Yap1-Regulated Glutathione Redox System Curtails Accumulation of Formaldehyde … · the reduced form (GSH), as the oxidized form (GSSG) (9) is converted rapidly by Glr. In yeast

EUKARYOTIC CELL, Apr. 2009, p. 540–549 Vol. 8, No. 41535-9778/09/$08.00�0 doi:10.1128/EC.00007-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Yap1-Regulated Glutathione Redox System Curtails Accumulation ofFormaldehyde and Reactive Oxygen Species in Methanol

Metabolism of Pichia pastoris�†Taisuke Yano,1 Emiko Takigami,1 Hiroya Yurimoto,1 and Yasuyoshi Sakai1,2*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku,Kyoto 606-8502, Japan,1 and CREST, Japan Science and Technology Agency, 5, Sanbancho,

Chiyoda-ku, Tokyo 102-0075, Japan2

Received 5 January 2009/Accepted 10 February 2009

The glutathione redox system, including the glutathione biosynthesis and glutathione regeneration reaction,has been found to play a critical role in the yeast Pichia pastoris during growth on methanol, and this regulationwas at least partly executed by the transcription factor PpYap1. During adaptation to methanol medium,PpYap1 transiently localized to the nucleus and activated the expression of the glutathione redox system andupregulated glutathione reductase 1 (Glr1). Glr1 activates the regeneration of the reduced form of glutathione(GSH). Depletion of Glr1 caused a severe growth defect on methanol and hypersensitivity to formaldehyde(HCHO), which could be complemented by addition of GSH to the medium. Disruption of the genes for theHCHO-oxidizing enzymes PpFld1 and PpFgh1 caused a comparable phenotype, but disruption of the down-stream gene PpFDH1 did not, demonstrating the importance of maintaining intracellular GSH levels. Absenceof the peroxisomal glutathione peroxidase Pmp20 also triggered nuclear localization of PpYap1, and althoughcells were not sensitive to HCHO, growth on methanol was again severely impaired due to oxidative stress.Thus, the PpYap1-regulated glutathione redox system has two important roles, i.e., HCHO metabolism anddetoxification of reactive oxygen species.

Glutathione (L-�-glutamyl-L-cysteinylglycine) assumes piv-otal roles in bioreduction, protection against oxidative stress,detoxification of xenobiotics and endogenous toxic metabo-lites, transport, enzyme activity, and sulfur and nitrogen me-tabolism (28). Its biological significance comes from the freesulfhydryl moiety of the cysteine residue, which confers uniqueredox (E0� � �240 mV for thiol disulfide exchange) and nu-cleophilic properties (30). In cells, glutathione mainly exists inthe reduced form (GSH), as the oxidized form (GSSG) (9) isconverted rapidly by Glr.

In yeast methanol metabolism, methanol is first oxidized toformaldehyde (HCHO) by a peroxisomal enzyme, alcohol ox-idase. This reaction results in high levels of hydrogen peroxide(H2O2) (Fig. 1), which, like other reactive oxygen species(ROS) in methanol-induced peroxisomes, is scavenged by twoperoxisomal antioxidant enzymes, Pmp20 (glutathione peroxi-dase) and catalase (1, 14, 15).

HCHO is an intermediate located at the branching pointbetween the assimilation and dissimilation pathways (51). Inthe former, HCHO is fixed to xylulose 5-phosphate by dihy-droxyacetone synthase within peroxisomes (33). Otherwise,HCHO nonenzymically reacts with GSH to form S-hydroxy-methylglutathione, and subsequently, HCHO is oxidized toCO2 via the dissimilation pathway (44, 51). S-Hydroxymethyl-

glutathione formed in peroxisomes may be exported to thecytosol (14), where it acts as a substrate for GSH-dependentHCHO dehydrogenase. S-Formylglutathione produced byHCHO dehydrogenase is then hydrolyzed to GSH and formicacid by S-formylglutathione hydrolase. Finally, formic acid isoxidized to CO2 by formate dehydrogenase.

Therefore, GSH is assumed to play critical roles during yeastmethanol metabolism in two distinct functions, i.e., (i) protec-tion against ROS and (ii) detoxification and oxidation ofHCHO. Although these studies suggested a tight link betweenmethanol metabolism and glutathione, the glutathione redoxsystem, including its synthesis and recycling, especially in theregulatory aspect of the glutathione redox ratio (GSH/GSSG),is poorly understood (31).

In Saccharomyces cerevisiae, several genes of the glutathioneredox system, such as the glutathione reductase gene (GLR1),the glutathione peroxidase gene (GPX2), and the gene thatencodes the rate-limiting enzyme of the glutathione synthesispathway (GSH1), are induced by the transcriptional regulatorScYap1, which responds to oxidative stress (8, 10, 11, 12, 16,23, 35, 40, 48). ScYap1, a basic leucine zipper (bZIP) DNAbinding protein of the AP-1 family, is activated upon exposureto peroxides or to thiol-modifying drugs such as diamide by amechanism that acts at the level of subcellular localization (5,6, 19, 20, 21, 22, 46, 50). ScYap1 is restricted to the cytosol, butupon exposure to ROS, Yap1 rapidly accumulates in the nu-cleus (21, 50). However, since cells were exposed to extracel-lularly supplemented oxidants in previous studies, the physio-logical role of Yap1 during normal cell growth has not beenelucidated.

In this study, we pursued the regulatory mechanism of theglutathione redox system and its physiological significance dur-

* Corresponding author. Mailing address: Division of Applied LifeSciences, Graduate School of Agriculture, Kyoto University, Kitash-irakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81-75-753-6385. Fax: 81-75-753-6454. E-mail: [email protected].

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 27 February 2009.

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ing methanol metabolism by Pichia pastoris. We examined theintracellular level of glutathione together with its redox ratioand analyzed the phenotypes of various strains in which one ofthe genes involved in the glutathione redox system was dis-rupted. Furthermore, we isolated and characterized the P.pastoris homologue of YAP1 (PpYAP1) and shed light on howthe accumulation of HCHO and ROS is prevented by theglutathione redox system during yeast methanol metabolism.

MATERIALS AND METHODS

Strains and media. P. pastoris PPY12 (arg4 his4) (32) was used as the wild-typestrain. Escherichia coli DH10B was routinely used for plasmid propagation.

The P. pastoris strains were grown on either YPD medium (2% glucose, 1%Bacto yeast extract, 2% Bacto peptone) or YNB medium (0.67% yeast nitrogenbase without amino acids) supplemented, when required, with an appropriateamino acid(s) (200 �g/ml for arginine, 200 �g/ml for histidine) or Zeocin (50�g/ml; Invitrogen, Carlsbad, CA). The following were used as carbon sources inYNB medium: 2% (wt/vol) glucose (SD), 2% (vol/vol) methanol (SM), and 0.5%(vol/vol) oleate (SO). Tween 80 was added to the medium containing oleate at aconcentration of 0.05% (vol/vol). We used 2% methanol to clearly detect thephenotypic differences between the wild type and gene disruptants, althoughsimilar differences could also be observed at lower methanol concentrations (e.g.,0.7%). For organelle fractionation, YPM medium (2% methanol, 1% Bactoyeast extract, 2% Bacto peptone) was used. The initial pH of the medium wasadjusted to 6.0. Cultivation was performed aerobically at 28°C with reciprocal

shaking, and the growth of the yeast cells was monitored by measuring the opticaldensity at 610 nm (OD610).

E. coli was grown at 37°C in LB medium (1% Bacto tryptone, 0.5% Bacto yeastextract, 0.5% NaCl) supplemented with ampicillin (50 �g/ml).

Construction of disruption vectors and one-step disruption of P. pastorisgenes. A 1.5-kb upstream region of the PpYAP1 gene was amplified by PCR withprimers PpYAP1-up-NotI and PpYAP1-up-PstI (Table 1) with genomic DNA asthe template. Analogously, a 1.4-kb downstream region of the PpYAP1 gene wasamplified with primers PpYAP1-down-PstI and PpYAP1-down-XhoI. The PCRproducts were recombined as NotI-PstI and PstI-XhoI fragments, respectively,with a 4.9-kb NotI-XhoI fragment of plasmid SK�-Zeor which included theZeocin resistance (Zeor) gene (26), yielding the PpYAP1 disruption vectorpDyap1. After being linearized with PstI, pDyap1 was transformed into thewild-type strain by electroporation. Zeocin-resistant colonies were selected onSD medium supplemented with arginine, histidine, and Zeocin. Disruption of thePpYAP1 gene was confirmed by Southern blot analysis with EcoRI-digestedgenomic DNA of transformants and a 0.7-kb ClaI-NaeI fragment from thedownstream region of the PpYAP1 gene as the probe.

The isogenic Ppcta1�, Ppfld1�, Ppfgh1�, Ppfdh1�, Ppglr1�, Ppgpx1�, andPppmp20� mutant strains were generated by replacing the respective gene cod-ing regions with the fragment of plasmid SK�-Zeor as described above.

Expression of PpGlr1 and PpYap1 in P. pastoris. To visualize the localizationof PpGlr1, a strain expressing a yellow fluorescent protein (YFP)-tagged PpGlr1(PpGlr1-YFP) fusion protein was constructed as follows. First, a fragment withthe 1-kb 5� untranslated region and the coding region of PpGLR1 was obtainedby PCR with primers Pglr1-EcoRI-Fw and PpGLR1-SpeI-Rv (Table 1) by usinggenomic DNA as the template. Next, YFP was amplified by PCR with primers

FIG. 1. The glutathione redox system and yeast methanol metabolism. Aox, alcohol oxidase; Das, dihydroxyacetone synthase; Cta, catalase; Fld,HCHO dehydrogenase; Fgh, S-formylglutathione hydrolase; Fdh, formate dehydrogenase; Pmp20, peroxisomal glutathione peroxidase; Gpx,glutathione peroxidase; GS-CH2OH, S-hydroxymethylglutathione; GS-CHO, S-formylglutathione; Xu5P, xylulose 5-phosphate; DHA, dihydroxy-acetone; GAP, glyceraldehyde 3-phosphate; ROOH (where R is an aliphatic or aromatic organic group or simply hydrogen), alkyl hydroperoxide.Dashed arrow, hydroxyl radicals from H2O2 attack the peroxisomal membrane, resulting in the generation of ROOH (14). Yap1-targeted enzymesare boxed.

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YFP-SpeI-Fw and YFP-SphI-Rv by using vector pEYFP-N1 (Clontech) as thetemplate. These two fragments were cloned into promoter-free integration ex-pression vector pIB1 (36). The resultant plasmid was linearized with NcoI andthen introduced into the Ppglr1� mutant strain.

To visualize the localization of PpYap1, a strain expressing a DsRed-monomerfluorescent protein (mRed)-PpYap1 fusion protein was constructed as follows.First, with genomic DNA as the template, a 1.4-kb SpeI-SphI fragment contain-ing the PpYAP1 coding region and a 0.8-kb NdeI-EcoRI fragment with thePpYAP1 promoter (PYAP1) region were amplified by PCR with primer setsPpYAP1-SpeI-Fw/PpYAP1-SphI-Rv and Pyap1-NdeI-Fw/Pyap1-EcoRI-Rv, re-spectively. Then, a 0.7-kb EcoRI-SpeI fragment with the mRed coding regionwas amplified by PCR with primers mRed-EcoRI-Fw and mRed-SpeI-Rv byusing vector pDsRed-Monomer-N1 (Clontech) as the template. These threefragments were cloned into pIB1. The resultant plasmid was linearized with SalIand transformed into P. pastoris strains.

Quantitative real-time PCR (qRT-PCR). Cells were grown aerobically to mid-exponential phase in SM or SD medium. Total RNAs were isolated from cellswith an RNeasy mini kit (Qiagen) monitored by on-column DNase digestion.cDNAs were synthesized from 1 �g total RNA with Random Primers (Promega)and ReverTra Ace (Toyobo). After reverse transcription for 50 min at 42°C,samples were heated for 5 min at 99°C to terminate the reaction and 0.5 �l ofRNase H was added. qRT-PCR was performed in 20-�l mixtures in glass cap-illary tubes in a LightCycler (Roche Diagnostic). The PCR cycling reaction wasperformed with 1� SYBR Premix Ex Taq (Takara) according to the followingparameters: first cycle, 10 min of denaturation at 95°C; second cycle with 40repetitions, 95°C for 20 s, 55°C for 20 s, and 72°C for 20 s (all temperaturetransitions, 20°C s�1). The primers used for the reactions are listed in Table 1.As a negative control, PCR was done without ReverTra Ace. The relativeabundance of mRNAs was standardized against the levels for PpACT1.

Measurement of intracellular glutathione and enzyme assays. The amounts ofGSH and GSSG were determined as described by Tietze (41). The activities ofglutathione reductase, catalase, and mitochondrial cytochrome c oxidase weremeasured as described previously (2, 38, 42). Proteins were quantified by aBradford (3) assay kit (Bio-Rad) with bovine serum albumin as the standard.Cells were grown aerobically to mid-exponential phase in SM, SD, or SO me-dium.

Subcellular fractionation. For subcellular fractionation experiments, wild-typecells were grown aerobically to mid-exponential phase in YPM medium.Preparation of spheroplasts and cell homogenates, as well as the subsequentdifferential and Histodenz density gradient centrifugation, were performed asdescribed by Monosov et al. (25).

Fluorescence microscopy and image acquisition. P. pastoris cells carrying YFP-and cyan fluorescent protein (CFP)-tagged proteins were incubated from astarting OD610 of 0.5 in SM, SD, or SO medium and labeled with MitoTrackerRed CMXRos to visualize mitochondria. Nuclear staining was performed as

follows. Cells grown on SM medium were harvested, washed once, and fixed with1 ml of 70% ethanol for 30 min at room temperature. Fixed cells were thenwashed, resuspended in 15 �l sterilized water, and stained with 150 �l of 0.125�g/ml DAPI (4�,6�-diamidino-2-phenylindole; Dojin) solution. After 10 min ofincubation, cells were observed under an inverted fluorescence microscope(IX70; Olympus) equipped with a UPlan Apo 100�/1.35 oil iris objective lenswith a U-MWIG mirror/filter unit (Olympus) for DsRed-monomer and Mito-Tracker Red CMXRos (Molecular Probes), a U-MWU2 mirror/filter unit(Olympus) for DAPI, a U-MF2 (Olympus) filter set (XF114-2; Omega Optical,Inc.) for CFP, and a U-MF2 filter set (XF104-2; Omega Optical, Inc.) for YFP.Image data were captured by a charged-coupled device camera (SenSys; Photo-Metrics) with MetaMorph. 6.0 and saved as Photoshop files (Adobe). The TIFFimage files were optimized for contrast in Photoshop CS3 and compiled onPowerPoint (Microsoft).

Nucleotide sequence accession numbers. The sequences of the PpYAP1, PpCTA1,PpPMP20, PpGLR1, PpGPX1, PpFGH1, and PpFDH1 genes have been submittedto DDBJ and assigned accession numbers AB472084, AB472085, AB472086,AB472087, AB472088, AB472089, and AB472090, respectively.

RESULTS

Increase in glutathione levels and change in thiol redoxratio during growth on methanol. Not only methanol but alsooleate is known to induce peroxisomes in P. pastoris (34). Toinvestigate the role of glutathione in relation to peroxisomemetabolism, the total amounts of glutathione (GSH plus GSSG)were compared among cells grown on methanol, oleate, orglucose as a single carbon source. The total amount of gluta-thione was more than twofold higher in cells grown on meth-anol (SM) than in cells grown on glucose (SD) or oleate (SO)(Fig. 2A). In methanol-grown cells, the intracellular GSH/GSSG ratio, the best index of the cellular thiol redox balance(29), was 70 to 60% lower than that in glucose- or oleate-growncells (Fig. 2B), indicating that the intracellular redox state ofthiols in methanol-grown cells is more toward their oxidizedform. Since oleate metabolism did not alter the glutathioneredox state by more than 15%, the intracellular redox regula-tion observed during methanol metabolism does not seem tobe a general feature of peroxisome metabolism but appearsspecific to methanol metabolism. Therefore, glutathione me-tabolism during growth on methanol was studied further.

Adaptation to methanol culture elevated the total amount ofglutathione and decreased the intracellular GSH/GSSG ratio.From the P. pastoris genome sequence database, we identi-fied the PpGLR1, PpGPX1, and PpGSH1 genes by homologysearches. While three glutathione peroxidase genes (ScGPX1,ScGPX2, and ScGPX3) were found in S. cerevisiae (16), onlyone was found in P. pastoris (PpGPX1), as in Schizosaccharo-myces pombe (SpGPX1) (49). Next, we examined the transcrip-tional levels of PpGLR1, PpGPX1, and PpGSH1 during growthon methanol by qRT-PCR analysis. Among them, PpGPX1was found to be the most highly induced during growth onmethanol (Fig. 2C).

Growth properties of gene disruptants on methanol. Wehypothesized that mutants with the glutathione redox systemdeleted would show a growth defect on methanol. It has beenreported that GSH-deficient mutants of the methylotrophicyeast Hansenula polymorpha failed to grow on methanol due tothe accumulation of toxic amounts of HCHO (37).

To investigate the role of the glutathione redox system andits putative regulator PpYap1, the growth on methanol ofPpGLR1, PpGPX1, PpPMP20, and PpYAP1 mutant strains wascompared. The Pppmp20� mutant strain completely lost the

TABLE 1. Oligonucleotide primers used in this study

Primer Sequence (5�p 3 3�)a

PpYAP1-up-NotI ...............ATATGATTGCGGCCGCCGGATTTGATGTTTPpYAP1-up-PstI ................AACTGCAGCCTCCTGATCAAAGGAATTAGCPpYAP1-down-PstI............AACTGCAGAGGGATCAGGCAGTTCCCGCAAPpYAP1-down-XhoI .........CCGCTCGAGTTATGCTTTATATTTCAGGAAPglr1-EcoRI-Fw .................GGAATTCACAGAGTAGTAGAAATGGATTGGPpGLR1-SpeI-Rv ..............GACTAGTATTCTTCAGCACTGGTTGGATGGYFP-SpeI-Fw......................GACTAGTGTGAGCAAGGGCGAGGAGCTGTTYFP-SphI-Rv......................ACATGCATGCTTACTTGTACAGCTCGTCCAPpYAP1-SpeI-Fw ..............GACTAGTATGAGTGACGTGGTAAACAAGAGPpYAP1-SphI-Rv...............ACATGCATGCCTATTTAAACATGGAAAAATC

GACAACATCPyap1-NdeI-Fw ..................GGAATTCCATATGCAGTTGAATCGGACACCPyap1-EcoRI-Rv ................GGAATTCTGCTGTTTGTGTGATTGTTGCCmRed-EcoRI-Fw................GGAATTCATGGACAACACCGAGGACGTCAmRed-SpeI-Rv ...................GACTAGTCTGGGAGCCGGAGTGGCGGGCCTPpGLR1-RT-Fw ................TTGTGTCCATGTTCTATGCCATGTCCPpGLR1-RT-Rv.................TCTTCAGCACTGGTTGGATGGATAGPpGPX1-RT-Fw ................ACCAGTTTGGTCATCAGGAACCAGGPpGPX1-RT-Rv.................ACCTTTGAATCCGAGGAGACCAGACPpGSH1-RT-Fw ................CCGAAGAGGTTGTAAAGTGGCTATCPpGSH1-RT-Rv.................AGCTTCTGCGTCACTGTATGGAAACPpACT1-RT-Fw.................GCCGGTAGAGATTTGACCGACTACTTGATGPpACT1-RT-Rv.................GTAAGTGGTTTGGTCGATACCAGAAGCCTC

a The underlined nucleotide sequences are additional restriction enzyme rec-ognition sequences.

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ability to grow on methanol, as observed with the Cbpmp20�mutant strain (15; Fig. 3A). The lag phase of the Ppglr1�,Ppgpx1�, and Ppyap1� mutant strains was significantly pro-longed. The Ppglr1� mutant strain showed the most severegrowth defect. On the other hand, all of the strains tested grewwell on glucose or oleate (data not shown).

Intracellular redox states of gene disruptants. Cells werecultivated to exponential phase, and the intracellular GSH/GSSG ratio, together with the total amount of glutathione, wasanalyzed. The total amount of glutathione was twofold higherand the GSH/GSSG ratio was decreased by 30 to 60% in cellsof all of the disruptants tested when they were grown on meth-anol compared to those of cells cultured on glucose or oleate(Fig. 3B and C). During growth on methanol, all of the dis-ruptants tested showed lower GSH/GSSG ratios than wild-typecells did. Especially in the Ppglr1� mutant strain, the GSHratio was drastically decreased. No glutathione reductase ac-tivity was detected in the Ppglr1� mutant strain (see Fig. S1 inthe supplemental material), indicating that there is only onegene that encodes Glr in P. pastoris and that GSSG could not

be reduced to GSH by another pathway. Interestingly, the totalamount of glutathione in the Ppglr1� mutant strain was signif-icantly higher than in other strains, suggesting that de novoGSH synthesis in the Ppglr1 disruptant partially complementedthe lack of regeneration of GSH from GSSG. The long lagphase of this strain when transferred to methanol (Fig. 3)indicated that this compensatory mechanism took a while tobecome fully active under the new growth conditions. Indeed,when Ppglr1� mutant cells in mid-exponential phase on meth-anol were diluted to fresh methanol medium, the lag phase wasshorter for the second culture (data not shown), suggesting ahigher level of GSH at the exponential phase of methanol-glucose-grown Ppglr1� mutant cells. We tested whether thegrowth defect of the Ppglr1� mutant cells on methanol couldbe complemented by adding GSH or GSSG to the culturemedium (Fig. 3D, left panel). The retarded growth of thePpglr1� mutant was partially complemented by the presence ofGSH (50 �g/ml) but not by the presence of GSSG (50 �g/ml).In the presence of GSH (200 �g/ml), the growth of the Ppglr1�mutant strain was comparable to that of wild-type cells (datanot shown). On the other hand, addition of GSH (50 �g/ml)did not affect its growth on glucose (Fig. 3D, right panel).These results indicate that GSH, not GSSG, plays a criticalrole during methanol metabolism in methylotrophic microor-ganisms.

Function of GSH during methanol metabolism. We nextasked whether the growth defect of the gene disruptantsmainly came from the failure to detoxify either HCHO orROS. First, the HCHO levels in the culture medium of thegene disruptants were determined (Fig. 4A). In the wild-typestrain, HCHO accumulation peaked after 20 to 30 h of incu-bation and then gradually decreased. The maximum HCHOlevels in the Ppgpx1�, Ppyap1�, and Ppglr1� mutant strainswere two- to fourfold higher than in wild-type cells, with thehighest peak level in the Ppglr1� mutant. Disruption ofPpPMP20, which encodes a glutathione peroxidase specificallyinvolved in the detoxification of ROS but not in that of HCHO,resulted in an almost constant high level of HCHO. As shownin Fig. 4B, this high HCHO level is owing to the remarkablysmall Pppmp20� mutant cell numbers, since this strain wasinviable on methanol due to ROS generated through methanolmetabolism. In contrast, the Ppglr1� mutant strain, which alsohad a severe growth defect on methanol, was hypersensitive toHCHO, suggesting that GSH, which has been found to func-tion mainly in the detoxification of HCHO during growth onmethanol (51), is not sufficiently abundant to sustain viabilityunder these growth conditions. This is in line with the highHCHO levels (Fig. 4A) and low GSH concentrations (Fig. 3C)detected in this strain.

Intracellular localization of the glutathione regenerationsystem in P. pastoris. Our present data indicate that Glr1-catalyzed regeneration of GSH has a critical role in HCHOdetoxification during methanol metabolism. On the otherhand, the glutathione peroxidase activity of PpPmp20 requiresGSH within peroxisomes for the detoxification of ROS (14). Inother yeasts, such as S. cerevisiae and S. pombe, glutathionereductase showed a bimodal distribution between mitochon-dria and the cytosol (27, 39). In addition, this enzyme wasreported to be localized in peroxisomes of pea (Pisum sativum)leaves (7, 18). Therefore, we examined the localization of

FIG. 2. Effects of carbon sources on the glutathione system. Wild-type P. pastoris was grown on methanol (SM), glucose (SD), or oleate(SO) medium to the mid-exponential phase. (A) Intracellular levels oftotal glutathione (GSH plus GSSG). (B) Intracellular glutathione re-dox ratio (GSH/GSSG). (C) Relative transcriptional levels of genesinvolved in the glutathione redox system on methanol compared tothose on glucose. The relative abundance of these mRNAs was stan-dardized against the levels for PpACT1. Results are means standarddeviations (n � 3).


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PpGlr1 during the methylotrophic growth of P. pastoris. Weexpressed PpGlr1 C-terminally tagged with YFP (PpGlr1-YFP) under the control of its native promoter (PGLR1) in thePpglr1� mutant strain and compared the fluorescence to thatobtained for a CFP-tagged peroxisomal marker, SKL (25).PpGlr1-YFP localized to the mitochondria and the cytosolduring growth on methanol (Fig. 5A), glucose, or oleate (datanot shown). The localization of PpGlr1 was also analyzed inthe wild-type strain by subcellular fractionation, which sepa-rated the intracellular components into a cytosolic supernatant

and an organelle pellet consisting mainly of peroxisomes andmitochondria. The glutathione reductase activity colocalizedwith the mitochondrial marker protein cytochrome c oxidase(4) on a Histodenz gradient (Fig. 5B) but not with peroxisomalcatalase. Previously, we showed that methanol-induced peroxi-somes purified from the methylotrophic yeast Candida boidiniialso did not exhibit any detectable glutathione reductaseactivity (14). Therefore, we found no evidence of the pres-ence of a glutathione regeneration system within peroxi-somes of methylotrophic yeasts.

FIG. 3. Phenotypes of strains carrying disruptions of genes involved in the glutathione redox system. (A) Growth properties of the wild type(WT) and the Ppglr1�, Ppgpx1�, Pppmp20�, and Ppyap1� disruptants on methanol (SM). The intracellular total glutathione levels (B) and theintracellular GSH/GSSG ratios (C) in the wild type and the Ppglr1�, Ppgpx1�, Pppmp20�, and Ppyap1� disruptants grown on methanol (SM),glucose (SD), or oleate (SO) medium are also shown. Results are means standard deviations (n � 3). (D) Growth of the wild type and thePpglr1� disruptant on minimal methanol (SM, left panel) or glucose (SD, right panel) medium not supplemented or supplemented with 50 �g/mlGSH or GSSG.


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PpYap1 transiently localized to the nucleus and positivelyregulated the expression of genes related to the glutathioneredox system during adaptation to methanol. In S. cerevisiae,the nuclear enrichment of transcription factor ScYap1 in re-sponse to oxidative stress (21, 50) resulted in the transcrip-tional activation of genes that encode enzymes related to theglutathione redox system, such as ScGLR1, ScGPX2 (one ofthe three glutathione peroxidase genes), and ScGSH1, as wellas numerous genes that are involved in preventing oxidativedamage to the cells (17, 19, 35, 40, 43). To investigate the roleand behavior of Yap1 in P. pastoris, we have cloned thePpYAP1 gene.

We expressed a DsRed-monomer–PpYap1 fusion protein(mRed-PpYap1) under the control of the native promoter(PYAP1) in the Ppyap1� mutant strain and confirmed this byWestern blot analysis (data not shown). The Ppyap1� mutantstrain was hypersensitive to various oxidative stresses, and un-der oxidative stress conditions, mRed-PpYap1 accumulated inthe nucleus (T. Yano and Y. Sakai, unpublished results). Theseexperiments confirmed that activation of mRed-PpYap1 canbe monitored in real time by its accumulation in the nucleus, asobserved in S. cerevisiae.

The Ppyap1� mutant strain also exhibited a prolonged lagphase (Fig. 3A) and accumulated a considerable amount ofHCHO (Fig. 4A), suggesting that PpYap1 is involved in the

detoxification of HCHO during growth on methanol. To seethe activation of PpYap1 under physiological conditions, themRed-PpYap1-expressing Ppyap1� mutant cells were grownto exponential phase in glucose medium (SD) and then shiftedto methanol or oleate medium. mRed-PpYap1 was predomi-nantly localized to the cytosol before the shift. At 3 to 14 hafter a shift to methanol medium, mRed-PpYap1 was tran-siently localized to the nucleus (Fig. 6A and B). In contrast,mRed-PpYap1 stayed in the cytosol during growth on glucoseor oleate (data not shown).

This transient nuclear localization of PpYap1 in methanol-grown cells appears to activate the expression of genes relatedto the glutathione redox system (PpGLR1, PpGPX1, andPpGSH1); as in the Ppyap1� mutant, the transcriptional acti-vation of these genes was suppressed (Fig. 6C). The promotersof these genes contain various putative Yap1 response ele-ments (13; data not shown). These results are consistent withthe finding that the total glutathione amount and the GSH/GSSG ratio were lower in the Ppyap1� mutant strain duringgrowth on methanol than in the wild-type strain (Fig. 3B andC). The retarded growth of the Ppyap1� mutant strain onmethanol and HCHO accumulation may be due to the re-tarded expression of these genes, especially PpGLR1.

Activation of PpYap1 in various mutants. Since we identi-fied a novel physiological role for PpYap1 in HCHO detoxifi-cation, we studied how PpYap1 is activated during adaptationto methanol in various gene disruptants. mRed-PpYap1-ex-pressing strains were precultured in glucose medium (SD) andshifted to methanol medium (SM containing 0.05% yeast ex-

FIG. 4. HCHO sensitivity. (A) HCHO level in culture mediumduring growth on methanol. Samples were obtained from the culturemedium of Fig. 3A. (B) Cell viability on HCHO or methanol. Cellswere incubated in YNB medium containing HCHO (2 mM) or meth-anol (2%) for the indicated times, spotted onto an SD plate, and grownfor 2 days at 28°C. WT, wild type.

FIG. 5. Glutathione reductase localizes to mitochondria and cy-tosol in P. pastoris. (A) Strains carrying PpGlr1-YFP and CFP-labeledperoxisomes were analyzed during growth on methanol. The mergedimages comprise PpGlr1 (green) and peroxisome (Ps, blue) signals andmitochondrion (Mt, red) signals stained with MitoTracker RedCMXRos (Molecular Probes). The right panel is the differential inter-ference contrast (DIC) image. Bar, 2 �m. (B) The organellar pelletobtained after centrifugation at 27,000 � g was further fractionated byHistodenz equilibrium density gradient centrifugation. Fractions wereassayed for relative catalase (E), cytochrome c oxidase (F), and glu-tathione reductase (Œ) activities.


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tract to support the growth of the mutants) to monitor thelocalization of mRed-PpYap1 periodically (time, 1 to 24 h).

First, we tested mutations affecting the GSH-dependentHCHO oxidation pathway (Ppfld1�, Ppfgh1�, and Ppfdh1�).In cells with these mutations, the intracellular HCHO level wasexpected to increase without enhancing the generation of ROS(Fig. 1), which made the Ppfld1� and Ppfgh1� mutants (butnot the Ppfdh1� mutant) hypersensitive to HCHO in the me-dium (see Fig. S2B in the supplemental material). As shown inFig. 7, in both the Ppfld1� and Ppfgh1� mutant strains, mRed-PpYap1 accumulated in the nucleus at earlier time points thanin the wild-type strain when these strains were grown on meth-

anol. This nuclear PpYap1 localization could not be released inPpfld1� and Ppfgh1� mutant cells growing on glucose mediumto which HCHO was added (see Fig. S2A in the supplementalmaterial). Moreover, both the Ppfld1� and Ppfgh1� mutantstrains showed HCHO sensitivity but the Ppfdh1� mutantstrain did not (see Fig. S2B in the supplemental material).These results indicate that HCHO toxicity or the deficiency ofGSH triggers the nuclear localization of PpYap1 in thePpfld1� and Ppfgh1� mutant strains and suggested activation ofPpYap1 by HCHO. We also confirmed that HCHO itself couldinduce the nuclear accumulation of mRed-PpYap1 (Yano andSakai, unpublished).

P. pastoris has two peroxisomal antioxidant enzymes, i.e.,peroxisomal catalase Cta1 and Pmp20 glutathione peroxidase;a homologue of cytosolic catalase CTT1 could not be found inthe P. pastoris genome. The growth defect of the Pppmp20�mutant was more severe on methanol medium than that of thePpcta1� mutant (Fig. 4B; see Fig. S2C in the supplementalmaterial), as was the case for their C. boidinii homologues (15).In the Pppmp20� mutant strain, mRed-PpYap1 localized tothe nucleus 1 to 2 h after a shift to methanol medium, whereasthis took another hour in Ppcta1� mutant and wild-type cells(Fig. 7). Thus, increased levels of peroxisomal ROS resultingfrom the lack of PpPmp20 activity cause a more rapid activa-tion of PpYap1 than H2O2 accumulation, which could only bedetected in the absence of PpCta1 (data not shown). HCHO byitself did not cause a growth defect in the Pppmp20� mutant,but methanol as a carbon source did (Fig. 4B). It is the con-version of methanol to HCHO that causes the generation ofROS as a by-product (Fig. 1), which is thus most damaging toPppmp20� mutant cells. Taken together, our results show thatthe absence of PpPmp20 made the cells highly susceptible tooxidative stress.

DISCUSSION

Previously, a GSH biosynthetic mutant was reported to beimpaired in growth on methanol (31). However, with such amutant it has been difficult to show that GSH has two distinctphysiological roles, i.e., (i) elimination of ROS and (ii) detox-ification of HCHO, because the mutant simultaneously lostboth functions. Our analyses of various mutations involved inGSH-related metabolism show that GSH indeed has these twodistinct roles and have revealed a novel role for yeast Yap1,which is normally associated with oxidative stress, under phys-iological condition in HCHO detoxification.

We detected a significant level of HCHO in the culturemedium of methanol-grown P. pastoris, which increased in allof the mutants tested. In the Ppglr1� mutant strain, a mutantdeficient in GSH regeneration from GSSG, the highest level ofHCHO was detected at an early stage of growth in methanol(Fig. 4A). Although GSH nonenzymatically reacts with HCHOin vitro (51), this and other experiments confirm in vivo thatGSH, not GSSG, functions in HCHO detoxification and thatboth the presence of GSH and its Glr1-catalyzed regenerationfrom GSSG are essential for methanol metabolism in yeast.

Pmp20, a peroxisomal glutathione peroxidase, rather thancatalase, has been shown to be the main scavenger of ROSduring methanol metabolism by P. pastoris. In vivo, methanol isconverted in a peroxisomal alcohol oxidase-catalyzed reaction

FIG. 6. mRed-PpYap1 is transiently localized to the nucleus andpositively regulates the transcription of genes related to the glutathi-one redox system during methanol metabolism. (A) Subcellular local-ization of mRed-PpYap1. The Ppyap1� mutant strain expressingmRed-PpYap1 was grown in glucose (SD) medium to exponentialphase and shifted to methanol (SM) medium. (B) Nuclear accumula-tion of mRed-PpYap1 during growth on methanol. mRed-PpYap1-expressing cells incubated on methanol for 6 h were stained withDAPI. Bars, 2 �m. (C) Relative transcriptional levels of PpGLR1,PpGPX1, and PpGSH1 in the Ppyap1� mutant compared to those inthe wild type. The relative abundance of these mRNAs was standard-ized against the levels for PpACT1. Results are means standarddeviations (n � 3).


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to HCHO and H2O2 (1). Subsequently, H2O2 causes the for-mation of ROS. As the Pppmp20� mutant strain was sensitiveto methanol but not to HCHO (Fig. 4B), the growth defect ofthe Pppmp20� mutant is due to the failure to detoxify ROS.On the other hand, the Ppglr1� mutant strain was not onlysensitive to methanol but also very sensitive to HCHO (Fig.4B), indicating the importance of the regeneration of GSHfrom GSSG. Therefore, in addition to ROS elimination, GSHhas a significant role in the detoxification of methanol-derivedHCHO.

We determined and compared the redox ratio of glutathioneamong various cells. The intracellular GSH/GSSG ratio be-comes more oxidized in methanol-grown cells than in oleate-or glucose-grown cells. Moreover, the total glutathione levelwas higher in methanol-grown cells (Fig. 2A). Since much ofGSH is assumed to be trapped by HCHO (Fig. 1) for furthermetabolism, the shifted intracellular redox state of methanol-grown cells may result in the upregulation of de novo GSHsynthesis. The Ppglr1� mutant strain exhibited a remarkablylow GSH/GSSG ratio due to the block in the GSH regenera-tion reaction, but its total glutathione level was enhanced,indicating that de novo GSH synthesis supported the slowgrowth (Fig. 3). These results show that the regulation of theglutathione redox system plays important roles during adapta-tion to methanol.

PpYap1-dependent activation of the glutathione redox sys-tem, e.g., PpGlr1, PpGpx1, and PpGsh1, was found to occurduring methanol adaptation and also in the Ppglr1� mutantstrain. Indeed the Ppyap1� mutant strain exhibited a pro-longed lag phase and accumulated a considerable amount ofHCHO (Fig. 3A and 4A). Distinct from PpGPX1, expressionof PpGLR1 and PpGSH1 was not completely abolished in the

Ppyap1� mutant strain. Activation of the latter two genesmight be mediated by some other factors, e.g., the Met4 ho-molog in P. pastoris (47).

Activation of PpYap1 in P. pastoris can be monitored by itslocalization to the nucleus (Yano and Sakai, unpublished). Weobserved that mRed-PpYap1 transiently localized to the nu-cleus after 4 to 12 h of methanol adaptation (Fig. 6A). Inter-estingly, the nuclear localization of mRed-PpYap1 was ob-served at earlier time points in HCHO oxidation pathwaymutants, the Ppfld1� mutant (after 1 h of adaptation) and thePpfgh1� mutant (after 3 h of adaptation). Moreover, in both ofthese mutant strains, HCHO accumulated to a considerablyhigher level than in the wild-type strain (see Fig. S2D in thesupplemental material), with the HCHO level in the Ppfld1�mutant being higher than that in the Ppfgh1� mutant. Becauseboth enzymes have not been shown to be involved in ROSelimination, their disruption stalls the regeneration of GSH asthe intermediate S-hydroxymethylglutathione or S-formylglu-tathione cannot be processed (Fig. 1). Thus, either depletion ofGSH or accumulation of HCHO is toxic for the cells and couldtrigger the activation of PpYap1 in these gene disruptants.

Cta1 and Pmp20 are the major peroxisomal antioxidantenzymes. Our present and preceding studies revealed thatpmp20� mutant strains exhibited more severe phenotypes thancta1� mutant strains (15; see Fig. S2C in the supplementalmaterial). PpYap1 activation, as indicated by its nuclear accu-mulation, which occurred at an earlier time point, suggestingthat oxidative stress caused by ROS that could not be removedby Pmp20 activity, was more severe than that imposed by H2O2

accumulating in the absence of catalase activity. In otheryeasts, Yap proteins have been identified as transcription fac-

FIG. 7. Subcellular localization of mRed-PpYap1 in various mutants on methanol. Mutants carrying mRed-PpYap1 were grown in glucose(SD) medium to exponential phase and then shifted to methanol (SM) medium containing yeast extract (0.05%). Bar, 2 �m. WT, wild type.


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tors that are induced by oxidative stress and that play a role inthe removal of ROS (24).

The GSH-dependent HCHO oxidation pathway exists exclu-sively in the cytosol because three enzymes, PpFld1, PpFgh1,and PpFdh1, localized to the cytosol (45, 51; data not shown).Although GSH was necessary within peroxisomes for detoxifi-cation of HCHO and ROS, we could not find evidence of thepresence of a GSH regeneration system within peroxisomes.Therefore, HCHO is supposed to be trapped in peroxisomesby GSH and exported to the cytosol to be a substrate forPpFld1 (51) and where GSH is regenerated (Fig. 5), suggestingthe presence of a transporter for at least GSH, GSSG, andS-hydroxymethylglutathione on the peroxisomal membrane.

In summary, the intracellular redox state maintained by glu-tathione metabolism has been found to be finely regulatedduring the growth of P. pastoris on methanol. GSH/GSSGredox becomes more oxidized in cells growing in methanolthan in cells growing in glucose or oleate. PpYap1 transientlylocalizes to the nucleus and activates the expression ofPpGLR1 and other target genes, and upregulated PpGlr1 re-generates GSH (Fig. 8). Therefore, the glutathione redox sys-tem has critical physiological roles during growth on methanol,i.e., detoxification and oxidation of HCHO and elimination ofROS. Yeast methanol metabolism provides a good model sys-tem to study the physiological role and regulation of glutathi-one metabolism together with its redox homeostasis.

ACKNOWLEDGMENTS

This research was supported in part by Grant-in-Aid for ScientificResearch (B) 19380048 to Y.S.; Grant-in-Aid for Scientific Research onPriority Areas 18076002 to Y.S.; a Grant-in-Aid for Young Scientists(B-type grant) from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan to H.Y. (19780058); and CREST, JST.

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