Antioxidant System within Yeast Peroxisome

Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using thePMP20-deleted strain of C. boidinii(pmp20Δ strain). The His6-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activityin vitro toward alkyl hydroperoxides and H2O2. Catalytic activity and dimerization of His6-CbPmp20 depended on the only cysteine residue corresponding to Cys53. The pmp20Δ strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20Δ growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20Δ strain had a more severe growth defect than the cta1Δ strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, thecta1Δ strain accumulated H2O2, whereas the pmp20Δ strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface,e.g. lipid hydroperoxides, rather than to decompose H2O2. In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.

The peroxisome is a ubiquitous organelle found in most eukaryotic cells in which various kinds of oxidative metabolism occur through at least one H 2 O 2 -generating peroxisomal oxidase (1,2). These oxidative metabolisms yield reactive oxygen species (ROS), 1 which can cause damage to all cellular constituents, i.e. nucleic acids, proteins, lipids, etc. In mammalian cells, treatment with peroxisome proliferators or constitutive expression of peroxisomal oxidases (acyl-CoA oxidase and urate oxidase) were shown to result in neoplastic transformation (3), and these and other studies indicate that ROS generation within peroxisomes is related to carcinogenesis.
The peroxisomal matrix and membranes are assumed to be exposed to a high level of ROS. Therefore, as in other ROSgenerating organelles, such as mitochondria (4), peroxisomes are assumed to have defensive enzyme activities against ROS toxicity (5). One such activity is peroxisomal catalase, which decomposes H 2 O 2 . It is distributed from lower to higher organisms, and as such, it is also used as a marker enzyme for peroxisomes. In mammalian peroxisomes, two other antioxidant enzymes are also present, i.e. superoxide dismutase (6 -8) and glutathione peroxidase (GPX) (9). Pmp20 (20-kDa peroxisomal membrane protein) was initially identified as a peroxisomal membrane protein of unknown function in the methylotrophic yeast Candida boidinii (10). Recently, Pmp20 family proteins have been suggested to detoxify ROS within human and murine peroxisomes (11,12). Subsequently, putative Pmp20 homologs, having a putative peroxisome targeting signal type 1 (PTS1) at their C terminus, were found to be widely distributed in various eukaryotic cells from yeasts and mammalian cells, e.g. Saccharomyces cerevisiae (ScPmp20; also designated as Ahp1p or type II thioredoxin peroxidase (TPX)) (13,14), Lipomyces kononenkoae (15), human (HsPmp20), and mouse (MmPmp20) (11).
Immunocytochemical and biochemical studies have clearly demonstrated that C. boidinii Pmp20 (CbPmp20) is a peroxisomal membrane-associated protein present also in the peroxisome matrix (16,17). However, the physiological significance and function of this and other Pmp20 family proteins seem to be more complex than initially expected. For example, a green fluorescent protein (GFP)-ScPmp20 fusion protein was localized to mitochondria despite the presence of a putative PTS1 sequence (18). Depletion of Pmp20 in S. cerevisiae did not affect its growth on oleate medium where normal peroxisomal metabolism is necessary for its growth. 2 Although the binding of HsPmp20 to the PTS1 receptor Pex5p was shown, up to 50% of epitope-tagged HsPmp20 was also detected in the cytosolic fraction of HeLa cells (11). More recently, a long form of HsPmp20, designated as AOEB166, having a mitochondrial sorting signal at its N terminus and peroxisome targeting signal at its C-terminus, showed bimodal distribution of AOEB166 in these organelles (12).
Recently, the Pmp20 family proteins were recognized from their primary amino acid sequence to belong to a new antioxidant family, the peroxiredoxins (Prx) (19). HsPmp20 and ScPmp20 exhibited antioxidant activity in vitro (11,14), and both MmPmp20 (20) and ScPmp20 (21) were reported to have TPX activity. However, the presence of thioredoxin molecule within peroxisomes has not been demonstrated. Furthermore, bimodal localization of Pmp20 proteins in peroxisomes and mitochondria has made it difficult for us to investigate the physiological function of Pmp20 proteins in relation to the ROS protection system within peroxisomes.
To answer these questions, we initiated a study of the biochemical and physiological functions of CbPmp20. The rationale for using CbPmp20 in the methylotrophic yeast C. boidinii are as follows: 1) Because CbPmp20 is specifically induced in methanol-containing medium and is exclusively localized within peroxisomes, we can specify the function of CbPmp20 within methanol-induced peroxisomes (17,22). 2) The molecular genetics (23,24) and organelle fractionation techniques (25,26) have been well established with C. boidinii. Through this study we have demonstrated that the Pmp20-antioxidant system indeed functions within peroxisomes as GPX and that the antioxidant function of CbPmp20 is biochemically and physiologically distinct from that of peroxisomal catalase.

EXPERIMENTAL PROCEDURES
Microorganisms and Growth Conditions-C. boidinii strain S2 was the original haploidal strain for construction of the genomic library. C. boidinii strain TK62 (ura3) (24) was used as the host for transformation. C. boidinii strain GC (27), which was derived from strain TK62 via gene conversion with URA3 fragment (24), was routinely used as the control wild type strain. C. boidinii strain cta1⌬, which is the disruptant of the peroxisomal catalase-encoding gene (CTA1), will be described elsewhere. 3 Synthetic MI medium was used as the basal medium on which C. boidinii was cultivated (26). One or more of the following was used as the carbon source in each experiment: 1% (w/v) glucose, 1% (v/v) methanol. The initial pH of the medium was adjusted to 6.0. The complex YP medium containing 2% bactopeptone and 1% yeast extract (Difco Laboratories, Detroit, MI) was also used as the basal medium in some experiments. YPD contained 2% glucose, and YPMGy contained 0.5% methanol and 0.5% glycerol as the carbon source(s). The C. boidinii strains were incubated aerobically at 28°C under reciprocal shaking, and the growth of the yeast was followed by measuring the OD 610 . Escherichia coli DH5␣ (28) was routinely used for plasmid propagation. LB medium (0.5% yeast extract, 1% bactopeptone, and 1% NaCl) (28) was used for bacterial expression of the recombinant CbPmp20s.
Cloning of Pmp20-encoding Gene from C. boidinii Strain S2-Two PCR primers, PMP20N1 and PMP20R1 (Table I), were designed based on the conserved DNA sequences between CbPmp20A and CbPmp20B (10), and synthesized. These primers were used to amplify an approx-imately 500-base pair fragment that encoded a partial Pmp20 sequence from C. boidinii strain S2. This PCR-amplified fragment was gel purified, 32 P-labeled according to the method of Feinberg and Vogelstein (29), and used for further cloning experiments. A pool of BamHI-digested genomic DNA of ϳ3.8 kb was gel purified and ligated into the BamHI site of pBluescript II KSϩ (Strategene, San Diego, CA). E. coli transformants were transferred onto a Biodyne nylon membrane (Pall Bio Support, New York, NY). After lysis of the bacteria, the liberated DNA was bound to the nylon membrane, and these blots were then used for colony hybridization under high stringency hybridization conditions using Church-Gilbert buffer (1% bovine serum albumin, 1 mM EDTA, 0.25 M NaCl, 0.25 M Na 3 PO 4 , pH 7.2, 7% SDS) (30). Hybridization was performed at 65°C overnight, and then the membranes were washed six times in 0.2ϫ SSC (1ϫ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at the same temperature. Five clones that showed strong positive signals were found to harbor a reactive 3.8-kb BamHI fragment. The nested deletion mutants were derived as previously described (31), and the entire PMP20 gene was sequenced in both directions using a 7-deaza sequencing kit (Thermo Sequence fluorescent labeled primer cycle sequencing kit) from Amersham Pharmacia Biotech and DNA sequencer model DSQ-2000L from SHIMADZU Co. Ltd. (Kyoto, Japan).
Construction of the Disruption Cassette and One-step Gene Disruption of PMP20 -pMP200, carrying 3.8-kb BamHI fragment harboring the C. boidinii PMP20 gene, was digested with BglII and StyI to delete a 1.7-kb fragment that included 5Ј half of PMP20 gene. The remaining fragment was gel purified, blunted with T4 DNA polymerase, and ligated to the SacI-XhoI fragment of pSPR (C. boidinii URA3 gene having repeated sequence at the 5Ј-and 3Ј-franking regions) (23). The ligation reaction generated the C. boidinii PMP20 disruption vector pD20SPR. After propagation of pD20SPR in E. coli, the insert DNA fragment was liberated following HindIII digestion and was used to transform C. boidinii TK62 (ura3) to uracil prototrophy. The disruption of PMP20 was confirmed by genomic Southern analysis with HindIIIdigested DNA from a Ura ϩ transformant, using the 1.0-kb StyI-HindIII fragment from pMP200 as the probe. The PMP20 disruptant strain pmp20⌬, obtained with pD20SPR, was reverted to uracil auxotrophy, yielding strain pmp20⌬ ura3, using our previously described procedure (23).
Expression of CbPmp20 and Its Derivatives in C. boidinii-NotI sites were added to both ends of the PMP20 coding sequence by PCR using two primers NOT20N and 20CNOT (Table I) and PMP20 DNA as the template. To replace the conserved Cys 53 residue of CbPmp20 with serine, at first, two fragments were PCR-amplified using either set of primers, primers NOT20N and C53S-R, or, primers C53S-F and C53S-R (Table I). Each fragment was purified and used as the templates of the second PCR, where primers NOT20N and 20CNOT were used. Each of the wild type or C53S-mutagenized PMP20-fragments amplified was introduced into the C. boidinii expression vector pNoteI (32), and the constructed plasmids were named pNot20 and pNot20C53S, respectively. pNoteI harbored the C. boidinii AOD1 promoter and terminator sequences with a unique NotI site to insert coding sequences for expression (32). pNot20-⌬AKL, in which the C-terminal 3 amino acids of CbPmp20 were deleted, was constructed in the same way with the use of primers NOT-20-N and 20dAKL-C (Table I). These constructed plasmids were linearized by BamHI and introduced into C. boidinii strain pmp20⌬ ura3 by the modified lithium acetate method (33). The expression plasmid, pGFP-AKL containing GFP tagged with the Pmp20 C-terminal -AKL sequence (34), was also introduced into strain pmp20⌬ ura3, yielding strain GFP-AKL/pmp20⌬. Strain GFP-AKL/wt (34) was used as the control wild type strain.
Preparation and Purification of His 6 -CbPmp20 Proteins-CbPmp20 or CbPmp20 C53S-coding protein was expressed in E. coli BL21(DE3) (Novagen, Madison, WI) as a His 6 fusion protein in pRSET A (Invitrogen, Carlsbad, CA) containing six histidine residues at their N termi-  ATAGTTTAGCGGCCGCCTATAATTTAGCAATAATCTTTTG 20dAKL-C ATAGTTTAGCGGCCGCCTAAATAATCTTTTGAGCAGTAGAAA C53S-F TTACACCACCATCTACTGAACAACATTT C53S-R AAATGTTGTTCAGTAGATGGTGGTGTAA nus. pNot20 and pNot20C53S was each digested with NotI, and the insert DNAs was blunt-ended and ligated to the blunt-ended BamHI site of pRSET A, yielding pRSET20 and pRSET20C53S, respectively. The proper in-frame integration was confirmed by DNA sequencing. A single colony was inoculated in 100 ml of LB medium containing ampicillin (50 g/ml) and incubated at 28°C with reciprocal shaking until the OD 610 reached ϳ0.6. The recombinant protein production was induced by adding isopropyl ␤-D-thiogalactopyranoside to a 1 mM final concentration and cultured further for 5 h. The cells were harvested by centrifugation at 5000 ϫ g for 5 min at 4°C, resuspended in 25 ml of cold 50 mM Tris-HCl, pH 7.5, and recentrifuged. The cell pellet was resuspended in 25 ml of the same buffer, frozen and thawed six times, and sonicated for 30 s three times. Solubilized proteins were recovered by centrifugation at 15,000 ϫ g for 15 min. The obtained proteincontaining supernatant was applied on the nickel-nitrilotriacetic acidagarose column (Qiagen, Chatsworth, CA), and proteins were purified through a protein nondenaturation procedure as recommended by the manufacturer. Purified proteins were dialyzed against 50 mM Tris-HCl, pH 7.5, and subjected to the biochemical analyses.
Enzyme Assays-GPX activity was determined by measuring consumption of NADPH in the presence of GSH (Wako, Osaka, Japan) and GSH reductase (Oriental Yeast, Tokyo, Japan) using cumene hydroperoxide, tert-butyl hydroperoxide, and hydrogen peroxide as substrates. The standard reaction mixture (1 ml) was 20 g of purified His 6 -tagged CbPmp20, 70 M NADPH, 0.1 mM GSH, and 0.4 units/ml GSH reductase in 50 mM Tris-HCl, pH 7.5. The reaction was carried out at 30°C and was started by adding peroxide on a double beam spectrophotometer UV 2200-A (Shimadzu Co. Ltd., Kyoto, Japan). In the thioredoxin system, 4 M thioredoxin (Oriental Yeast, Tokyo, Japan) and 0.4 units/ml thioredoxin reductase (Oriental Yeast, Tokyo, Japan) were substituted for GSH and GSH reductase. Enzyme activity was expressed as mol of NADPH oxidized/min/mg protein. The amount of GSH and GS-SG was determined according to Tietze (35). GSH reductase (GR) activity was determined by the method of Casalone et al. (36). Catalase and cytochrome c oxidase activities were determined as described previously (37,38). Protein quantitation was performed using the method of Bradford (39) with a protein assay kit (Bio-Rad) using bovine serum albumin as the standard.
Protein Methods and Antibody-Standard 9% Laemmli gels (40) with a separating gel of pH 9.2 were used. Immunoblotting was performed by the method of Towbin et al. (41) using the ECL detection kit (Amersham Pharmacia Biotech). The VA9 monoclonal anti-Pmp20 antibody and anti-alcohol oxidase were kindly provided by Dr. J. M. Goodman (University of Texas Southwestern Medical Center, Dallas, TX).
O 2 Uptake-O 2 dissolved in aqueous solution was measured polarographically at 37°C in 50 mM Hepes-NaOH, pH 7.4, using a Clark electrode placed in a water-jacketed cell assembly from Rank Brothers Ltd. (model 0646). The calibration of the O 2 monitor was performed as described before (42). O 2 uptake was initiated by the addition of the reducing agent (ascorbate or DTT), unless specifically noted. The profile of DTT oxidation is comprised of a lag phase of several minutes, followed by a rapid O 2 uptake step when the maximal rate is reached. The maximum rate was determined from the slope of the steepest, nearly linear segment of the O 2 uptake curve.
H 2 O 2 Concentration-Fluorescent measurement was employed in the horseradish peroxidase-catalyzed oxidation of homocanillic acid by H 2 O 2 (43,44). Because thiols are substrates for horseradish peroxidase (45), sulfhydryl groups were alkylated with N-ethylmaleimide, prior to the determination of H 2 O 2 concentration. Calibration curves were generated with known amounts of H 2 O 2 .
Organelle Fractionation-Wild type C. boidinii cells were grown on YPMGy medium overnight, spheroplasted with Zymolyase 100T (Seikagaku Co., Tokyo Japan), and osmotically lysed according to the method of Goodman et al. (46), as described previously (26). Unlysed cells, nuclei, and other cell debris were removed carefully from the lysate by centrifugation at 1,000 ϫ g at 4°C for 10 min. The supernatant was subjected to centrifugation at 20,000 ϫ g at 4°C for 20 min to obtain a crude pellet containing mainly peroxisomes and mitochondria.
To prepare a continuous Nycodenz (Sigma) gradient solution, a step gradient of 10.6 ml (1.3 ml of 60% Nycodenz, 2 ml of 50% Nycodenz, 4 ml of 35% Nycodenz, and 3.3 ml of 28% Nycodenz (w/v)), was frozen once in liquid nitrogen, and then thawed. Then, the organellar suspension was layered on top of the 10.6-ml gradient, and the samples were spun at 100,000 ϫ g for 2 h at 4°C in a VTi 65.1 vertical rotor (Beckman Instruments, Inc., Palo Alto, CA). The gradients were fractionated from the bottom.
Fluorescence Microscopy-Cells representing GFP fluorescence were placed on a microscope slide and examined under a Carl Zeiss Axioplan 2 Fluorescence Microscope (Oberkochen, Germany), and set at the fluorescein isothiocyanate channel. Images were acquired using a CCD camera (Carl Zeiss ZVS-47DE) and a CG7 Frame Grabber (Scion Corp., Frederick, MD).
Electron Microscopy-C. boidinii strain pmp20⌬ cells were incubated in MI medium containing 0.5% yeast extract for 10 h. Cells were fixed with 2% glutaraldehyde and postfixed sequentially with 1.5% KMnO 4 and 1.5% uranyl acetate as described previously (47). The material was dehydrated in a graded acetone series and then embedded in Spurr resin (Polysciences, Inc., Warrington, PA). Ultrathin sections were prepared by using a diamond knife for cutting and observed under an electron microscope (JEOL model C100).
GenBank Accession Number-The nucleotide sequence of PMP20 gene published here has been submitted to GenBank and is available under accession number AB055180.

RESULTS
Gene Structure of CbPmp20 in C. boidinii Strain S2-From the deduced encoded amino acid sequence of the gene cloned FIG. 1. Amino acid sequence alignment of CbPmp20 with other Pmp20 family proteins. The cysteine residues responsible for catalysis in Prx is boxed with a solid line. The putative PTS1 sequences at the C terminus are boxed with a dashed line. The residues with asterisks represent residues conserved in all sequences, and those with dots represent conserved similar amino acids. from strain S2 of C. boidinii, CbPmp20 is a protein of 167 amino acids with a molecular mass of 18,083 Da. Fig. 1 shows a multiple alignment of several Pmp20 family proteins. All family members contain a putative PTS1 sequence at their Cterminus. The amino acid identity of CbPmp20 based on deduced amino acid sequences with CbPmp20B, CbPmp20A, HsPmp20, MmPmp20, and ScPmp20 was 99.4% (C. boidinii ATCC32195), 96.4% (C. boidinii ATCC32195), 25.7% (human), 23.4% (mouse), and 18.6% (S. cerevisiae), respectively. BLAST analysis revealed that these Pmp20 family proteins exhibited significant sequence similarities with other Prx family proteins, especially in the region around Cys 53 of CbPmp20. The most unique feature of the C. boidinii Pmp20 proteins is that CbPmp20 has only one cysteine residue, whereas mammalian and S. cerevisiae Pmp20s have more than two cysteine residues. Therefore, CbPmp20 was classified as 1-Cys Prx, whereas other Pmp20 proteins belonged to 2-Cys Prx (48 -50). From genomic Southern analysis (data not shown) and gene disruption analysis (see below), C. boidinii strain S2 was found to contain a sole gene encoding for CbPmp20. On the other hand, C. boidinii strain ATCC32195 contained two genes, CbPMP20A and CbPMP20B (10).
Antioxidant Activity of His 6 -tagged CbPmp20 -Prx family proteins are known to exert their antioxidant activity through an active site cysteine residue resulting in the formation of a homodimeric form by disulfide linkage through the cysteine residues (13,50). To study the biochemical characteristics of CbPmp20, a His 6 -tagged version of CbPmp20 (His 6 -CbPmp20) and His 6 -CbPmp20 C53S in which the cysteine 53 residue corresponding to CbPmp20 was replaced by serine were overproduced in E. coli under the control of the T7 promoter. The resistance to tert-butyl hydroperoxide of E. coli cells producing His 6 -CbPmp20 or His 6 -CbPmp20 C53S was then determined ( Fig. 2A). A filter paper containing tert-butyl hydroperoxide was placed on a lawn of E. coli cells, and growth inhibition was evaluated by the size of the clear zone surrounding the filter paper. The cells producing His 6 -CbPmp20 became more resistant to tert-butyl hydroperoxide than the cells producing His 6 -CbPmp20 C53S ( Fig. 2A). This indicated that His 6 -CbPmp20 exhibited its anti-oxidant activity in E. coli and that the Cys-53 residue of CbPmp20 is necessary for its anti-oxidant activity. Similarly, overproduction of ScPmp20 (Ahp1p) in S. cerevisiae was reported to result in yeast resistance to alkyl hydroperoxides in a thioredoxin-dependent manner (13). However, overproduction of CbPmp20 in S. cerevisiae did not show such a resistance, even though a considerable amount of CbPmp20protein was detected by Western analysis (data not shown).
His 6 -CbPmp20 and His 6 -CbPmp20 C53S were purified to homogeneity judged on SDS-PAGE through nickel-nitrilotriacetic acid column as described under "Experimental Procedures" (Fig. 2B). His 6 -CbPmp20 prepared in E. coli showed two bands corresponding to the monomeric and dimeric size under the nonreducing conditions (Fig. 2B, lane 1). Upon the reduction by 10 mM DTT, the upper band disappeared (Fig. 2B, lane  2). Therefore, we assumed that CbPmp20 dimerized through disulfide linkage between a single cysteine residue corresponding to Cys 53 of the monomeric form. Indeed, His 6 -CbPmp20 C53S was present only as a a monomeric form in E. coli cell- free extract under nonreducing conditions (data not shown). The His 6 -CbPmp20 C53S protein was also purified to homogeneity via the same procedure for His 6 -CbPmp20 and used for further analyses (Fig. 2B, lane 3).
The thiol specificity of His 6 -CbPmp20 in antioxidant activity was studied by monitoring the consumption of O 2 using a metal-catalyzed oxidation system (51). This oxidation system is comprised of Fe 3ϩ , O 2 , and electron donors. When DTT was used as an electron donor, O 2 was consumed slowly in several minutes (lag phase) (Fig. 2C). During this lag phase, H 2 O 2 accumulated, and propagation of radical chain reaction lead to very rapid consumption of O 2 (Fig. 2C, no addition). The rapid O 2 consumption was prevented by His 6 -CbPmp20 (Fig. 2C). The lack of this inhibitory effect of His 6 -CbPmp20 added at 16 min (Fig. 2C) indicated that CbPmp20 prevented the accumulation of H 2 O 2 at the lag phase. In the ascorbate system, the lag phase of O 2 consumption is shorter than the DTT system, and His 6 -CbPmp20 did not inhibit the O 2 consumption (Fig. 2D) (51). Therefore, purified His 6 -CbPmp20 prevented the O 2 consumption using a thiol-metal-catalyzed oxidation system (DTT/ Fe 3ϩ /O 2 ) (Fig. 2B) but did not prevent the O 2 consumption in a non-thiol-metal-catalyzed oxidation system (ascorbate/Fe 3ϩ / O 2 ) (Fig. 2D). On the other hand, catalase prevented the O 2 consumption in both systems (Fig. 2, C and D). These results indicated that His 6 -CbPmp20 does carry thiol-specific peroxidase activity. Furthermore, because purified His 6 -CbPmp20 C53S could not prevent O 2 uptake (Fig. 2C), Cys 53 of CbPmp20 is considered to be essential for thiol peroxidase activity.
Glutathione Peroxidase Activity of His 6 -CbPmp20 -Some thiol-specific antioxidants which reduce ROS have been shown to be themselves reduced by electron donors, e.g. glutathione, thioredoxin, and tryparedoxin, to recycle oxidized thiol groups (19,52). CbPmp20 belongs to the 1-Cys Prx subfamily, which has been suggested to use glutathione as an electron donor (19). Therefore, we tested glutathione-dependent peroxidase activity of purified His 6 -CbPmp20. The rate of cumene hydroperoxide reduction catalyzed by purified His 6 -CbPmp20 was measured by monitoring the decrease in A 340 (attributable to the oxidation of NADPH) in a glutathione-dependent system. In the presence of GSH, GR, and NADPH, His 6 -CbPmp20 reduced cumene hydroperoxide efficiently (Fig. 3, column 1). Only background activity was detected when either CbPmp20 (column 2) or GSH (column 3) or GR (column 4) was omitted from the reaction. When His 6 -CbPmp20 was replaced with His 6 -Cb-Pmp20 C53S (column 5), the GPX activity was lost. Our biochemical results established His 6 -CbPmp20 as a functional GPX. On the other hand, thioredoxin has recently been identified as a biochemical hydrogen donor for HsPmp20 and ScPmp20 (12,13,20). We assessed the effect of thioredoxin on the peroxidase activity of purified His 6 -CbPmp20. In this system, however, CbPmp20 did not exhibit a thioredoxin-dependent peroxidase activity (Fig. 3, column 6).
As shown in Fig. 2B, the dimeric form of His 6 -CbPmp20 was converted to the monomeric form upon reduction by 10 mM DTT. Because His 6 -CbPmp20 could use GSH as an electron donor, we asked whether a dimeric form of His 6 -CbPmp20 could be reduced by GSH (Fig. 4A). We found that GSH indeed reduced the oxidized dimeric form of His 6 -CbPmp20 protein into the reduced monomeric form and that the reduction was in dose-dependent manner. The physiological level of GSH, ϳ2 mM, was sufficient to complete reaction (Fig. 4B).
Next, we measured the initial rates of NADPH oxidation at various concentrations of cumene hydroperoxide, tert-butyl hydroperoxide, and H 2 O 2 (Table II). Double-reciprocal plots of GPX activity versus substrate concentration were linear for these peroxides (data not shown). Because GPX activity did not show saturation with GSH, apparent kinetic constants were determined at 0.1 mM GSH (Table II)  CbPmp20 Is Necessary for the Methylotrophic Growth of C. boidinii-CbPmp20 is specifically induced when C. boidinii cells are grown in methanol medium (16,22,34). To study the physiological function of CbPmp20 during methylotrophic growth, gene disruption of CbPMP20 was carried out as described in Fig. 5A, and the growth of the disrupted strain (strain pmp20⌬) on methanol was compared with both the wild type strain, and the cta1⌬ strain, in which the peroxisomal catalase-encoding gene was disrupted. 3 Proper gene disruption and subsequent excision of the URA3 sequence were confirmed by Southern analysis with HindIII-digested genomic DNAs from the transformant, using the 1.0-kb StyI-HindIII fragment from pMP200 as the probe (Fig. 5B). The 3.0-kb hybridizing band in the host strain (Fig. 5B, lane 1) shifted to 6.0-kb in the pmp20⌬ strain (Fig. 5B, lane 2), and this 6.0-kb band shifted to 2.4-kb upon the regeneration of uracil auxotrophy (Fig. 5B,  lane 3). In addition, immunoblotting with an anti-Pmp20 antibody confirmed the loss of the signal in the pmp20⌬ strain (data not shown).
The pmp20⌬ strain lost the capacity for methylotropic growth (Fig. 6A), but it could grow normally in other peroxisome-inducing carbon sources, such as oleate or D-alanine (data not shown). On the other hand, the cta1⌬ strain could grow on methanol, although its growth rate was retarded (Fig. 6A).
The growth defect of the pmp20⌬ strain, which was more severe than that of the cta1⌬ strain, was also demonstrated by another experiment. During the incubation of each strain in methanol medium, the viability on YPD medium-plate (Fig.  6B) and the H 2 O 2 concentration in the medium (Fig. 6C) 3. GSH-dependent peroxidase activity of purified His 6 -CbPmp20. Assays were performed with the purified His 6 -CbPmp20 (20 g) in the presence of the GSH system (columns 1-4) or the thioredoxin system (column 6) using 1 mM cumene hydroperoxide as the substrate. Column 5, His 6 -CbPmp20 was replaced by His 6 -CbPmp20 C53S in the complete GSH system. Experiments were done in triplicates as described under "Experimental Procedures," and the error bars indicate standard deviations.
in the medium was not detected for the pmp20⌬ strain. Nevertheless, the viability of the pmp20⌬ strain decreased drastically, whereas that of the cta1⌬ strain was not affected (Fig.  6B). These results show that H 2 O 2 generated during the methylotrophic growth is mainly decomposed by peroxisomal catalase and also suggest that CbPmp20 is more involved in decomposition of ROS which is more toxic than H 2 O 2 . The in vitro activity of His 6 -CbPmp20 toward alkylhydroperoxides and the association of CbPmp20 with the inner side of peroxisomal membrane lead us to speculate that CbPmp20 reduces and detoxifies lipidperoxides generated in peroxisomal membrane. However, the peroxisomal location of Pmp20 family proteins has not been previously demonstrated to be necessary for their physiological function. To obtain evidence that Cb-Pmp20 indeed executes its physiological antioxidant function within peroxisomes, we asked whether peroxisomal targeting of CbPmp20 is necessary to complement the growth defect of strain pmp20⌬.
Peroxisomal Targeting of CbPmp20 and Complementation of the Growth Defect in Strain pmp20⌬-Pmp20 family proteins contain PTS1 sequences at their C-terminal ends (11, 12, 16 -18). At first, we introduced GFP tagged with the C-terminal PTS1 of CbPmp20, -AKL, to visualize the peroxisomes in strain pmp20⌬. Previous studies showed that the C-terminal -AKL   sequence was sufficient for targeting cytosolic GFP to peroxisomes (34). As shown in Fig. 7A, transport of GFP-AKL to peroxisomes was normal in the pmp20⌬ strain.
Next, three C-terminal amino acids, -AKL, were deleted from CbPmp20 (CbPmp20⌬AKL), and CbPmp20 and CbPmp20⌬AKL were expressed in the pmp20⌬ strain to determine whether the peroxisomal localization of CbPmp20 was necessary to complement the growth defect of the pmp20⌬ strain. Whereas CbPmp20 could complement the growth defect of the pmp20⌬ strain comparable with the level of the wild type strain, the Pmp20 protein without PTS1 sequence, CbPmp20⌬AKL, could not (Fig. 7B).
The localization of CbPmp20 and CbPmp20⌬AKL was analyzed by subjecting each strain (the rCbPmp20 strain and the rCbPmp20⌬AKL strain, respectively) to differential centrifugation, which separated the intracellular components into a cytosolic supernatant (S) and an organelle-pellet fraction (P) consisting mainly of peroxisomes and mitochondria (Fig. 8A). More than 80% of CbPmp20 and peroxisomal alcohol oxidase was detected in the organelle-pellet fraction (Fig. 8A), and CbPmp20 protein colocalized with a peroxisomal marker protein catalase on Nycodenz gradient (Fig. 8B). On the other hand, CbPmp20⌬AKL was found in the cytosolic supernatant fraction (Fig. 8A). Therefore, deletion of the C-terminal -AKL sequence from CbPmp20 lead to mistargeting of CbPmp20 from peroxisomes to the cytosol. Furthermore, because rCbPmp20⌬AKL strain showed a 9.8-fold higher level of cytosolic GPX activity (7.90 units/mg protein) when compared with the rCbPmp20 strain (0.808 unit/mg protein), CbPmp20⌬AKL is considered to be present in the cytosol in an enzymatically active form.
As demonstrated with the biochemical in vitro experiment, an antioxidant activity or GPX activity was executed through the Cys 53 residue of CbPmp20. Therefore, CbPmp20 C53S was introduced in pmp20⌬ (the rCbPmp20 C53S strain) to see whether this anti-oxidant activity CbPmp20 was necessary for its physiological function. As expected, CbPmp20 C53S could not complement the growth of C. boidinii pmp20⌬ on methanol (Fig. 7B), although CbPmp20 C53S was targeted to peroxisomes (data not shown). These growth complementation and subcellular fractionation experiments demonstrate that Cb-Pmp20 exerts its physiological function through its antioxidant activity specifically within peroxisomes.
Glutathione System in Peroxisomes-Our results demonstrated that His 6 -CbPmp20 had GPX activity in vitro and that CbPmp20 exerted its physiological activity within peroxisomes. Glutathione is an abundant cellular thiol compound that has been implicated in many cellular processes, but its existence within peroxisomes has not previously been established. We detected a dimeric oxidized form of His 6 -CbPmp20 in E. coli, the formation of which was stimulated by addition of tert-butyl hydroperoxide into the culture medium (data not shown). How-ever, the dimeric oxidized form of CbPmp20 could not be detected in C. boidinii cells even when the cells were exposed to highly ROS-stressed conditions, e.g. in the cta1⌬ strain or cells grown under high methanol concentrations (data not shown). Therefore, we speculated that a dimeric form of CbPmp20 is rapidly re-reduced into its monomeric form via reduced glutathione present within peroxisomes of C. boidinii. In addition, His 6 -tag might alter the conformational change of CbPmp20 and hinder the TPX activity of CbPmp20.
Next, we asked whether peroxisomes contain GPX (and/or TPX), and the other components of the GPX system, i.e. GR and glutathione. The highly purified peroxisomal fractions (Fig. 8B, fraction 4 from the rCbPmp20 strain; fraction 3 from the rCbPmp20⌬AKL strain), which contained no mitochondrial contamination as judged by cytochrome c oxidase activity, were tested for further analyses. As expected, the purified peroxisomal fraction from the rCbPmp20 strain exhibited both GPX activity (3.78 units/mg protein) and catalase activity (2080 units/mg protein), but neither TPX nor GR activity could be detected. On the other hand, the purified peroxisomal fraction from the rCbPmp20⌬AKL strain contained a catalase activity (1460 units/mg protein) bud did not contain a detectable GPX activity. These results show that peroxisomal GPX is CbPmp20 and that CbPmp20 with or without His 6 -tag sequence (cf. Fig.  3) did not have TPX activity. Fig. 8C shows the glutathione and protein concentration throughout the fractionated fractions from the rCbPmp20 strain. Glutathione showed a small peak around the peroxisomal fraction (fraction 4) in proportion to the amount of protein (Fig. 8C). Glutathione in fraction 4 was found to be in the reduced form (GSH) (0.950 g/mg protein), and the oxidized form of glutathione (GS-SG) was less than the detectable level (0.05 g/mg protein). Therefore, GSH is found to be present in peroxisomes at least at the physiological level.
Morphology of Peroxisomes in C. boidinii pmp20⌬ Cells-Recently, Schrader et al. (54) reported that ROS could induced a tubular form of peroxisomes in HepG2 cells. To determine whether ROS generation caused by CbPmp20-depletion induced abnormal peroxisomal morphology, methanol-induced cells of strain pmp20⌬ were observed under electron microscopy. Electron microscopic observation confirmed normal morphology of peroxisomes in pmp20⌬ cells (Fig. 9).

DISCUSSION
Three antioxidant enzymes, i.e. catalase, CuZn superoxide dismutase, and GPX, were described previously as antioxidant enzymes in mammalian peroxisomes and were assumed to play important roles in the detoxification of ROS. In contrast to higher organisms, catalase was until recently the only known anti-oxidant enzyme in yeast peroxisomes. Recently described Pmp20 family members are novel candidate peroxisomal antioxidant proteins. Thus far, the anti-oxidant activity of ScPmp20 and mammalian Pmp20 has been demonstrated only in vitro, and their in vivo function has been unclear. In this study, we have characterized the biochemical and physiological function of CbPmp20 in the methylotrophic yeast C. boidinii, taking advantage of its well characterized molecular genetics and cell fractionation technique and its methylotrophic growth.
The most important fact revealed in this study is that Cb-Pmp20 performs its physiological anti-oxidant function within peroxisomes as a GPX, which can use alkyl hydroperoxides and H 2 O 2 as substrates. Interestingly, the knock-out defect caused by CbPmp20 depletion was much more severe than that induced by depletion of peroxisomal catalase. Moreover, the viability of pmp20⌬ cells decreased during the incubation in methanol medium, whereas that of the cta1⌬ strain did not, even though a considerable level of H 2 O 2 accumulation was observed with the cta1⌬ strain. During methylotrophic growth, the majority of H 2 O 2 generated by alcohol oxidase may be mainly decomposed by catalase within peroxisomes. However, because the affinity of catalase toward H 2 O 2 is low (K m for H 2 O 2 , 25 mM) (55), a trace amount of H 2 O 2 will not be eliminated by catalase alone. Therefore, CbPmp20 having higher affinity against H 2 O 2 (K m for H 2 O 2 , 2.86 mM; Table II) may also have a role in eliminating H 2 O 2 at lower H 2 O 2 concentrations. However, we think that CbPmp20 has a more important role in decomposing lipid hydroperoxides. A major peroxisomal enzyme alcohol oxidase contains FAD (or flavin semiquinone) (56), and peroxisomal catalase contains heme as a cofactor (55). When these cofactors or iron molecules are released from the protein, they could catalyze formation of hydroxyl radicals from H 2 O 2 (57,58), which then attack peroxisomal membrane, resulting in the generation of lipid hydroperoxides. Accumulation of lipid hydroperoxides will further accelerate oxidative decomposition of membrane lipids through radical chain reactions (59). Therefore, it may be reasonably speculated that CbPmp20 is present and functions at the inner side of peroxisomal peripheral membranes. The main physiological function of CbPmp20 is most likely the elimination of lipid hydroperoxides generated in peroxisomal membranes so that cells can maintain the integrity of peroxisomal membrane to avoid cell death.
Although other Pmp20 family members (HsPmp20, ScPmp20) contain more than two cysteine residues and belong to the 2-Cys Prx subfamily, CbPmp20 is unique among Pmp20 family proteins in having only one conserved cysteine, 1-Cys Prx. Many 2-Cys Prxs are known as TPXs, and the catalytic model involving 2-Cys residues for TPX activity has also been postulated (50,60). In contrast, the role of GSH as the reductant for 1-Cys Prx protein has been controversial (49, 53, 61). In FIG. 8. The C-terminal -AKL in Cb-Pmp20 is necessary for peroxisomal transport. A, methanol-induced cells were lysed by osmotic shock, and cell debris was removed by centrifugation at 500 ϫ g. The resultant supernatant was separated into pellettable fraction (P) at 20000 ϫ g including peroxisomes and mitochondria and its supernatant (S) and dissolved in the same volume of the buffer. The same aliquots were analyzed by Western analysis using anti-alcohol oxidase or anti-CbPmp20. B, the organellar pellet fraction after centrifugation at 20,000 ϫ g was further fractionated on Nycodenz equilibrium density gradient centrifugation. The relative activity 100% for catalase (open circle) corresponds to 2210 units/ml for the rCbPmp20 strain and 1430 units/ml for the rCbPmp20⌬ AKL strain, and that for cytochrome c oxidase (closed circle) corresponds to 0.297 unit/ml for the rCbPmp20 strain and 0.104 unit/ml for the rCbPmp20⌬ AKL strain. Peroxisomal localization of CbPmp20 was confirmed on Western blot using anti-Pmp20 monoclonal antibody with the rCbPmp20⌬ strain C, peroxisomal fraction contained glutathione. Open square, protein amount; bars, glutathione amount. this study, we have shown that 1-Cys Prx CbPmp20 can utilize GSH as a reductant for both the catalytic reaction and monomerization from oxidized dimeric form in vitro. In contrast, yeast thioredoxin did not replace GSH, which fits with the model requiring both Cys residues for the catalysis of thioredoxin reductase (50). However, because a physiological level of GSH was detected in a peroxisomal fraction and a dimeric form of CbPmp20 was not detected in C. boidinii, GSH may be a native reductant for CbPmp20 within peroxisomes.
The presence of GPX and glutathione within peroxisomes has raised another question regarding whether the glutathione regeneration system is present in peroxisomes. However, we could not detect GR activity in the peroxisomal fraction of C. boidinii. Because of the fragile nature of the isolated peroxisomes, early studies led to the hypothesis that peroxisomal membrane are freely permeable to compounds of low molecular weight (62). However, recent studies indicated that some metabolites are unable to permeate the peroxisomal membrane and that there are transporters for low molecular weight compounds (26,63,64). Likewise, there may be some glutathione transport system in peroxisome membrane to import GSH and to export GS-SG. In methanol metabolism, formaldehyde produced by peroxisomal alcohol oxidase should be hydrated spontaneously to form S-hydroxymethyl glutathione, being a substrate for cytosolic glutathione-dependent formaldehyde dehydrogenase (65). Therefore, S-hydroxymethyl glutathione might be formed in peroxisomes and then exported to cytosol via the glutathione transport system in peroxisomal membrane.
Among Pmp20 family members, CbPmp20 has another unique feature, i.e. CbPmp20 is exclusively localized in peroxisomes, whereas AOEB166 (a long form of HsPmp20) and ScPmp20 seem to show bimodal distribution between peroxisomes and mitochondria (12,18). CbPmp20 may have evolved in a unique pathway such that the Pmp20 molecule specifically protects the peroxisome membrane from ROS generated during the methanol metabolism. In fact, CbPmp20 was induced specifically in methanol medium but not in other peroxisomeinducing carbon sources, e.g. oleate or D-alanine (22). Although oleate-and D-alanine metabolism also generate H 2 O 2 within peroxisomes through acyl-CoA oxidase and D-amino acid oxidase, respectively, CbPmp20 is not necessary for their metabolism. Why then does methanol-metabolism specifically require Pmp20? Methanol-induced peroxisomes harbor a high amount of FAD protein alcohol oxidase, which can amount to nearly 40% of the total soluble proteins (66), whereas intracellular protein contents of acyl-CoA oxidase and D-amino acid oxidase were low (less than 1%) (34). Alcohol oxidase contains FAD in both quinone and semiquinone forms (67), and these quinone cofactors could be released from the alcohol oxidase protein during the methylotrophic growth (68). These released quinone compounds may catalyze radical chain reaction together with generated H 2 O 2 and induce highly oxidative conditions within peroxisomes as described above (57,58). Therefore, C. boidinii may have retained Pmp20 molecule during the evolution to protect against these highly oxidative conditions in the methanol metabolism. In contrast, ScPmp20 might have lost its physiological function in peroxisomes during the evolution, because S. cerevisiae can grow on oleate medium without ScPmp20.
Our experiments, using highly purified peroxisomes, indicate that methanol-induced peroxisomes of C. boidinii contain a GPX activity catalyzed by CbPmp20 (Fig. 9). Singh and Shichi (61) reported that peroxisomes purified from rat liver had a GPX activity and the peroxisomal fraction showed a cross-reacting band around 20 kDa with antibody raised against cytosolic GPX (1-Cys Prx). In human cells, HsPmp20 or another Pmp20 family protein may have peroxisomal GPX activity, which will play an antioxidant role similar to CbPmp20.
In summary, ROS in methanol-induced peroxisomes of C. boidinii is scavenged by two anti-oxidant enzymes: CbPmp20 and peroxisomal catalase. Our results show that ROS generated in peroxisomes could cause severe injury that leads to cell death, and these ROS species are eliminated by CbPmp20 having a GPX activity at the peroxisomal membrane surface. This is the first report that clarifies the physiological role of Pmp20 family proteins as the anti-oxidant system within a ROS generating organelle, the peroxisome.