae appears more similar to that of NF-B. Oxidants strongly induce yAP-1 transactivation while evidence has been obtained by others that antioxidants suppress yAP-1 transactivation (37).
The regulation of yAP-1 by oxidative stress exhibits both positive and negative components. These differing components are defined mutationally by the relative response of a given mutant to either diamide or H 2 O 2 exposure. The complex nature of the regulation of yAP-1 in response to oxidative stress is most clearly illustrated by the yAP-1⌬220 -335 protein. This mutant protein exhibits greater than wild-type levels of diamide tolerance and ARE-TRP5-lacZ expression. However, the same mutant factor exhibits the opposite behavior in response to H 2 O 2 . The yAP-1⌬220 -335 factor is less effective than wildtype yAP-1 in correcting the H 2 O 2 hypersensitivity of a ⌬yap1 strain and shows a defect in stimulating ARE-TRP5-lacZ expression in response to H 2 O 2 . These data indicate that this segment of the yAP-1 protein serves as a site for negative control of function during diamide-induced stress but acts as a positive regulatory site upon H 2 O 2 -induced oxidative challenge.
A second region of yAP-1 also exhibits this differential regulatory functionality. The site 3 CSE mutant (CSE629AAA) is more effective than the wild-type protein in terms of conferring diamide tolerance to the ⌬yap1 strain and activating diamideinduced ARE-TRP5-lacZ expression. However, this same mutant protein is defective in its ability to correct the H 2 O 2 hypersensitivity of a ⌬yap1 strain although still producing high levels of ARE-TRP5-lacZ-dependent ␤-galactosidase activity. There are two likely reasons for this discrepancy between H 2 O 2 resistance and transactivation mediated by the CSE629AAA mutant protein. Since the H 2 O 2 -dependent transactivation of the CSE629AAA mutant factor is assessed after 1.5 h of exposure to the oxidant, it is possible that this is insufficient time for the high levels of pre-existing ␤-galactosidase to turn over and exhibit the expected reduction in activity relative to the normal factor. Owing to the extremely stable nature of E. coli ␤-galactosidase expressed in yeast (t1 ⁄2 Ͼ 20 h (34)), this is a strong possibility. A second possibility is that the CSE629AAA mutant protein is not normally able to activate transcription of FIG. 5. Oxidative stress tolerance of CSE mutants. SM13 cells were transformed with the low-copy plasmids expressing the various CSE alanine scanning mutant forms of yAP-1. Selected transformants were grown in minimal medium and processed for spot test analysis as described in the legend to Fig. 3. The CSE mutation present in each form of yAP-1 is listed to the left. Oxidative stress agents were added to YPD medium at the indicated concentrations.  the relevant target gene(s) for H 2 O 2 resistance although it is capable of activating the ARE-TRP5-lacZ reporter gene. We view this as less likely since all mutant yAP-1 derivatives we have examined exhibit strong correlation between the ability to activate the ARE-TRP5-lacZ reporter gene and the ability to confer resistance to cadmium, cycloheximide, diamide, and H 2 O 2 , with the exception of the CSE629AAA mutant protein and H 2 O 2 resistance. 4 Discrimination between these and other possible explanations requires further experimentation.
An unexpected outcome of these studies is the finding that the same mutant derivative of yAP-1 behaves differently in response to two different oxidants, H 2 O 2 and diamide. This result suggests that the stresses generated by each type of oxidant are perceived differently by yAP-1. An important issue raised by this observation is the nature of the signal that leads to activation of yAP-1 during oxidative stress. Diamide acts to both deplete glutathione pools and oxidize thiol groups (38) while H 2 O 2 has several effects including lipid peroxidation, protein oxidation, and DNA damage (39). Although both of these oxidants elicit oxidative stress, their respective intracellular effects are non-identical. Perhaps the transcriptional regulatory properties of yAP-1 vary in response to the type of oxidative stress to which the cell is subjected. Confirmation of this suggestion awaits identification of the relevant downstream target genes of yAP-1.
The possibility that cysteine residues are involved in control of yAP-1 function is interesting in light of studies in E. coli where several different oxidant-sensitive transcription factors use cysteines as part of their redox sensing apparatus. Both SoxR and FNR contain cysteine-bound iron-sulfur clusters that are essential for their function (40 -43). OxyR contains six cysteine residues, only one of which is required for regulation by oxidative stress (33). Our mutagenesis of the CSE repeats in yAP-1 indicates that loss of any of these cysteine-containing sequences alters the function of the protein. The possibility that the carboxyl-terminal cysteine residues are involved in the response to oxidative stress is attractive as a mechanism of redox control of yAP-1 function. However, since multiple amino acids were replaced in the alanine scanning mutations, we cannot ascribe consequences of these lesions to the specific loss of cysteine. We have recently constructed a C629A derivative of yAP-1 which, similar to the CSE629AAA protein, exhibits highlevel constitutive expression of the ARE-TRP5-lacZ fusion and enhanced diamide tolerance. 5 This finding is consistent with the notion that the cysteine residues in the yAP-1 carboxyl terminus play a major role in the regulation of the factor by oxidative stress.
A central issue that remains to be clarified is the mechanism of yAP-1 regulation. The data presented here demonstrates that the sequence requirements for response to oxidative stress generated by H 2 O 2 are more extensive than those for response to diamide. Additionally, at least two different domains (⌬220 -335, CSE629AAA) of the protein can be mutationally altered to give rise to an enhanced diamide but reduced H 2 O 2 resistance phenotype. These findings strongly suggest that the mechanisms underlying the response of yAP-1 to diamide or H 2 O 2induced oxidative stress will not be the same. A central goal of future work will be to elucidate the molecular details behind oxidative stress activation of yAP-1.