A Novel Membrane-bound Glutathione S-Transferase Functions in the Stationary Phase of the Yeast Saccharomyces cerevisiae

The glutathione S-transferases (GSTs) represent a significant group of detoxification enzymes that play an important role in drug resistance in all eukaryotic species. In this paper we report an identification and characterization of the twoSaccharomyces cerevisiae genes, GTT1 and GTT2 (glutathionetransferase 1 and 2), coding for functional GST enzymes. Despite only limited similarity with GSTs from other organisms (∼50%), recombinant Gtt1p and Gtt2p exhibit GST activity with 1-chloro-2,4-dinitrobenzene as a substrate. Both Gtt1p and Gtt2p are able to form homodimers, as determined by two hybrid assay. Subcellular fractionation demonstrated that Gtt1p associates with the endoplasmic reticulum. Expression of GTT1 is induced after diauxic shift and remains high throughout the stationary phase. Strains deleted for GTT1 and/or GTT2 are viable but exhibit increased sensitivity to heat shock in stationary phase and limited ability to grow at 39 °C. The glutathione S-transferases (GSTs) represent a significant group of detoxification enzymes that play an important role in drug resistance in all eukaryotic species. In this paper we report an identification and characterization of the twoSaccharomyces cerevisiae genes, GTT1 and GTT2 (glutathionetransferase 1 and 2), coding for functional GST enzymes. Despite only limited similarity with GSTs from other organisms (∼50%), recombinant Gtt1p and Gtt2p exhibit GST activity with 1-chloro-2,4-dinitrobenzene as a substrate. Both Gtt1p and Gtt2p are able to form homodimers, as determined by two hybrid assay. Subcellular fractionation demonstrated that Gtt1p associates with the endoplasmic reticulum. Expression of GTT1 is induced after diauxic shift and remains high throughout the stationary phase. Strains deleted for GTT1 and/or GTT2 are viable but exhibit increased sensitivity to heat shock in stationary phase and limited ability to grow at 39 °C. glutathioneS-transferase glutathione S-conjugate transporting ATPase thiolate anion ATP-binding cassette transport protein 1-chloro-2,4-dinitrobenzene S-(2,4-dinitrophenyl)glutathione stress response element post-diauxic shift element polymerase chain reaction kilobase pair(s) base pair(s). Cellular metabolism and detoxification of xenobiotics (carcinogens, toxins, environmental pollutants, and drugs) occur in three stages. In stage I, toxins are activated by oxidation, reduction, or hydrolysis to introduce a functional group. In stage II, the functional group is conjugated with glutathione (GSH), glucuronic acid, or glucose. In particular, S-conjugates of GSH are formed by cytosolic glutathione S-transferase (GST)1 enzymes. In stage III, GSH conjugates are eliminated from the cytosol by a GS-X pump located in the plasma membrane of animal cells and the vacuolar membranes of yeast cells (1Ishikawa T. Trends. Biochem. Sci. 1992; 17: 463-468Abstract Full Text PDF PubMed Scopus (570) Google Scholar). The level of expression of GSTs and their biochemical properties are crucial factors in determining cellular resistance to carcinogens, antitumor drugs, environmental pollutants, and products of oxidative stress (2Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar, 3Moscow J.A. Dixon K.H. Cytotechnology. 1993; 12: 155-170Crossref PubMed Scopus (47) Google Scholar, 4Branum G.D. Selim N. Liu X. Whalen R. Boyer T.D. Biochem. J. 1998; 330: 73-79Crossref PubMed Scopus (14) Google Scholar, 5Hubatsch I. Ridderstrom M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (314) Google Scholar). All eukaryotes possess multiple GST isoenzymes, each displaying distinct catalytic as well as noncatalytic binding properties (6Mannervik B. Danielson U.H. Crit. Rev. Biochem. Mol. Biol. 1988; 23: 283-337Crossref Scopus (1688) Google Scholar, 7Listowsky I. Abramovitz M. Homma H. Niitsu Y. Drug. Metab. Rev. 1988; 19: 305-318Crossref PubMed Scopus (239) Google Scholar). The functional basis for the catalytic activity of GSTs is their ability to bind GSH and lower the pK aof its sulfhydryl group (-SH) from 9.0 to about 6.5. Once the thiolate anion (GS-) is formed in the binding site for GSH, it is capable of reacting spontaneously by nucleophilic attack with electrophilic xenobiotics in close proximity. Therefore, catalysis by GST occurs through the combined ability of the enzyme to (i) bind GSH and promote the formation of GS-, and (ii) bind hydrophobic electrophilic compounds at a nearby site. In contrast to the GSH-binding site, which exhibits a high specificity, the second substrate-binding site displays a broad specificity toward hydrophobic compounds (8Mannervik B. Adv. Enzymol. Relat. Areas Mol. Biol. 1985; 57: 357-417PubMed Google Scholar). The yeast Saccharomyces cerevisiae is an excellent model for studying detoxification pathways due to the ease of its genetic manipulation. Indeed, a large number of genes mediating the resistance to toxic xenobiotics have been identified in yeast. The gene products described so far fall into two major classes: (i) membrane transport proteins belonging to the ATP-binding cassette (ABC) superfamily, such as Snq2p, Pdr5p, and Ycf1p, and (ii) factors regulating the expression of these membrane transport proteins, including Pdr1p, Pdr3p, Pdr7p, yAP1, and yAP2 (9Balzi E. Goffeau A. J. Bioenerg. Biomembr. 1995; 27: 71-76Crossref PubMed Scopus (229) Google Scholar). The detoxification pathways involving GST enzymes have not been studied in yeast, because gene(s) coding for GST in S. cerevisiaehave not been identified (10Wu A.-L. Hallstrom T.C. Moye-Rowley W.S. J. Biol. Chem. 1996; 271: 2914-2920Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 11Sheehan D. Casey J.P. Comp. Biochem. Physiol. 1993; 104B: 7-13Google Scholar, 12Casalone E. Di Ilio C. Federici G. Polsinelli M. Antonie van Leeuwenhoek. 1988; 54: 367-375Crossref PubMed Scopus (17) Google Scholar). However, overexpression of Issatchenkia orientalis GST in S. cerevisiaeelevated the resistance to o-dinitrobenzene (10Wu A.-L. Hallstrom T.C. Moye-Rowley W.S. J. Biol. Chem. 1996; 271: 2914-2920Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), and expression of human Alpha or Pi GST in S. cerevisiaeresulted in a marked increase in resistance to the anticancer drugs adriamycin and chlorambucil (13Black S.M. Beggs J.D. Hayes J.D. Bartoszek A. Muramatsu M. Sakai M. Wolf C.R. Biochem. J. 1990; 268: 309-315Crossref PubMed Scopus (128) Google Scholar). The GSH conjugates are transported to the vacuole by the yeast GS-X pump Ycf1p (product of YCF1gene; Refs. 14Li Z.-S. Szczypka M. Lu Y.-P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 15Li Z.-S. Lu Y.-P. Zhen R.-G. Szczypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar, 16Wemmie J.A. Moye-Rowley W.S. Mol. Microbiol. 1997; 25: 683-694Crossref PubMed Scopus (44) Google Scholar). In this paper we report an identification of the first two S. cerevisiae genes, GTT1 and GTT2, coding for functional GST enzymes. We expressed both Gtt1p and Gtt2p in E. coli, purified the proteins, and determined that they both exhibit GST activity. Transcription from GTT1 promoter is induced by osmotic stress and xenobiotics, and, most significantly, after diauxic shift. Subcellular fractionation experiments show that Gtt1p associates with the endoplasmic reticulum. GTT1 and GTT2 are not essential to the cell, but gtt1Δ, gtt2Δ, and gtt1Δgtt2Δ mutant strains exhibit significantly reduced thermotolerance in the stationary phase and limited ability to grow at 39 °C. Yeast strains used in this study (listed in Table I) were grown in rich medium (YPD; 1% yeast extract, 2% Bacto-peptone, 2% glucose) or under selection in synthetic minimal medium (SD) supplemented with appropriate nutrients. Yeasts were manipulated as described previously (17Vancura A. Sessler A. Leichus B. Kuret J. J. Biol. Chem. 1994; 269: 19271-19278Abstract Full Text PDF PubMed Google Scholar).Table IYeast strains used in this studyStrainGenotypeSource1-aSources: 1, Rothstein laboratory (Columbia University, New York, NY); 2, this study; 3, CLONTECH.W303–1aMATa ade2–1his3–11,15 leu2–3,112 trp1–1 ura3–1 ssd1-d2 can1–1001Ishikawa T. Trends. Biochem. Sci. 1992; 17: 463-468Abstract Full Text PDF PubMed Scopus (570) Google ScholarJC100W303–1agtt1::TRP12Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google ScholarJC101W303–1agtt1::URA32Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google ScholarJC102W303–1agtt2::URA32Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google ScholarJC103W303–1agtt1::TRP1 gtt2::URA32Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google ScholarJC104W303–1a [pYX242-HT-T7-GTT1]2Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google ScholarJC105W303–1a [pSEYC102-GTT1]2Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google ScholarCG1945MATa ura3–52 his3–200 lys2–801 trp1–901 ade2–101 leu2–3,112 gal4–542 gal80–538 LYS::GAL1-HIS3 cyhr2 URA3::GAL4 17-mers)3-CYC1-lacZ3Moscow J.A. Dixon K.H. Cytotechnology. 1993; 12: 155-170Crossref PubMed Scopus (47) Google Scholar1-a Sources: 1, Rothstein laboratory (Columbia University, New York, NY); 2, this study; 3, CLONTECH. Open table in a new tab Plasmid pYX242-HT-T7 was constructed by ligatingNcoI-SmaI fragment from plasmid pET28 (Novagen) into NcoI-SmaI-digested pYX242 plasmid (Novagen; yeast shuttle plasmid with 2μ, LEU2, and TPI(triose-phosphate isomerase) promoter). This introduces His tag and T7 epitope at the N terminus of a protein expressed from this plasmid. The coding region of GTT1 was isolated by PCR amplification using total yeast (strain W303–1a) DNA as a template and Vent DNA polymerase. The GTT1–5A oligonucleotide (5′-GCGCGGATCCTGTCGTTGCCAATTATCAAAGTCC-3′) introduced aBamHI site; the GTT1–3A oligonucleotide (5′-GCGCGAGCTCTTATTGGGCCCTGAAATTGCTACCTAAAGCACGC-3′) introduced aSacI site. The resulting 0.7-kb DNA fragment was digested with BamHI and SacI and ligated in pYX242-HT-T7 or pCITE-4C plasmids restricted with the same enzymes to yield pYX242-HT-T7-GTT1 or pCITE-4C-GTT1, respectively. For construction of pET23b-GTT1, pET14b-GTT1, pGBT9-GTT1, and pGAD424-GTT1, the GTT1 coding sequence was generated by PCR as described above with oligonucleotides GTT1–5B (5′-GCGCGGATCCATATGTCGTTGCCAATTATCAAAGTCC-3′) and GTT1–3B (5′-GCGCGGATCCTCACTCGAGGAAATTGCTACCTAAAGCACGC-3′). Primer GTT1–5B introduces NdeI and BamHI sites and GTT1–3B introduces XhoI and BamHI sites. The PCR-derived GTT1 fragment was digested with NdeI and XhoI and ligated in NdeI-XhoI sites of pET23b (Novagen) and in NdeI-BamHI sites of pET14b (Novagen). The resulting plasmids, pET23b-GTT1 and pET14b-GTT1, express N- and C-terminal Gtt1p fusions with His tag in E. coli, respectively. The above GTT1 fragment (generated with GTT1–5B and GTT1–3B primers) was digested with BamHI and ligated in BamHI site of pGBT9 and pGAD424, to create pGBT9-GTT1 and pGAD424-GTT1, respectively. To construct pET23d-GTT2, pGBT9-GTT2, and pGAD424-GTT2,BamHI and NcoI sites were first added to the 5′ end of GTT2's coding region, and XhoI and BamHI sites were added to the 3′ end of the coding region by PCR using oligonucleotides GTT2–5 (5′-GCGCGGATCCCCATGGATGGCAGAGGTTTCCTGATTTAC-3′) and GTT2–3 (5′-GCGCGGATCCTTACTCGAGCGAGGATTTTGAACGGATTTC-3′), respectively. The fragment was either digested with NcoI-XhoI and ligated into the same sites of pET23d to construct pET23d-GTT2, or it was digested with BamHI and ligated in BamHI site of pGBT9 or pGAD424 to create pGBT9-GTT2 and pGAD424-GTT2, respectively. The plasmids for expression and two-hybrid assay with Ure2p were built by first adding BamHI and NdeI sites to the 5′ end of the coding region of URE2, and XhoI and BamHI sites to the 3′ end of the coding region by PCR with oligonucleotides URE2–5 (5′-GCGCGGATCCATATGATGAATAACAACGGCAACC-3′) and URE2–3 (5′-GCGCGGATCCTCACTCGAGTTCACCACGCAATGCCTTGATG-3′), respectively. The fragment was digested with HindIII (internalHindIII site within URE2) and XhoI and ligated in HindIII-XhoI-digested pET23b. This plasmid was subsequently digested with NdeI and ligated withNdeI-digested PCR fragment of URE2 to create pET23b-URE2. The PCR fragment of URE2 was also digested withBamHI and ligated in BamHI site of pGBT9 or pGAD424 to create pGBT9-URE2 and pGAD424-URE2, respectively. In addition, plasmid pET23b-URE2(ΔN) for expression of Ure2p(ΔN) (N-terminal truncation of Ure2p) was constructed as described above for pET23b-URE2; however, the PCR fragment was generated with oligonucleotide URE2–5A (5′-GCGCGGATCCATATGAGTCACGTGGAGTATTCC-3′), which anneals 279 bp downstream from the ATG codon, and oligonucleotide URE2–3. To construct plasmid for expression of GTT1-lacZfusion, a 1854-bp fragment of GTT1 promoter region was amplified by PCR using oligonucleotide GTT1–5C (5′-GCGCGAATTCTGCAGCATATCCAGGACTAATACATCCAC-3′) that hybridizes 1836 bp upstream of the ATG start codon and introduces EcoRI site, and GTT1–3C (5′- GCGCGGATCCTTGATAATTGGCAACGACATAATGC-3′) that hybridizes 19 bp downstream of the ATG start codon and introducesBamHI site. The amplified 1.9-kb fragment was digested withEcoRI and BamHI and ligated in EcoRI-BamHI-digested pSEYC102 (CEN4, URA3, lacZ) to create pSEYC102-GTT1. The 2.6-kb DNA fragment encompassing 1.9 kb of the promotor and 0.7 kb of the coding region of GTT1 was generated by PCR using GTT1–5C and GTT1–3A oligonucleotides, and inserted in pYX242 to yield pYX242-GTT1.SalI-BamHI TRP1 gene fragment was excised from pJJ248 (19Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (327) Google Scholar) and ligated in XhoI-BglII sites of GTT1 gene in pYX242-GTT1 to create pYX242-GTT1-TRP1. The GTT1–5C and GTT1–3A oligonucleotides were used to generate a 2.1-kb PCR fragment containingTRP1 gene flanked by 0.3 and 0.5 kb of GTT1sequences. This fragment was used to direct homologous recombination at the GTT1 locus of W303–1a, creating strain JC100. Another disruption allele of GTT1 was constructed by a fusion PCR strategy (18Amberg D.C. Botstein D. Beasley E.M. Yeast. 1995; 11: 1275-1280Crossref PubMed Scopus (117) Google Scholar). The primers GTT1–5C and GTT1-KO2 (5′-GTCGTGACTGGGAAAACCCTGGCGTCGCGTGTCATCGTGCATAGC-3′) were used to amplify promotor region (540 bp) of GTT1, and GTT1-KO1 (5′-TCCTGTGGTAAATTGTTATCCGCTGTAGAAGGTTCCTTGCAACCA CC-3′) and GTT1–3A were used to generate 3′-terminal region (380 bp) of GTT1 gene. The URA3 selective marker was amplified with M13F (5′-CGCCAGGGTTTTCCCAGTCACGAC-3′) and M13R (5′-AGCGGATAACAATTTCACACAGGA-3′) primers, using pJJ244 as a template (20Rose M. Botstein D. Methods Enzymol. 1983; 101: 167-180Crossref PubMed Scopus (273) Google Scholar). The promotor fragment was first fused to the URA3marker by fusion PCR with GTT1–5C and M13R primers. The 1.8-kb PCR product was isolated and fused to the 3′-terminal fragment, using GTT1–5C and GTT1–3A primers. The final 2.2-kb PCR product was then gel-purified and used to transform strain W303–1a to create strain JC101. To construct deletion in GTT2 gene, URA3 fragment was excised from pJJ244 plasmid (19Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (327) Google Scholar) asBamHI-PvuII fragment and was ligated intoBamHI-SmaI-digested pSK(−) to create pSK-URA3. A 333-bp fragment from the promotor region of GTT2 was amplified with oligonucleotides that hybridized 370 bp (GTT2-C1; 5′-GCGCGAATTCAGGAACAGTAGTG CAATTGGG-3′) and 37 bp (GTT2-C2; 5′-GCGCGAATTCGAGCTCTCTATCCTTTAGCAGGAAGCAC-3′) upstream of the ATG codon, and EcoRI sites were introduced. The fragment was ligated into EcoRI site of pSK-URA3 to construct pSK-URA3-GTT2–5. A 254-bp fragment corresponding to the 3′-terminal region of GTT2 was amplified with oligonucleotides that hybridized 445 bp (GTT2-C3; 5′-GCGCGGATCCGAGCTCAAGAGTGGGGACTTCGCCAG-3′) and 699 bp (GTT2–3; 5′-GCGCGGATCCTTACTCGAGCGAGGATTTTGAACGGATTTC-3′) downstream of the ATG codon and introduced BamHI sites. The fragment was ligated into BamHI site of pSK-URA3-GTT2–5 plasmid to construct pSK-URA3-GTT2–5,3. Primers GTT2-C1 and GTT2–3 were used to generate DNA fragment of URA3 flanked byGTT2 sequences. The PCR-generated DNA fragment was transformed into W303–1a and JC100 cells to create strains JC102 and JC103, respectively. Strain JC105 was grown in SD-Ura medium at 30 °C overnight and then diluted in YPD medium to an A 600 nm of 0.15. Cells were grown to anA 600 nm of 1.3 before treatment with various stress conditions for 1 h. In heat shock experiments, cells were grown at 23 °C to an A 600 nm of 1.5, and then shifted to 37 °C for 1 h. Cells were harvested and disrupted by vortexing with glass beads, and the β-galactosidase activity was assayed usingo-nitrophenyl-β-d-galactoside essentially as described previously (20Rose M. Botstein D. Methods Enzymol. 1983; 101: 167-180Crossref PubMed Scopus (273) Google Scholar). Protein concentration was determined using the Coomassie Plus protein assay kit (Pierce). BL21 (DE3) cells harboring pET23b-GTT1, pET14b-GTT1, pET23d-GTT2, pET23b-URE2, and pET23b-URE2(ΔN) were grown in LB medium containing ampicillin (100 μg/ml) at 37 °C to anA 600 nm of 0.6, at which point isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm. After 3 h of induction, cells were harvested by centrifugation (15 min at 3000 ×g; 4 °C) and stored at −70 °C until used. All subsequent steps were carried out at 4 °C. Frozen cells were thawed, resuspended in three volumes of lysis buffer (20 mm Tris, pH 7.5, 0.5 m NaCl, 10% glycerol, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml each of leupeptin, aprotinin, and pepstatin) and ruptured by sonication (10 × 30 s). The homogenate was centrifuged (10 min at 5000 × g; 4 °C), supernatant removed, and the pellet resuspended in the lysis buffer, sonicated (10 × 30 s) and centrifuged again. The supernatants were combined and centrifuged (1 h at 170,000 ×g, 4 °C). The clear supernatant was loaded onto a Ni2+-agarose column (2 ml) preequilibriated with lysis buffer. The column was washed with 20 ml of the lysis buffer and then eluted with 4-ml aliquots of the lysis buffer containing 10, 20, 50, 100, 200, and 500 mm imidazole. Fractions with high GST activity were pooled and dialyzed against storage buffer (50% glycerol, 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm dithiothreitol, 0.1 mm EDTA, 0.01% Brij 35). The GST activity of Gtt1p and Gtt2p was assayed spectrophotometrically (21Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar). The reaction mixture contained 0.1m potassium phosphate buffer, pH 6.5, 1 mmreduced glutathione (GSH), and 1 mm1-chloro-2,4-dinitrobenzene (CDNB). The reaction mixture was preincubated at 25 °C (5 min), and the reaction was initiated by an addition of the enzyme. The increase of A 340 nmwas recorded, and the activity was calculated from the difference in A 340 nm changes in the reaction mixtures with and without the enzyme. JC104 cells were grown, lysed, and fractionated by differential centrifugation into 10,000 × g pellet (P1), 170,000 × gpellet (P2), and supernatant (S) fractions as described previously (17Vancura A. Sessler A. Leichus B. Kuret J. J. Biol. Chem. 1994; 269: 19271-19278Abstract Full Text PDF PubMed Google Scholar,22Wang X. Hoekstra M. DeMaggio A. Dhillon N. Vancura A. Kuret J. Johnston G. Singer R. Mol. Cell. Biol. 1996; 16: 5375-5385Crossref PubMed Scopus (82) Google Scholar). P1 and P2 pellets were then subjected to density gradient fractionation (17Vancura A. Sessler A. Leichus B. Kuret J. J. Biol. Chem. 1994; 269: 19271-19278Abstract Full Text PDF PubMed Google Scholar). The gradients were fractionated from the top (600-μl fractions) and labeled sequentially as fractions 1–20. All fractions were assayed in duplicate for protein and sucrose concentration. Epitope-tagged HT-T7-Gtt1p was assayed by immunoblotting with monoclonal T7-Tag® antibody. The following marker proteins were assayed by immunoblotting with specific antibodies: vanadate-sensitive plasma membrane ATPase (assayed with Pma1 polyclonal antibody provided by Dr. A. Chang), Kex2p (assayed with KXR-B6 polyclonal antibody provided by Dr. R. Fuller), vacuolar H+-ATPase subunit (V-ATPase; assayed with 10D7-A7-B2 monoclonal antibody, Molecular Probes), mitochondrial porin (assayed with 16G9-E6 monoclonal antibody, Molecular Probes), dolichol-phosphate mannose synthase (assayed with 5C5-A7 monoclonal antibody, Molecular Probes). Immunoreactivity was measured by enhanced chemiluminescence using sheep anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Pharmacia Biotech). Images were collected on x-ray film and quantified by laser densitometry. A Blast search of GenBank and Saccharomyces Genome Database revealed three open reading frames whose putative protein products exhibited similarity to known mammalian and plant GST enzymes. The first uncharacterized open reading frame, YIRO38c, codes for a protein of 234 amino acids (named Gtt1p: glutathionetransferase 1). Since Gtt1p exhibits only a limited sequence homology with GSTs from other organisms, it was not clear whether it also encodes a protein with GST activity. When we overexpressed HT-T7-Gtt1p (N-terminal fusion with His tag and T7 epitope) in JC104 strain, the GST activity in the cell lysate was elevated only 2-fold compared with the W303–1a strain. Synthesis of Gtt1p by in vitro transcription-translation yielded similar results, but the GST activity in the reticulocyte lysate was elevated only 50% compared with control. Since these results do not unambiguously show that GTT1 codes for a protein with GST activity, we expressed Gtt1p in E. coli as a C-terminal or N-terminal fusion with His tag. After induction with isopropyl-1-thio-β-d-galactopyranoside, we detected high GST activity in E. coli lysate. This GST activity co-eluted with the induced protein during a subsequent chromatography on Ni2+-agarose (Fig. 1 A, Table II). Enzyme characterization was performed with purified Gtt1p with C-terminal His tag.K m values for GSH and 1-chloro-2,4-dinitrobenzene (CDNB; the model substrate for GST) are 0.6 mm and 3.3 mm, respectively, and V max is 500 nmol/min·mg. These results confirm that GTT1 codes for a protein with GST activity.Table IIPurification of recombinant Gtt1pFractionProteinTotal activitySpecific activityPurificationYieldmgnmol/minnmol/min·mg-fold%1. Extract before inductionND2-aND, not determined.—2-bThe GST activity in the extract before induction was not detectable.———2. Extract after induction305.4353.41.211003. Ni2+-agarose7.2234.132.527662-a ND, not determined.2-b The GST activity in the extract before induction was not detectable. Open table in a new tab The second gene with sequence similarity to GSTs is also an uncharacterized open reading frame (YLL060c; GenBank Accession numberZ73165). Again, after we had confirmed that the protein product of YLL060C exhibits GST activity (described below), we named itGTT2 (glutathionetransferase 2). We expressed Gtt2p in E. coli as a C-terminal fusion with His tag, purified the protein on a Ni2+-agarose column, and determined its GST activity (Fig. 1 B, Table III). The results confirm that Gtt2p is also a GST enzyme; however, itsK m values (0.2 mm for CDNB and 5.0 mm for GSH), as well as its V max(340 nmol/mg·min), differ from Gtt1p.Table IIIPurification of recombinant Gtt2pFractionProteinTotal activitySpecific activityPurificationYieldmgnmol/minnmol/min·mg-fold%1. Extract before inductionND3-aND, not determined.—3-bThe GST activity in the extract before induction was not detectable.———2. Extract after induction107.6390.63.611003. Ni2+-agarose1.660.337.210153-a ND, not determined.3-b The GST activity in the extract before induction was not detectable. Open table in a new tab The third gene whose protein product exhibited limited similarity to GST enzymes is URE2, which functions as a transcriptional regulator of genes involved in nitrogen metabolism (23Coschigano P.W. Magasanik B. Mol. Cell. Biol. 1991; 11: 822-832Crossref PubMed Scopus (222) Google Scholar, 24Blinder D. Coschigano P.W. Magasanik B. J. Bacteriol. 1996; 178: 4734-4736Crossref PubMed Google Scholar). We also expressed Ure2p in E. coli. Although induction of Ure2p synthesis can be easily detected on SDS-polyacrylamide gels, it did not result in any detectable GST activity in the E. coliextract. Since Ure2p contains an N-terminal extension that is not shared by Gtt1p, Gtt2p, or other GST enzymes, we constructed, expressed, and purified (Fig. 1 C) a truncated form of Ure2p (Ure2p(ΔN)), which has this N-terminal extension (93 amino acids) deleted. Ure2p(ΔN) also did not exhibit any detectable GST activity. This result, however, is not entirely surprising. Coschigano and Magasanik (23Coschigano P.W. Magasanik B. Mol. Cell. Biol. 1991; 11: 822-832Crossref PubMed Scopus (222) Google Scholar) and Blinder et al. (24Blinder D. Coschigano P.W. Magasanik B. J. Bacteriol. 1996; 178: 4734-4736Crossref PubMed Google Scholar) found that Ure2p directly, and in catalytic fashion, inhibits the ability of zinc finger protein Gln3p to activate transcription. Given the sequence similarity of Ure2p with GST enzymes, these authors speculated about possible Ure2p-catalyzed glutathiolation and inactivation of Gln3p, but they did not detect any GST activity associated with Ure2p. Pairwise protein sequence alignment (CLUSTAL W program with PAM250 weight table; Ref. 25Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar) revealed that Gtt1p and Gtt2p exhibit only 43% sequence similarity (11% identity and additional 32% similarity), and both Gtt1p and Gtt2p display 38% similarity with Ure2p. Blast search of GenBank identified GST from maize (form III and IV), Silene vulgaris (plant Phi class), Arabidopsis, Manducta sexta, silkworm, and house fly as most similar GST enzymes (about 50% similarity with both Gtt1p and Gtt2p). The conserved amino acid residues are clustered in the N-terminal region (Fig. 2), which corresponds to the GSH binding site (26Sinning I. Kleywegt G.J. Cowan S.W. Reinemer P. Dir H.W. Huber R. Gilliland G.L. Armstrong R.N. Xinhua J. Board P.G. Olin B. Mannervik B. Jones A.T. J. Mol. Biol. 1993; 232: 192-212Crossref PubMed Scopus (415) Google Scholar, 27Dirr H. Reinemer P. Huber R. J. Mol. Biol. 1994; 243: 79-92Crossref Scopus (84) Google Scholar). The cytosolic GST enzymes in all vertebrate species comprise two subunits, and exist as either homodimers or heterodimers, with each subunit in the dimeric protein functioning independently (2Hayes J.D. Pulford D.J. Crit. Rev. Biochem. Mol. Biol. 1995; 30: 445-600Crossref PubMed Scopus (3267) Google Scholar). To determine whether Gtt1p, Gtt2p, and Ure2p are also able to form homodimers or heterodimers, we performed a two-hybrid assay (28Fields S. Song O. Nature. 1989; 340: 245-247Crossref PubMed Scopus (4880) Google Scholar, 29Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1224) Google Scholar). The coding regions of GTT1, GTT2, and URE2 were inserted in both GAL4 DNA binding domain plasmid and GAL4 activation domain plasmid, and strains containing both constructs and corresponding control plasmids were streaked on selective medium containing 10 mm 3-amino-1,2,4-triazole (Fig. 3). The results show that both Gtt1p and Gtt2p are able to form homodimers in vivo, but they do not form Gtt1p-Gtt2p, Gtt1p-Ure2p, and Gtt2p-Ure2p heterodimers. We could not test the Ure2p ability to dimerize, since when expressed from GAL4 DNA binding domain plasmid, it nonspecifically activated transcription of the reporter genes. Cell lysate, prepared from JC104 strain which expresses HT-T7-Gtt1p fusion, was subjected to differential centrifugation as described previously (17Vancura A. Sessler A. Leichus B. Kuret J. J. Biol. Chem. 1994; 269: 19271-19278Abstract Full Text PDF PubMed Google Scholar). The amounts of HT-T7-Gtt1p and organelle specific markers in individual fractions were determined by Western blotting and chemiluminescence detection. The majority of Gtt1p (63%) was found in 10,000 × gpellet, 22% sedimented at 170,000 × g, and 12% was recovered in the soluble fraction (Table IV). This distribution closely parallels the distribution pattern of dolichol-phosphate mannose synthase, a marker for endoplasmic reticulum, while distribution profiles of the other markers were clearly different. To increase the resolution of the analysis, the 10,000 × g pellet was subjected to sucrose density gradient fractionation (17Vancura A. Sessler A. Leichus B. Kuret J. J. Biol. Chem. 1994; 269: 19271-19278Abstract Full Text PDF PubMed Google Scholar). Again, the fractionation profile of HT-T7-Gtt1p is nearly identical to the distribution of dolichol-phosphate mannose synthase, and distinct from distribution of other markers (Fig. 4).Table IVDifferential centrifugation analysis of Gtt1p localizationMarker proteinP1P2SPM ATPase (plasma membrane)4-aPM, plasma membrane; Dol-P-Man, dolichol-phosphate mannose.28644Kex2p (Golgi)315311V-ATPase (vacuoles)283137Porin (mitochondria)8711<1Dol-P-Man synthase (endoplasmic reticulum)65264HT-T7-Gtt1p632212JC104 cells were lysed and subjected to differential centrifugation as