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• W1992931605 abstract "We have identified three genes,gst1 +, gst2 +, andgst3 +, encoding θ-class glutathioneS-transferases (GSTs) in Schizosaccharomyces pombe. The gst1 + andgst2 + genes encode closely related proteins (79% identical). Our analysis suggests that Gst1, Gst2, and Gst3 all have GST activity with the substrate 1-chloro-2,4-dinitrobenzene and that Gst3 has glutathione peroxidase activity. Although Gst1 and Gst2 have no detectable peroxidase activity, all three gst genes are required for normal cellular resistance to peroxides. In contrast, each mutant is more resistant to diamide than wild-type cells. Thegst1Δ, gst2Δ, and gst3Δ mutants are also more sensitive to fluconazole, suggesting that GSTs may be involved in anti-fungal drug detoxification. Bothgst2 + and gst3 + mRNA levels increase in stationary phase, and all three gst genes are induced by hydrogen peroxide. Indeed, gst1 +,gst2 +, and gst3 + are regulated by the stress-activated protein kinase Sty1. The Gst1 and Gst2 proteins are distributed throughout the cell and can form homodimers and Gst1-Gst2 heterodimers. In contrast, Gst3 is excluded from the nucleus and forms homodimers but not complexes with either Gst1 or Gst2. Collectively, our data suggest that GSTs have separate and overlapping roles in oxidative stress and drug responses in fission yeast. We have identified three genes,gst1 +, gst2 +, andgst3 +, encoding θ-class glutathioneS-transferases (GSTs) in Schizosaccharomyces pombe. The gst1 + andgst2 + genes encode closely related proteins (79% identical). Our analysis suggests that Gst1, Gst2, and Gst3 all have GST activity with the substrate 1-chloro-2,4-dinitrobenzene and that Gst3 has glutathione peroxidase activity. Although Gst1 and Gst2 have no detectable peroxidase activity, all three gst genes are required for normal cellular resistance to peroxides. In contrast, each mutant is more resistant to diamide than wild-type cells. Thegst1Δ, gst2Δ, and gst3Δ mutants are also more sensitive to fluconazole, suggesting that GSTs may be involved in anti-fungal drug detoxification. Bothgst2 + and gst3 + mRNA levels increase in stationary phase, and all three gst genes are induced by hydrogen peroxide. Indeed, gst1 +,gst2 +, and gst3 + are regulated by the stress-activated protein kinase Sty1. The Gst1 and Gst2 proteins are distributed throughout the cell and can form homodimers and Gst1-Gst2 heterodimers. In contrast, Gst3 is excluded from the nucleus and forms homodimers but not complexes with either Gst1 or Gst2. Collectively, our data suggest that GSTs have separate and overlapping roles in oxidative stress and drug responses in fission yeast. All organisms have evolved protective mechanisms and programmed responses to limit cellular damage from exposure to toxic compounds in their environment. Glutathione S-transferases (GSTs) 1The abbreviations used are: GST, glutathioneS-transferase; DAPI, 4′,6-diamidino-2-phenylindole; ROS, reactive oxygen species; GSH, reduced glutathione; CDNB, 1-chloro-2,4-dinitrobenzene; PIPES, 1,4-piperazinediethanesulfonic acid; hGSTT1, human glutathione S-transferase θ; tBOOH, tetrabutylhydroperoxide. are evolutionarily conserved enzymes that are important in the detoxification of many xenobiotic compounds. These enzymes catalyze the conjugation of glutathione to electrophilic substrates, producing compounds that are generally less reactive and more soluble. This facilitates their removal from the cell via membrane-based glutathione conjugate pumps. The broad substrate specificity of GSTs allows them to protect cells against a range of toxic chemicals (1Salinas A.E. Wong M.G. Curr. Med. Chem. 1999; 6: 279-309PubMed Google Scholar). However, GST activity can sometimes be deleterious to the cell. For example, dihaloalkanes are bioactivated by conjugation with glutathione, generating more genotoxic metabolites (2Sherratt P.J. Manson M.M. Thomson A.M. Hissink E.A. Neal G.E. van Bladeren P.J. Green T. Hayes J.D. Biochem. J. 1998; 335: 619-630Crossref PubMed Scopus (69) Google Scholar). In mammalian cells, amino acid sequence analysis has identified several subgroups of soluble GSTs that appear to have evolved from the θ-prototype found in vertebrates, insects, plants, and bacteria (1Salinas A.E. Wong M.G. Curr. Med. Chem. 1999; 6: 279-309PubMed Google Scholar,3Pemble S.E. Taylor J.B. Biochem. J. 1992; 287: 957-963Crossref PubMed Scopus (180) Google Scholar). Mammalian cells also contain a distinct microsomal GST family of enzymes, but these endoplasmic reticulum membrane-associated enzymes have evolved separately from the soluble GSTs (4Hayes J.D. Strange R.C. Pharmacology. 2000; 61: 154-166Crossref PubMed Scopus (829) Google Scholar). The prevalence of GSTs, together with the evidence suggesting their importance in bioactivation and detoxification of cytotoxic and genotoxic compounds, have stimulated many investigations into the potential role of these enzymes in disease. For example, epidemiological studies have established the absence of certain GST isozymes as an indicator of susceptibility to specific cancers (5Strange R.C. Fryer A.A. IARC Sci. Publ. 1999; 148: 231-249PubMed Google Scholar), and GSTpi null mice are more susceptible to carcinogen-induced tumorigenesis (6Henderson C.J. Smith A.G. Ure J. Brown K. Bacon E.J. Wolf C.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5275-5280Crossref PubMed Scopus (349) Google Scholar). There is also much interest in the regulation of cellular GST activity as increased expression of GSTs in tumor cells is frequently associated with multidrug resistance (1Salinas A.E. Wong M.G. Curr. Med. Chem. 1999; 6: 279-309PubMed Google Scholar). As a consequence of aerobic growth, organisms are exposed to damaging reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and hydrogen peroxide. GSTs have long been suspected to be important in protecting cells from oxidative stress by detoxifying some of the secondary ROS produced when ROS react with cellular constituents. For example, GSTs are able to conjugate glutathione to the toxic reactive compounds, 4-hydroxynonenal and cholesterol α-oxide, which are generated during the oxidation of membranes (7Hubatsch I. Ridderstrom M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (314) Google Scholar, 8Danielson U.H. Esterbauer H. Mannervik B. Biochem. J. 1987; 247: 707-713Crossref PubMed Scopus (127) Google Scholar). The glutathione peroxidase activity of some GST proteins also suggests that they may be important in the detoxification of organic hydroperoxides (9Tan K.L. Board P.G. Biochem. J. 1996; 315: 727-732Crossref PubMed Scopus (59) Google Scholar, 10Sherratt P.J. Pulford D.J. Harrison D.J. Green T. Hayes J.D. Biochem. J. 1997; 326: 837-846Crossref PubMed Scopus (142) Google Scholar, 11Zhao T. Singhal S.S. Piper J.T. Cheng J. Pandya U. Clark-Wronski J. Awasthi S. Awasthi Y.C. Arch. Biochem. Biophys. 1999; 367: 216-224Crossref PubMed Scopus (108) Google Scholar). However, although several studies have correlated high GST levels with increased resistance to oxidative stress, there is little direct evidence establishing that GSTs are important for the protection of cells from oxidative stress (12Hayes J.D. McLellan L.I. Free Rad. Biol. Med. Res. 1999; 31: 273-300Crossref PubMed Scopus (1269) Google Scholar). Cells respond to oxidative stress by inducing the expression of genes whose products protect the cell, for example, by repairing stress-related damage or by inactivating ROS. In the fission yeastSchizosaccharomyces pombe, the regulation of oxidative stress-responsive genes is mediated by the Sty1 stress-activated protein kinase (also known as Spc1) through its downstream target, the Atf1 transcription factor, and by the redox-sensitive Pap1 transcription factor (13Toone W.M. Jones N. Genes Cells. 1998; 3: 485-498Crossref PubMed Scopus (123) Google Scholar). Interestingly, recent work (14Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (966) Google Scholar) has established that GSTs may have a wider role in the response to cellular stress beyond their enzymatic activity. In particular, GSTs have been shown to act as stress-sensitive inhibitors of the mammalian stress-activated protein kinase c-Jun NH2-terminal kinase, which help maintain c-Jun NH2-terminal kinase in an inactive form in unstressed cells (14Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R.J. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (966) Google Scholar). In this report, we have identified three GSTs, Gst1, Gst2, and Gst3, inS. pombe. We show that each of these proteins has associated GST activity. We also demonstrate that the expression of thegst1 +, gst2 +, andgst3 + genes is induced by hydrogen peroxide (H2O2), whereas the expression ofgst2 + and gst3 + is strongly induced in stationary phase. All three GSTs are important for cellular resistance to oxidative stress; however, they have different roles in response to different types of oxidative stress. The S. pombe strains used in this study are shown in TableI. Yeast were grown at 30 °C in rich medium (YE5S) or in synthetic minimal medium (EMM2) as described previously (15Moreno S. Klar A. Nurse P. Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3148) Google Scholar, 16Alfa C.E. Gallagher I.M. Hyams J.S. Methods Cell Biol. 1993; 37: 201-222Crossref PubMed Scopus (23) Google Scholar). Experiments were carried out using exponentially growing (mid-log) cells (A 600nm = 0.2–0.5) unless otherwise stated. Cells were grown for 3 days after reaching mid-log to achieve stationary phase.Table IS. pombe strains used in this studyStrainGenotypeSourceCHP428h+ leu1–32 ura4-D18 his7–366 ade6–210Laboratory stockCHP429h− leu1–32 ura4-D18 his7–366 ade6–216Laboratory stockEV22h+ leu1–32 ura4-D18 his7–366 ade6–210 gst1∷ura4+This studyEV23h+ leu1–32 ura4-D18 his7–366 ade6–210 gst2∷ura4+This studyEV24h− leu1–32 ura4-D18 his7–366 ade6–216 gst1∷ura4+This studyEV25h+ leu1–32 ura4-D18 his7–366 ade6 gst1∷ura4+ gst2∷ura4+This studyEV26h− leu1–32 ura4-D18 his7–366 ade6 gst3∷ura4+This study972h−D. ChenDMatf1h− atf1∷ura4+D. ChenDMpap1h− pap1∷ura4+D. ChenDMsty1h− sty1∷ura4+D. Chen Open table in a new tab The deletion of thegst1 + and gst2 + genes was achieved by replacing the wild-type copy of each gene with theura4 + gene in CHP429 and CHP428 cells. Thegst1Δgst2Δ mutant was obtained by matinggst1Δ and gst2Δ strains. Thegst3Δ mutant was obtained by sporulation of a CHP428/CHP429 diploid in which one copy of the gst3 +gene was replaced with ura4 +. Each of the gst1 +,gst2 +, and gst3 + open reading frames was amplified from genomic DNA by PCR using an N-terminal-specific oligonucleotide primer containing anNdeI restriction site in-frame with the ATG start codon and a C-terminal-specific oligonucleotide primer containing aBamHI restriction site. PCR products were digested withNdeI and BamHI and ligated into theNdeI and BamHI sites of pRep1 (17Maundrell K. Gene (Amst.). 1993; 123: 127-130Crossref PubMed Scopus (931) Google Scholar), pRep41FLAG (a gift of Simon Whitehall), and pRep42HMN (18Craven R.A. Griffiths D.J. Sheldrick K.S. Randall R.E. Hagan I.M. Carr A.M. Gene (Amst.). 1998; 221: 59-68Crossref PubMed Scopus (200) Google Scholar). Mid-log cells (A 600nm = 0.2–0.5) were diluted 10-, 100-, and 1000-fold. 5 μl of each dilution and undiluted cells was spotted onto YE5S plates containing different concentrations of oxidizing agent. Plates were incubated at 30 °C for 2–3 days until sufficient growth had taken place to allow discrimination of any changes in sensitivity. Mid-log cells were harvested and flash-frozen. Thawed cell pellets were washed then resuspended in 200 μl of protein lysis buffer (50 mm Tris-Cl, pH 7.5, 150 mm NaCl, 0.5 g/100 ml Nonidet P-40 with 2 μg/ml pepstatin A, 2 μg/ml leupeptin, 100 mg/ml phenylmethylsulfonyl fluoride, 1 ml/100 ml aprotinin). Cells were lysed by vortexing with 2.5 ml glass beads for 45 s, cooling on ice, and then vortexing for an additional 45 s. Proteins were washed from the beads with an additional 500 μl of protein lysis buffer. Cell extracts were clarified by centrifugation at 12,000 × g for 30min. Protein concentrations were estimated using Coomassie assay protein reagent (Pierce), and equivalent amounts of protein were used in all experiments. GST activity was measured using assays based on the methods of Habig et al. (19Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). 25–50 μl of freshly prepared cell lysate was mixed in a cuvette with 1 ml of GST assay buffer containing either 1 mm 1-chloro-2,4-dinitrobenzene (CDNB) and 1 mm reduced glutathione [GSH] or 1 mm 4-nitrobenzylchloride and 5 mm GSH in 0.11 m sodium phosphate, pH 6.5, and incubated at room temperature. The absorbance (CDNB, 340 nm; 4-nitrobenzylchloride, 310 nm) was measured at timed intervals, and the specific GST activity of each lysate was determined from the increase in absorbance, the time between measurements (min) and the amount of protein (μg). Glutathione peroxidase activity was measured using a modification of the methods of Lawrence and Burk (20Lawrence R.A. Burk R.F. Biochem. Biophys. Res. Commun. 1976; 71: 952-958Crossref PubMed Scopus (2937) Google Scholar) and Avery et al. (21Avery A.M. Avery S.V. J. Biol. Chem. 2001; 276: 33730-33735Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). 50 μl of freshly prepared cell lysate was mixed in a cuvette with 1 ml of glutathione peroxidase assay buffer (50 mm potassium phosphate buffer, pH 7.0, 1 mm EDTA, 1 mm NaN3, 0.2 mm NADPH, 1 EU/ml glutathione reductase (Sigma), and 1 mm GSH). After a 5-min equilibration, the reaction was started by the addition of 20 μl of 69 mm cumene hydroperoxide. The oxidation of NADPH was monitored by recording the absorbance at 340 nm at timed intervals after starting the reaction. The specific glutathione peroxidase activity of each lysate was determined from the decrease in NADPH divided by the time between measurements (min) and the amount of protein (μg). Controls omitting protein were used to correct for the non-enzymatic degradation of NADPH. Cells from mid-log and stationary phase cultures in YE5S media were treated as indicated, and then 25 ml harvested at each time point. RNA was prepared by a hot phenol method essentially as described by White et al. (22White J.H. Green S.R. Barker D.G. Dumas L.B. Johnston L.H. Exp. Cell Res. 1987; 171: 223-231Crossref PubMed Scopus (54) Google Scholar). 5 μg of total RNA was denatured with glyoxal, separated by agarose gel electrophoresis, and transferred to a GeneScreen hybridization membrane (PerkinElmer NEN Life Sciences). Gene-specific probes were prepared from PCR-generated fragments by labeling with 32P using a DNA Megaprime labeling kit (Amersham Biosciences). Hybridization conditions were as described in the GeneScreen protocol. A probe for hmg1 + was used as a loading control. FLAG-tagged proteins were partially purified from cell lysates using anti-FLAG (M2) antibody-conjugated agarose (Sigma). Immunoprecipitated proteins were separated by SDS-PAGE and then transferred to ProtranTMnitrocellulose membrane (Schleicher & Schuell). Nonspecific interactions were blocked with 10 g/100 ml milk in TTBS (20 mm Tris-Cl, pH 7.6, and 0.1 ml/100 ml Tween 20) for 30 min at room temperature. The blocked membrane was incubated with monoclonal anti-Myc (9E10) or anti-FLAG (M2) mouse antibodies (Sigma) diluted 1/1000 with TTBS containing 5 g/100 ml bovine serum albumin for 2 h at room temperature. The membrane was washed with TTBS before incubation with peroxidase-conjugated polyclonal anti-mouse IgG antibodies (Sigma) diluted 1/2000 with TTBS containing 5 g/100 ml bovine serum albumin for 1 h at room temperature. Finally, the membrane was washed with TTBS and then incubated with ECL™ (AmershamBiosciences) chemiluminescent substrates and exposed to x-ray film (Fuji) for an appropriate length of time. Fixation and immunofluorescence were carried out using a modification of the methods of Hagan and Ayscough (23Hagan I. Ayscough K.R. Allan V.J. Protein Localization by Fluorescence Microscopy. Oxford University Press, New York2000: 179-206Google Scholar). Mid-log and stationary phase cells were fixed by agitation with 3.7% paraformaldehyde in PEM (100 mm PIPES, pH 7.0, 1.0 mm EGTA, and 1.0 mmMgSO4) for 30 min at 30 °C. Fixed cells were washed and then resuspended in PEM containing 1.2 m sorbitol and 20 mg/ml zymolyase and incubated at 37 °C for 70 min to spheroplast. Spheroplasted cells were resuspended in 1.2 m sorbitol, 1% Triton X-100 in PEM for 2 min, washed with PEM, and then blocked with PEMBAL (PEM containing 100 mm lysine, 1% bovine serum albumin, and 0.1% sodium azide) for 30 min with rotation at room temperature. The cells were resuspended in monoclonal anti-Myc (9E10) antibodies (Sigma) diluted 1/1000 with PEMBAL and agitated overnight at room temperature. Cells were washed with PEMBAL and then incubated for 1 h with polyclonal fluorescein isothiocyanate-conjugated anti-mouse IgG antibodies (Sigma) diluted 1/50 in PEMBAL. The immunostained cells were washed with PEM and then dried onto a glass microscope slide. The cells were fixed to the slide by immersion in −20 °C methanol for 6 min followed by immersion in −20 °C acetone for 30 s and then mounted in VectashieldTMcontaining 1.5 μg/ml DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories). DAPI-stained nuclei and anti-Myc immunofluorescence were visualized by excitation at 365 nm (DAPI) and 450–490 nm (fluorescein isothiocyanate) under a ×63 oil immersion lens using a Zeiss Axioscope fluorescence microscope and digital imaging system (Axiovision). Sequence analysis of the recently completedS. pombe genome (24Wood V. Gwilliam R. Rajandream M.A. Lyne M. Lyne R. Stewart A. Sgouros J. Peat N. Hayles J. Baker S. Basham D. Bowman S. Brooks K. Brown D. Brown S. Chillingworth T. Churcher C. Collins M. Connor R. Cronin A. Davis P. Feltwell T. Fraser A. Gentles S. Goble A. Hamlin N. Harris D. Hidalgo J. Hodgson G. Holroyd S. Hornsby T. Howarth S. Huckle E.J. Hunt S. Jagels K. James K. Jones L. Jones M. Leather S. McDonald S. McLean J. Mooney P. Moule S. Mungall K. Murphy L. Niblett D. Odell C. Oliver K. O'Neil S. Pearson D. Quail M.A. Rabbinowitsch E. Rutherford K. Rutter S. Saunders D. Seeger K. Sharp S. Skelton J. Simmonds M. Squares R. Squares S. Stevens K. Taylor K. Taylor R.G. Tivey A. Walsh S. Warren T. Whitehead S. Woodward J. Volckaert G. Aert R. Robben J. Grymonprez B. Weltjens I. Vanstreels E. Rieger M. Schafer M. Muller-Auer S. Gabel C. Fuchs M. Fritzc C. Holzer E. Moestl D. Hilbert H. Borzym K. Langer I. Beck A. Lehrach H. Reinhardt R. Pohl T.M. Eger P. Zimmermann W. Wedler H. Wambutt R. Purnelle B. Goffeau A. Cadieu E. Dreano S. Gloux S. Lelaure V. Mottier S. Galibert F. Aves S.J. Xiang Z. Hunt C. Moore K. Hurst S.M. Lucas M. Rochet M. Gaillardin C. Tallada V.A. Garzon A. Thode G. Daga R.R. Cruzado L. Jimenez J. Sanchez M. del Rey F. Benito J. Dominguez A. Revuelta J.L. Moreno S. Armstrong J. Forsburg S.L. Cerrutti L. Lowe T. McCombie W.R. Paulsen I. Potashkin J. Shpakovski G.V. Ussery D. Barrell B.G. Nurse P. Nature. 2002; 415: 871-880Crossref PubMed Scopus (1251) Google Scholar) revealed three open reading frames that encoded proteins with significant homology to human glutathioneS-transferase θ (hGSTT1). We have named these genesgst1 + (SPCC191.09c), gst2 +(SPCC965.07c), and gst3 + (SPAC688.04c) (Fig.1). Notably, gst1 + andgst2 + encode very closely related proteins (79% identical). A comparison of the sequences of these three putative GSTs fromS. pombe with the consensus sequence derived for the θ-class GST enzymes (Fig. 1, GSTθ consensus) (25Rossjohn J. Board P.G. Parker M.W. Wilce M.C. Protein Eng. 1996; 9: 327-332Crossref PubMed Scopus (30) Google Scholar) revealed conservation at 31 and 32 of the 34 amino acid positions in Gst1 and Gst2, respectively. The GSTθ consensus sequence contains six amino acids that are only conserved within the θ-class GSTs (Fig. 1, shown in red). These six amino acids include the serine that is involved in glutathione conjugation in Lucilia cuprinaGSTθ (26Board P.G. Coggan M. Wilce M.C. Parker M.W. Biochem. J. 1995; 311: 247-250Crossref PubMed Scopus (123) Google Scholar, 27Caccuri A.M. Antonini G. Nicotra M. Battistoni A. Bello M.L. Board P.G. Parker M.W. Ricci G. J. Biol. Chem. 1997; 272: 29681-29686Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and probably in other GSTs. The spacing between this serine and the rest of the GSTθ consensus sequence is variable (25Rossjohn J. Board P.G. Parker M.W. Wilce M.C. Protein Eng. 1996; 9: 327-332Crossref PubMed Scopus (30) Google Scholar). Indeed, although Gst1 and Gst2 contain all six of the residues that are specific for the θ-family of GSTs, the conserved serine and the first three hydrophobic residues of the GSTθ consensus sequence are four amino acids further from the rest of the GSTθ consensus than they are in hGSTT1 (Fig. 1). Gst3 matched the GST consensus at 28 of the 34 amino acid positions and contained 3 of the 6 amino acid residues that are only conserved among θ-class GSTs. However, Gst3 has a serine residue at position 46 instead of the asparagine present at this position in all other GSTθ enzymes, and the residue in Gst3 that is most likely to be involved in glutathione conjugation is unclear. The similarity of Gst1, Gst2, and Gst3 to GSTs from other organisms suggests that they have GST activity. However, studies of Ure2 from Saccharomyces cerevisiae, which also has extensive homology with other GSTs (28Coschigano P.W. Magasanik B. Mol. Cell. Biol. 1991; 11: 822-832Crossref PubMed Scopus (222) Google Scholar) and Gst1 and Gst2 from S. pombe, have been unable to detect any GST activity (data not shown) (29Choi J.H. Lou W. Vancura A. J. Biol. Chem. 1998; 273: 29915-29922Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Hence, to examine whether Gst1, Gst2, or Gst3 had GST activity, gst1 +,gst2 + , and gst3 + were ligated into the pRep1 plasmid to allow high levels of expression inS. pombe. GST activity assays were then performed using lysates prepared from wild-type cells containing these plasmids. The overexpression of Gst1, Gst2, or Gst3 significantly increased the GST activity of cell lysate when assayed with the model GST substrate CDNB (Fig. 2 A). However, the same cell lysates did not have increased cellular GST activity toward the substrate 4-nitrobenzylchloride (Fig. 2 A). Certain θ-class GST enzymes including hGSTT1 have been shown to have glutathione peroxidase activity (26Board P.G. Coggan M. Wilce M.C. Parker M.W. Biochem. J. 1995; 311: 247-250Crossref PubMed Scopus (123) Google Scholar). Hence, we also investigated whether the overexpression of Gst1, Gst2, or Gst3 increased the glutathione peroxidase activity of cell lysate. Interestingly, only the overexpression of Gst3 caused any substantial increase in the peroxidase activity of cell lysate against cumene hydroperoxide (Fig.2 A). Controls omitting glutathione revealed that this peroxidase activity was specific to glutathione (data not shown). Taken together, these data suggest that Gst1, Gst2, and Gst3 all have GST activity and that Gst3 has glutathione peroxidase activity. To assess the contributions of Gst1, Gst2, and Gst3 to cellular GST activity, the GST-encoding genes were individually deleted to give gst1Δ, gst2Δ, and gst3Δ strains. The fact that Gst1 and Gst2 are 79% identical suggested that they might have overlapping functions, so agst1Δgst2Δ double mutant was also generated. The GST activities of cell lysates prepared from thegst1Δ, gst2Δ, gst3Δ, andgst1Δgst2Δ mutant strains were analyzed and compared with the GST activity of wild-type lysate. The lysates prepared from exponentially growing cells were found to have very low GST activity, preventing accurate comparisons; therefore, no significant difference between the GST activity of any of the mutant strains and the wild-type could be detected (data not shown). However, consistent with the increased GST expression reported in stationary phase S. cerevisiae cells (29Choi J.H. Lou W. Vancura A. J. Biol. Chem. 1998; 273: 29915-29922Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the GST activity of lysate prepared from stationary phase wild-type S. pombe was 2–3-fold higher than that of lysates from mid-log cells (data not shown). However, the GST activities of stationary phasegst2Δ- and gst1Δgst2Δ-derived lysates were reproducibly lower than those of the wild-type (Fig.2 B). A preliminary analysis suggests that this is because there is no increase in GST activity in gst2Δ andgst1Δgst2Δ cells as they enter stationary phase (data not shown). The GST activity of stationary phasegst3Δ cell lysate was slightly reduced compared with the wild-type, whereas the GST activities of gst1Δ cell lysates were consistently slightly higher than those of wild-type cell lysates. These data demonstrate that the increase in cellular GST activity in stationary phase is predominately dependent on Gst2. The observed increase in cellular GST activity in stationary phase wild-type cells prompted us to investigate whether this reflected changes in the expression of thegst genes. Hence, RNA was extracted from exponentially growing (mid-log) and stationary phase cells and analyzed by Northern blotting. This analysis revealed that gst2 + andgst3 + mRNA are both at much higher levels in stationary phase cells (Fig. 3), whereasgst1 + mRNA levels, although difficult to detect, are relatively unchanged (Fig. 3 and data not shown). Together with the analysis of GST activity of stationary phase wild-type andgst mutant cells, these data suggest that the increased GST activity in stationary phase cells is because of increased expression of gst2 + and gst3 +. To determine whether the expression of individual gst genes was affected by the mutation of other gst genes,gst mRNA levels were examined in wild-type andgst mutant cells. This examination revealed that the changes in GST activity observed in gst1Δ, gst2Δ,gst1Δgst2Δ, and gst3Δ cell lysates (Fig.2 B) are unlikely to be attributed to indirect effects on the expression of the remaining gst genes. Eukaryotic cytosolic GST enzymes are usually found as homodimers, although heterodimers between different GST enzymes have also been reported. Hence, we examined whether each of the GST proteins could form homodimers and/or heterodimers with the other GSTs. Potential in vivo protein-protein interactions between the GST proteins were examined by immunoprecipitation experiments. Anti-FLAG antibody-conjugated agarose was used to immunopurify FLAG-tagged Gst1, Gst2, or Gst3 from wild-type cells co-expressing different combinations of FLAG and Myc epitope-tagged GST proteins. These immunoprecipitates were analyzed by Western blotting with anti-Myc antibodies to determine whether the co-expressed Myc-tagged GST was present in the immunocomplex with the FLAG-tagged GST. These analyses revealed that Gst1 and Gst2 are able to form homodimers and also Gst1-Gst2 heterodimers. However, although Gst3 was able to form homodimers, Gst3 did not form heterodimers with either Gst1 or Gst2 (Fig. 4). The epitope-tagged GST proteins had mobilities consistent with those predicted from their primary amino acid sequences, and their expression also increased the GST activities of cell lysates, indicating that neither epitope tag affects the function of the protein (data not shown). The identity of the proteins producing the additional minor bands is unknown. It is possible that they are derived from immunoglobulins from the anti-FLAG-agarose or breakdown products of the Gst proteins. Previous studies have shown that GSTs localize to different parts of the cell. For example, Gtt1 in S. cerevisiae, is found to be associated with the endoplasmic reticulum (29Choi J.H. Lou W. Vancura A. J. Biol. Chem. 1998; 273: 29915-29922Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), the mammalian κ-family of soluble GSTs are located in mitochondria (30Pemble S.E. Wardle A.F. Taylor J.B. Biochem. J. 1996; 319: 749-754Crossref PubMed Scopus (265) Google Scholar), and murine GSTθ accumulates in the nucleus under certain stress conditions (12Hayes J.D. McLellan L.I. Free Rad. Biol. Med. Res. 1999; 31: 273-300Crossref PubMed Scopus (1269) Google Scholar). The data described above suggests that Gst1 and Gst2 may act together in the cell, whereas Gst3 has a separate role. Hence, to test this hypothesis further, we examined the cellular distribution of each GST protein. To determine the cellular localization of Gst1, Gst2, and Gst3, wild-type cells containing pRep42-based plasmids expressing each GST fused to an N-terminal Myc epitope were analyzed by indirect immunofluorescence microscopy using anti-Myc antibodies. This analysis revealed that Gst1 and Gst2 were found throughout the cell, whereas, in contrast, Myc-tagged Gst3 was excluded from the nucleus (Fig. 5 and data not shown). Similar localization patterns were observed in both exponentially growing and stationary phase cells, which suggests that it is unlikely that the increased GST activity in wild-type stationary phase cells reflects a change in the cellular distribution of any of these three proteins. The similar distributions of Gst1 and Gst2 are consistent with them being able to form heterodimers (Fig. 4). These data also support the hypothesis that Gst3 may have a separate role from Gst1 and Gst2. The expression of some mammalian GSTs increases in response" @default.
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