A New Antioxidant with Alkyl Hydroperoxide Defense Properties in Yeast*

To isolate new antioxidant genes, we have searched for activities that would rescue the tert-butyl hydroperoxide (t-BOOH)-hypersensitive phenotype of aSaccharomyces cerevisiae strain deleted for the gene encoding the oxidative stress response regulator Skn7. We report the characterization of AHP1, which encodes a 19-kDa protein similar to the AhpC/TSA protein family within a small region encompassing Cys-62 of Ahp1p and the highly conserved N-terminal catalytic AhpC/TSA cysteine. Ahp1p contains a peroxisomal sorting signal, suggesting a peroxisomal localization. AHP1 exerts strong antioxidant protective functions, as demonstrated both by gene overexpression and deletion analyses, and is inducible by peroxides in an Yap1- and Skn7-dependent manner. Similar to yeast Tsa1p, Ahp1p forms a disulfide-linked homodimer upon oxidation and in vivo requires the presence of the thioredoxin system but not of glutathione to perform its antioxidant protective function. Furthermore, in contrast to Tsa1p, which is specific for H2O2, Ahp1p is specific for organic peroxides. Therefore, with respect to substrate specificity, Ahp1p differs from Tsa1p and is similar to prokaryotic alkyl hydroperoxide reductase AhpC. These data suggest that Ahp1p is a yeast orthologue of prokaryotic AhpC and justifies its name of yeast alkyl hydroperoxide reductase.

The incomplete reduction of molecular oxygen during respiration and the lipid metabolism in peroxisomes leads to the formation of reactive oxygen species (ROS) 1 (1). ROS are potent oxidants and can damage all cellular constituents (2). They cause DNA base modifications and strand breaks and are therefore mutagenic. They can damage proteins and inactivate enzymes. Oxidation of membrane lipids can initiate free radical chain reactions, which alter cellular membranes and give rise to other very toxic reactive species such as lipid hydroperoxides, alkoxyl and peroxyl radicals, lipid epoxides, and aldehydes. To protect against the toxicity of ROS, aerobic organisms use an array of defense mechanisms (1,3,4). Among these, cytosolic and mitochondrial superoxide dismutases eliminate the O 2 . radical, cytosolic and peroxisomal catalases remove H 2 O 2 , and glutathione peroxidases reduce both H 2 O 2 and alkyl hydroperoxides. The AhpC/TSA family, also referred to as peroxiredoxin, is a large family of newly discovered peroxidases that are highly conserved from prokaryotes to eukaryotes (5) and act to reduce hydroperoxides with electrons donated by NADPH via thioredoxin or other thiol-containing intermediates (6 -8). Antioxidant defense mechanisms are induced as part of global cellular adaptive responses to oxidative stress (9 -12). In Saccharomyces cerevisiae, the cellular response to hydroperoxides is associated with the induction of a very large stimulon of at least 115 proteins (13), which involves, at least in part, the transcriptional regulators Yap1 (14,15) and Skn7 (16,17). Strains inactivated for either one of these regulators are hypersensitive to killing by H 2 O 2 and by several other oxidants (15,16,18). Yap1 controls the expression of GSH1-encoded ␥-glutamylcysteine synthetase (19) and of GLR1-encoded glutathione reductase (20) and co-operates with Skn7 to activate the expression of TRX2-encoded thioredoxin and TRR1-encoded thioredoxin reductase (17).
To gain insight into the molecular physiology of the yeast oxidative stress response, we have attempted to identify new genes involved in the cellular defense against alkyl hydroperoxides and possible targets of Yap1 and Skn7. As an approach to this question, we have used a genetic selection for identifying genes, which could rescue the alkyl hydroperoxide hypersensivity of an skn7 null strain. We report the identification of one of the genes isolated from this selection, which we have named AHP1 (see below). Evidence is provided which demonstrates that the AHP1 gene product is a yeast antioxidant related to the AhpC/TSA peroxiredoxin family with alkyl hydroperoxide defense properties.

Selection of Yeast cDNAs
A S. cerevisiae cDNA library under control of the repressible GAL1 promoter in the centromeric vector pRS316 (29) was transformed into strain yMT2 (skn7::TRP1). Transformed cells were plated onto SMG lacking uracil and containing 1 M sorbitol. URA3 transformants were pooled from each plate separately, diluted, and then plated onto SMGal lacking uracil and containing t-BOOH (0.5 mM). Plates were incubated at 30°C for 5-12 days. Plasmids recovered from viable colonies were analyzed by restriction digest and sequencing. Sequencing was performed using an automated Perkin-Elmer DNA sequencer according to the recommendations of the manufacturer.

Sensitivity Assays
Patch Assays-Aliquots (10 l) containing approximately 10 3 or the indicated cell number of an overnight culture were spotted on YPD plates containing oxidants at the indicated concentration. Plates were monitored after 3-6 days of incubation at 30°C.
Challenge Assay-Cells from an exponential growth culture were washed, resuspended in phosphate-buffered saline at a density of OD 600 of 0.1, and not exposed or exposed to peroxides at different concentrations for 1 h. Cells were then diluted and plated for colony survival.

Northern Blot Analysis
Yeast cells from overnight culture were diluted to an OD 600 of 0.01 in SC medium and incubated with shaking at 30°C until they reached an OD of 0.3. The cells were then aliquoted and incubated in the absence or in the presence of H 2 O 2 (0.2 mM) or t-BOOH (0.2 mM) for 20 min. Total RNA was prepared by the hot phenol method (30). For each condition tested, 20-g RNA samples were loaded per lane on an agarose gel containing formaldehyde, separated by electrophoresis, transferred to a Bio-Rad nylon membrane, and hybridized with an an AHP1 32 P-labeled DNA specific probe produced by random priming (Boehringer Mannheim). Hybridization of each blot with a small nuclear RNA U3 (SNR17A)-specific 32 P-labeled DNA probe served as a RNA loading control. Prehybridization, hybridization, and washes were carried out as described elsewhere (26). Hybridized membrane were exposed for autoradiography.

Two-dimensional Gel Electrophoresis and Protein Spot Identification
Two-dimensional gel electrophoresis and protein spot identification were performed as described elsewhere (13). Alkylation of protein free sulfhydryls before the second dimension was achieved as follows. The first dimensional gel rod was first reduced by a 10-min incubation in buffer A (30% glycerol, 6 M urea, 2.5% SDS, 0.001% bromphenol blue, 0.2 M Bis-Tris, 0.1 M HCl, and 67 mM dithiothreitol (DTT)). Alkylation was then performed by incubating the gel rod in buffer in which DTT was omitted, and iodoacetamide (222 mM) was added instead.

Selection of Genes
Increasing the Tolerance to t-BOOH of a skn7 Null Strain-A S. cerevisiae strain carrying a skn7 null mutation fails to grow on medium containing 0.5 mM of t-BOOH (a synthetic alkyl hydroperoxide), whereas the isogenic wild type strain will tolerate up to 1.5 mM of this oxidant. We have searched for genes whose overexpression would rescue the t-BOOH hypersensitivity of the skn7 null strain using a galactose-inducible yeast cDNA library (29). This library was used to bypass the possible Skn7 requirement for the expression of candidate genes. We expected to identify genes controlled by Skn7 and/or encoding alkyl hydroperoxide antioxidant defense activities. Strain yMT2 (skn7::TRP1) was transformed with the GAL1-controlled cDNA library. Forty thousand transformants were obtained and incubated on plates containing 0.5 mM t-BOOH. Viable colonies were recovered from these plates after 7 days and the corresponding library plasmids isolated. Five plasmids, able to increase the t-BOOH tolerance of the yMT2 strain upon retransformation, were shown to carry inserts of different sizes, but containing the same ORF. One of them, pY1, was further analyzed. pY1 was able to increase the tolerance to t-BOOH not only of yMT2 but also of yMT1 (a yap1 null strain) (Fig. 1). Therefore, the gene carried on the pY1 plasmid has antioxidant properties. This gene was called AHP1 for alkyl hydroperoxide reductase (see below) and further analyzed. The AHP1 gene is encoded at the YLR109W locus. Interestingly, its gene product has been recently identified as a 19-kDa protein by comparative two-dimensional gel electrophoresis in the search of yeast proteins induced by H 2 O 2 (13).
Similarity of Ahp1p with Peroxisomal-like Proteins and with the AhpC/TSA Protein Family-Ahp1p is a protein predicted to be 176 amino acids long with a molecular mass of 19,108 Da (Fig. 2). This predicted mass is in agreement with a molecular weight of 19,000 extrapolated from its mobility in denaturing polyacrylamide gels (13) (see also Fig. 6). Ahp1p contains 3 cysteine residues at positions 31 (Cys-31), 62 (Cys-62), and 120 (Cys-120). Ahp1p carries the C-terminal sequence AHL similar to the putative S(A)K(H/R)L(I) peroxisomal-like sorting signal sequence (31). A sequence comparison with the GenBank data base revealed that Ahp1p is highly similar throughout its entire sequence to five peroxisomal-like fungal proteins, and to three other prokaryotic proteins. Ahp1p has also a weak, yet significant similarity to the peroxiredoxin AhpC/TSA family.
The sequences of the proteins that are highly similar to Ahp1p are compared in Fig. 3. Of the three Ahp1p cysteines, Cys-62 is the only one conserved among these related proteins and occupies a region with a high density of amino acid identities. Cys-31 is conserved only in Lipomyces kononenkoae peroxisomal-like protein and in Aspergillus fumigatus Aspf3 protein. C. bondinii PMP20 A and B proteins (32) and the other related fungal protein sequences all contain a tripeptide peroxisomal-like sorting signal at their extreme C terminus. Inspection of these related sequences also shows that the Hae- The AHP1 gene partially rescues the t-BOOH-hypersensitive phenotype of skn7 and yap1 null strains. skn7 and yap1 null strains not carrying (⌬skn7, ⌬yap1) or carrying the pY1 plasmid (⌬skn7pGAL1AHP1, ⌬yap1pGAL1AHP1), which expresses the AHP1 gene under control of the GAL1 promoter and isogenic wild type strain, were compared by the patch assay for their ability to grow on SDGal solid medium containing t-BOOH at the indicated concentration. For each strain, 10 l of an overnight culture (OD 600 2-3) diluted to 20, 5, or 1 ϫ 10 3 cells (from left to right) were spotted on tester plates. mophilus influenzae Y572 protein has an additional C-terminal domain with a very strong similarity to the redox-active center of glutaredoxins (Fig. 3). C. bondinii PMP20 A and B are two isoforms of a protein that was identified as the most abundant peroxisomal proteins of methanol-exposed cells but was not assigned any specific function (32,33). None of the other proteins shown in Fig. 3 have an identified function. Nevertheless, the presence of a glutaredoxin-like domain in Y572 suggests that this protein might be involved in redox reactions.
The overall similarity between Ahp1p and the AhpC/TSA family is confined in a very short sequence surrounding Ahp1p Cys-62 and the highly conserved N-terminal catalytic cysteine of the AhpC/TSA homology (5,7). This similarity between Ahp1p and the AhpC/TSA family is shared among all the Ahp1p homologues shown in Fig. 3. The functional importance of the N-terminal cysteine in the peroxidase function of Tsa1p (7, 34) strongly suggests that Ahp1p and its homologues may have an equivalent function. We therefore analyzed in detail the in vivo antioxidant properties of Ahp1p.
Ahp1p Is an Antioxidant Specific for Organic Peroxides-A null mutation of the AHP1 gene that removes all its coding sequence was created. The viability of the resulting ahp1 null strain demonstrates that this gene is not essential. The ahp1 null strain does not have any particular growth phenotype on glucose, galactose, acetate, pyruvate, or oleate media or under anaerobic conditions. The effect of the ahp1 null mutation was then tested on the stress tolerance to t-BOOH by a patch assay. Compared with its isogenic wild type control, the ahp1 null strain is hypersensitive to t-BOOH (Fig. 4A). Conversely, high expression of AHP1 from a multicopy plasmid dramatically increases the resistance of the wild type strain toward t-BOOH (Fig. 4C). Note that the difference in t-BOOH sensitivity of wild type cells in panels A and C is related to the different strain backgrounds used in these experiments (see figure legend and "Experimental Procedures"). The role of AHP1 in the stress tolerance to H 2 O 2 was then examined. In sharp contrast to its role in t-BOOH tolerance, the ahp1 null mutation barely affects the cell tolerance to H 2 O 2 (Fig. 4B) and high expression of AHP1 does not improve the wild type strain tolerance to H 2 O 2 (Fig. 4C). Similar results were obtained by a challenge assay (data not shown). These results suggested that AHP1 is involved in the cellular defense against t-BOOH but not against H 2 O 2 . This prompted us to compare the antioxidant properties of Ahp1p with those of the structurally related Tsa1p/peroxiredoxin. An in vitro measurement of Tsa1p catalytic activity had shown that this enzyme is more efficient in reducing H 2 O 2 than t-BOOH (7). Significantly, the tsa1 null mutant is hypersensitive to killing by H 2 O 2 and has a wild type resistance to t-BOOH (Fig. 4, A and B).
The antioxidant properties of AHP1 were also tested toward several other oxidants and a metal (Fig. 4D). The ahp1 null mutation results in a slight decrease in the tolerance to the thioloxidant diamide and to cadmium and suprisingly to an increase in the resistance to the superoxide generating redox cycling drug menadione. However, high copy expression of AHP1 has no effect on the wild type strain tolerance to any of these compounds.
The Antioxidant Function of Ahp1p Is Independent of GSH and Dependent upon the Thioredoxin System-Tsa1p1 reduces peroxides with electrons donated by thioredoxin, thioredoxin reductase, and NADPH (7, 34), whereas AhpC reduces organic peroxides with electrons donated by the FAD-bound NADPHdependent AhpF reductase (6). We thus sought to determine the potential requirement of an electron donor system for the Ahp1p antioxidant function. The effect of overexpressing AHP1 on t-BOOH resistance was tested in strains deleted of thioredoxin 1 (TRX1), thioredoxin 2 (TRX2), or both TRX1 and TRX2 (Fig. 5A). The trx1 null strain has a normal tolerance toward t-BOOH, whereas the trx2 null mutant is slightly more sensitive than the wild type strain to this oxidant (15) (compare Fig.  5A with Fig. 4C). However, the trx1trx2 double null strain is hypersensitive to t-BOOH. In the trx1 null strain, overexpression of AHP1 results in a significant increase in the resistance to t-BOOH, similar to that seen in the wild type strain (compare Fig. 5A and 4C). In the trx2 null strain, the effect of overexpressing AHP1 can be seen only at the lower t-BOOH concentrations. In the trx1trx2 null strain, no effect can be seen upon overexpression of AHP1. A thioredoxin reductase (TRR1) deletion mutant also displayed a t-BOOH-hypersensitive phenotype that was not improved by overexpression of AHP1 (data not shown).
The effect of overexpressing AHP1 on t-BOOH resistance was also tested in strains deleted for ␥-glutamylcysteine synthase (GSH1), the rate-limiting enzyme in glutathione (GSH) biosynthesis (Fig. 5B). The gsh1 null strain appears slightly more resistant to t-BOOH than the wild type strain, suggesting that high levels of GSH are not required for the resistance to this oxidant (the gsh1 null strain is auxotroph for GSH and can only grow in the presence of at least minimal amounts of exogenous GSH supplement such as those present in YPD-rich medium; Ref. 35). In addition, the gsh1 null mutation does not affect the gain in t-BOOH resistance resulting from AHP1 overexpression. Taken together, these data show that thioredoxins but not GSH are important in the protection against t-BOOH and provide indirect evidence for the requirement of the thioredoxin system, but not of GSH, in the antioxidant protective function of Ahp1p.
Ahp1p Can Form a Disulfide-linked Homodimer in Vitro-Ahp1p was identified on two-dimensional gel electrophoresis of total yeast proteins as a H 2 O 2 -inducible spot migrating at a pI of 4.5 and a molecular weight of 19,000. Interestingly, an unidentified H 2 O 2 -induced spot with a pI identical to that of Ahp1p and a molecular weight of 40,000 was observed (Fig. 6). We suspected that this unknown spot could represent a dimeric form or other covalent modifications of Ahp1p. This spot was indeed identified as Ahp1p by peptide mapping using matrixassisted laser desorption ionization-time of flight/mass spectrometry (36). We next asked whether the 40-kDa spot could represent a disulfide-linked dimer of Ahp1p. Cellular extracts subjected to two-dimensional gel electrophoresis are initially reduced in a buffer containing 160 mM ␤-mercaptoethanol. However, some substrates could be potentially reoxidized dur- FIG. 4. Antioxidant and metal protective properties of Ahp1p. A and B, ahp1 null (⌬ahp1), tsa1 null (⌬tsa1), and isogenic wild type strains (WT) in the YPH98 background were compared for their ability to grow on YPD not containing any added chemical or containing either t-BOOH (A) or H 2 O 2 (B) at the indicated concentrations. C, a wild type strain (EMY60) not carrying (Ϫ) or carrying (؉) an episomal plasmid expressing AHP1 under control of its own promoter (pAHP1) were tested for resistance to t-BOOH or to H 2 O 2 at the indicated concentrations. D, an ahp1 null (⌬ahp1) and isogenic wild type strain (WT) (YPH98) not carrying or carrying an episomal plasmid expressing AHP1 under control of its own promoter (pAHP1) were compared for their ability to grow on YPD containing diamide (1.4 mM), menadione (0.1 mM), or cadmium (0.1 mM). For each strain, 10 l of an overnight culture (OD 600 2-3) diluted to 20, 5, or 1 ϫ 10 3 cells (from left to right) were spotted on tester plates. Growth was monitored after 3 days for all plates except for cadmium plates and after 6 days for cadmium plates.

FIG. 5. The antioxidant function of
Ahp1p is independent of GSH but dependent upon the thioredoxin system. Patch assays monitoring the sensitivity of genetically modified strains to peroxides. A, isogenic strains, derived from EMY60, with null deletions of TRX1 (⌬trx1), TRX2 (⌬trx2), or both TRX1 and TRX2 (⌬trx1⌬trx2), not carrying (Ϫ) or carrying (؉) an episomal plasmid expressing AHP1 from its own promoter (pAHP1) were tested on YPD solid media containing t-BOOH at the indicated concentrations. B, a wild type (WT) in the YPH98 background and isogenic strain with a GSH1 null deletion (⌬gsh1) not carrying or carrying an episomal plasmid expressing AHP1 from its own promoter (pAHP1) were tested on YPD medium containing t-BOOH at the indicated concentrations.
ing the second dimension, when all of the ␤-mercaptoethanol has been lost during electrophoresis. To test this hypothesis, the first dimension polyacrylamide rod was incubated in a buffer containing DTT to reduce any potentially reoxidized cysteine residues, and then treated in a buffer containing the cysteine alkylating agent iodoacetamide, which irreversibly blocks free sulfhydryls. As shown in Fig. 6, this procedure led to the complete disappearance of the 40-kDa spot. These data strongly suggest that the 40-kDa Ahp1p spot represents a disulfide-linked Ahp1p homodimer.
AHP1 Is Regulated by Yap1 and Skn7-Ahp1p is an abundant protein under non induced conditions, and its synthesis rate is increased by about 3-fold upon exposure to H 2 O 2 (13). We therefore analyzed whether the peroxide stimulation of Ahp1p was dependent upon Yap1 and Skn7. Northern blots were performed using an AHP1 specific probe and total RNA from exponentially growing wild type, yap1, and skn7 null cells that had not been treated or were treated with 0.2 mM amounts of either t-BOOH or H 2 O 2 . In wild type cells, AHP1 mRNA levels were significantly increased upon H 2 O 2 (Fig. 7A) or t-BOOH (Fig. 7B) exposure, confirming the results of the twodimensional gel analysis. In contrast, in yap1 or in skn7 null strains, both uninduced and t-BOOH-induced AHP1 mRNA levels were significantly decreased, although they could still be moderately induced by peroxide. However, this residual induction was not seen in the yap1skn7 double null strain, although some uninduced levels persisted (Fig. 7C). Consistent with a transcriptional control by Yap1, the 5Ј AHP1 flank contains the sequence TTAGTAA at position (Ϫ484) from the start codon, which perfectly matches a Yap1 response element (Fig. 1) (37). DISCUSSION Oxidative damage to lipids can lead to a variety of alkyl hydroperoxides, which are particularly detrimental due to their ability to initiate and propagate free radical chain reactions. Enzymes involved in the breakdown of alkyl hydroperoxides are thus important for protection against oxidative stress. We report here the characterization of AHP1, which encodes a yeast antioxidant important for the protection against alkyl hydroperoxides, and related to the AhpC/TSA peroxiredoxin family.
Gene deletion and overexpression analyses have demonstrated that AHP1 exerts strong antioxidant protective functions. Furthermore, the observation that AHP1 mRNA and protein levels are induced by peroxides in a Yap1-and Skn7dependent manner also supports the notion that Ahp1p is involved in protecting yeast cells from oxidative stress. Therefore, AHP1 can be added to the growing list of yeast antioxidant genes of the Yap1 and Skn7 regulons, which includes TRX2encoded thioredoxin (15), GSH1-encoded ␥-glutamylcysteine synthetase (19), GLR1-encoded glutathione reductase (20), and TRR1-encoded thioredoxin reductase (17).
Sequence analysis relates this antioxidant to the AhpC/TSA protein family. Although mostly confined to a small region surrounding Cys62 of Ahp1p and the highly conserved N-terminal cysteine of the AhpC/TSA proteins family (5), this homology is functionally important because the N-terminal cysteine of yeast Tsa1p is the catalytic center of its peroxidase function (7,34). The proposed catalytic mechanism for Tsa1p involves substrate peroxide reduction by Cys N terminus, which upon oxidation reacts with the Cys C terminus of another subunit to form an intermolecular disulfide. This disulfide is subsequently reduced with electrons donated by thioredoxin, thioredoxin reductase, and NADPH (7,34). It was observed that Ahp1p forms a disulfide-linked homodimer upon oxidation. Furthermore, genetic data suggested that the antioxidant protective function of AHP1 is independent of GSH but dependent upon both thioredoxin and thioredoxin reductase. Therefore, the structural similarity between Ahp1p and Tsa1p may be extended to a common mode of action, which suggests that a peroxidase function underlies the antioxidant properties of Ahp1p. Of the three Ahp1p cysteines, Cys-62 would be most likely to be involved in direct substrate peroxide reduction. However, it is not clear whether another cysteine would play a role in the putative Ahp1p catalytic function, nor which cys-FIG. 6. Ahp1p can form a disulfidelinked homodimer in vitro. Autoradiogram of two-dimensional gel electrophoresis performed with total cellular extracts from [ 5 S]methionine-labeled exponentially growing wild type YPH98 cells. The 19-kDa Ahp1p protein and the 40-kDa Ahp1p dimer identified by mass spectrometry are indicated by an arrow. A, extracts prepared from untreated cells. B, extracts prepared from cells exposed to H 2 O 2 (0.4 mM) for 15 min. C, alkylated extracts prepared from untreated cells. In this case, the two-dimensional gel electrophoresis procedure was modified by the following change; after the first-dimensional electrophoresis, the resulting polyacrylamide rod was incubated in a buffer containing DTT and then treated with iodoacetamide before subjecting it to the second electrophoretic dimension. teine(s) is (are) involved in homodimer disulfide bond formation. Site-directed mutagenesis of individual cysteines of Ahp1p will answer these questions.
The antioxidant protective function of AHP1 appears specific for organic peroxides. The slight increase in H 2 O 2 sensitivity, and perhaps the altered diamide tolerance phenotype seen in the ahp1 null mutant, could be related to the lipid hydroperoxides which might be generated upon exposure to these oxidants. Although not an oxidant, cadmium may also lead to the production of ROS through metal-catalyzed electron transfer (38,39), and its toxicity might therefore be exacerbated by the lack of AHP1. It is unlikely that cadmium is a direct substrate for Ahp1p. The menadione hyperresistance phenotype of the ahp1 null strain is not understood but could be related to a deregulated expression of superoxide defense genes. Interestingly, an in vivo comparison of the substrate specificities of Ahp1p and Tsa1p showed that, whereas Ahp1p activity is spe-cific for organic peroxides, Tsa1p activity is specific for H 2 O 2 . This observed in vivo substrate specificity of Tsa1p is fully consistent with in vitro measurements of its catalytic activity toward t-BOOH and H 2 O 2 (7). Therefore, with respect to its substrate specificity, Ahp1p differs from Tsa1p and appears related to the Escherichia coli and Salmonella typhimurium alkyl hydroperoxide reductase AhpC, which is specifically involved in the breakdown of lipid and other alkyl hydroperoxides, but not of H 2 O 2 (6).
Eukaryotic cells have several enzymes involved in the breakdown of lipid hydroperoxides. The structurally related selenoproteins glutathione peroxidase (40) and phospholipid hydroperoxide glutathione peroxidase (41,42) can reduce both alkyl hydroperoxides and H 2 O 2 . However, glutathione peroxidase is more active toward H 2 O 2 , whereas phospholipid hydroperoxide glutathione peroxidase is more specific for lipid hydroperoxides (41,43). Furthermore, only phospholipid hydroperoxide glutathione peroxidase can catalyze the reduction of lipid hydroperoxide derivatives of intact phospholipids. Glutathione S-transferase isozymes are also able to reduce lipid hydroperoxides with GSH (44,45). Another pathway for the reduction of lipid hydroperoxides is constituted by NADPH-dependent thioredoxin reductase (46). However, an eukaryotic orthologue of prokaryotic AhpC has not been described. We suggest, based on substrate specificity and structural homology, that Ahp1p is a peroxidase and the yeast orthologue of prokaryotic AhpC.
Ahp1p is highly similar to C. bondinii PMP20 A and B and to several other fungi and prokaryotic proteins (see Fig. 1B). C. bondinii PMP20 A and B are two isoforms of a protein identified as the most abundant peroxisomal protein of methanolexposed cells and suspected to play a role in methanol metabolism (32,33). However, these two proteins and their fungi and prokaryotic homologues (see Fig. 3A), which share with Ahp1p the same similarity to the AhpC/TSA family, may have also a peroxidase function. Consistent with this idea is the presence of a C-terminal glutaredoxin domain in the H. influenzae Y572, which may serve as an electron donor for the catalytic cysteine of this putative peroxidase. Based on immunostaining and cellular subfractionation experiments, PMP20 is a peroxisomal protein tightly associated with membranes (33). Consistent with this localization, PMP20 A and B contain a C-terminal peroxisomal sorting signal sequence (32). A similar peroxisomal sorting signal is present in Ahp1p and in the other PMP20related fungi proteins, suggesting that they may all be similarly localized in peroxisomes. However, the abundance of Ahp1p observed on two-dimensional gels, which only analyze soluble proteins, and its presence in high quantity in the supernatant of a stationary cell culture (data not shown), may indicate that this polypeptide is also present in the cytosol and is shed out of the cell in the medium. A detailed analysis of Ahp1p subcellular location will provide an answer to this question.
In summary, AHP1 encodes an AhpC/TSA-related antioxidant with apparent substrate specificity for alkyl hydroperoxides. These data suggest that this antioxidant is a yeast orthologue of prokaryotic AhpC and justifies its name of Ahp1p for yeast alkyl hydroperoxide reductase. AHP1 is coordinately controlled with TRX2 and TRR1 by Yap1 and Skn7 and requires the presence of these two enzymes to exert in vivo its antioxidant protective function. In vitro measurements of the antioxidant activity of wild type and Ahp1p cysteine substitution mutants will provide a detailed analysis of its peroxidase function and will confirm the role of the thioredoxin system in supporting the antioxidant activity of Ahp1p. Further studies aimed at analyzing the redox characteristics of Ahp1p and FIG. 7. AHP1 is an oxidative stress-inducible gene regulated by Yap1 and Skn7. Northern blot analysis of AHP1 transcription. A, total RNA was isolated from exponentially growing (OD 600 0.3) wild type cells (WT), and isogenic strains with null deletion of YAP1 (⌬yap1), SKN7 (⌬skn7), which had not been treated or were treated for 20 min with H 2 O 2 (0.2 mM) as indicated. The resulting RNA were subjected to Northern blot which was probed for AHP1 and U3 as a loading control, as described under "Experimental Procedures." B, same as in A, but cells were induced by t-BOOH (0.2 mM) instead of H 2 O 2 . C,total RNA was isolated from wild type (lane 1 to 3) and from an isogenic strain with a null deletion of Yap1 and Skn7 (⌬yap1⌬skn7). Cells were not treated (lanes 1 and 4) or were treated for 20 min by H 2 O 2 (0.2 mM) (lanes 2 and 5) or by t-BOOH (0.2 mM) (lanes 3 and 6). Densitometric quantification of the autoradiogram normalized to the U3 loading control; relative values are given for each lane: 1 (2.83), 2 (8.16), 3 (6.37), 4 (0), 5 (1.68), and 6 (1.77).
understanding the molecular basis of its substrate specificity will be of interest. In that respect, the propensity of Ahp1p to oxidize to a disulfide-linked homodimer during electrophoresis is quite striking and suggests a high reactivity to oxidation in vitro and probably also in vivo of one or more of its cysteines.