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Cell Biology International (2006) 30, 665671 (Printed in Great Britain)
Role of γ-glutamyltranspeptidase in detoxification of xenobiotics in the yeasts Hansenula polymorpha and Saccharomyces cerevisiae
Vira M. Ubiyvovka, Oleksandra V. Blazhenkoa, Daniel Gigotb, Michel Penninckxc and Andriy A. Sibirnyad*
aInstitute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street, 14/16, Lviv 79005 Ukraine
bUniversité Libre Bruxelles, Laboratory of Microbiology, Jean Wiame Institute, 1 Av E Gryson, B-1070 Brussels, Belgium
cUniversité Libre Bruxelles, Laboratory of Microbial Physiology and Ecology, 642 rue Engeland, B-1180 Brussels, Belgium
dRzeszów University, Department of Metabolic Engineering, Cwiklinskiej 2, 35-310 Rzeszów, Poland
GGT1 gene of the methylotrophic yeast Hansenula polymorpha appears to be a structural and functional homologue of Saccharomyces cerevisiae CIS2/ECM38 gene encoding γ-glutamyltranspeptidase (γGT). This is confirmed by the absence of the corresponding activity of γGT in the mutant with disrupted GGT1 gene. It was shown that γGT of both H. polymorpha and S. cerevisiae are involved in detoxification of electrophilic xenobiotics, as the corresponding mutants appeared to be defective in the disappearance of the fluorescent vacuolar complex of GSH with xenobiotic bimane and the further diffuse distribution of this complex in the cytosol. We hypothesize that metabolism of electrophilic xenobiotics in the yeasts H. polymorpha and S. cerevisiae occurs through a γGT-dependent mercapturic acid pathway of GSH-xenobiotic detoxification, similar to that known for mammalian cells, with cysteine-xenobiotics and/or N-acetylcysteine-xenobiotics as the end products.
Keywords: γ-glutamyltranspeptidase, Gene cloning, Yeast, Hansenula polymorpha, Saccharomyces cerevisiae, Xenobiotics, Detoxification.
*Corresponding author. Institute of Cell Biology, National Academy of Sciences of Ukraine, Molecular Genetics and Biotechnology Department, Drahomanov Street, 14/16, Lviv 79005, Ukraine. Tel.: +380 322 740 363; fax: +380 322 721 648.
Metabolism of methanol in yeasts requires GSH-dependent detoxification of toxic methanol oxidation intermediates, such as formaldehyde, hydrogen peroxide and alkyl hydroperoxides, which occurs in formaldehyde dehydrogenase, formaldehyde reductase and GSH peroxidase reactions (Sahm, 1977; Sibirny et al., 1990; Sakai et al., 2001). Methanol-induced cells of methylotrophic yeast wild type strains, and especially of formaldehyde dehydrogenase- and formaldehyde reductase-deficient mutants, accumulate elevated levels of both formaldehyde and GSH (Ubiyvovk and Trotsenko, 1986; Sibirny and Ubiyvovk, 1988; Sibirny et al., 1990). It is known that GSH non-enzymatically binds formaldehyde producing non-toxic S-hydroxymethylglutathione (Sahm, 1977).
In this communication, we describe the isolation and characterization of the mutant of the methylotrophic yeast H. polymorpha defective in γGT. It was shown that γGT is important for detoxification of electrophilic xenobiotics (monobromobimane and N-[1-pyrene]maleimide) in both H. polymorpha and S. cerevisiae. We hypothesize that GSH conjugates with the studied xenobiotics are metabolized through the mercapturic acid pathway. The obtained data could have implications for the study of GSH-dependent multidrug resistance in some mammalian tumours and detoxification of herbicides in plants.
2 Materials and methods
2.1 Yeast strains and media
H. polymorpha strains used were CBS4732 leu2-2 ura3-20 (kindly provided by K. Lahtchev, Bulgaria) and originated from it: CBS4732 leu2-2∷LEU2 ura3-20 wild type strain and null mutant ggt1∷LEU2 ura3-20 (preparation described later). The S. cerevisiae strains were: wild type – Y1000 MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0 (BY4774) and null mutant Y15209 MATα, his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YLR299w::kanMX4 (cis2; ecm38). The strains were grown in YPD medium (2% glucose, 2% bactopeptone, 1% yeast extract with or without 2% agar). Synthetic medium 1 used for fluorescence microscopy and electrophile transport studies of H. polymorpha and S. cerevisiae contained the following compounds (per litre): 30
2.2 Construction of HpGGT1 deleted cassette and null Hpggt mutant isolation
General DNA manipulation was performed as described in Sambrook et al. (1989). To create the Hpggt1∷LEU2 allele in which HpGGT1 gene was disrupted with ScLEU2 gene, the N′-end fragment (433
Oligonucleotides used as primers for PCR amplification
2.3 Construction of H. polymorpha CBS4732 leu2-2∷LEU2 ura3-20 wild type strain
H. polymorpha CBS4732 leu2-2∷LEU2 ura3-20 wild type strain was obtained by the electroporation of H. polymorpha CBS4732 leu2-2 ura3-20 wild type cells with vector pYT1 linearized in PstI restriction site. Leu+ integrants were selected on glucose synthetic medium 2 without leucine.
2.4 Growth of H. polymorpha cells on various sulfur and nitrogen sources
The regulation of γGT activity in H. polymorpha was studied in nitrogen-deficient synthetic medium 2 without (NH
2.5 γGT activity assay
The method of Payne and Payne (1984) was used with some modifications. Cell-free extracts (0.1–0.2
2.6 Fluorescence microscopy of H. polymorpha and S. cerevisiae cells
Cells (wild type strains and null ggt mutants) grown in synthetic medium 1 to OD
2.7 Transport of electrophiles out of H. polymorpha and S. cerevisiae cells
Cells (wild type strains and null ggt mutants) grown in synthetic medium 1 to OD
2.8 High-performance liquid chromatography (HPLC)
The derivatives were eluted from a C18μ Bondapak column (300
3 Results and discussion
3.1 Functional analysis of HpGGT1 gene
H. polymorpha genome was searched for the presence of the gene coding for γGT, involved in GSH catabolism. Two ORFs, HpGGT1 (putative product, γGT1) and HpGGT2 (putative product, γGT2), were identified based on their primary sequence homology to S. cerevisiae CIS2/ECM38/YLR299w gene.
Protein sequence analysis revealed that H. polymorpha putative γGT1 protein shares the strongest homology with S. cerevisiae Cis2p (42% identity and 59% similarity). H. polymorpha putative γGT2 protein possesses weaker homology to S. cerevisiae Cis2p (21% identity and 38% similarity) (Fig. 1).
Alignment of the deduced amino acid sequences of the putative γGT1 and γGT2 proteins from H. polymorpha (H. pol.) with the homologous protein Cis2p from S. cerevisiae (S. cer.).
It is well known that yeast γGT catalyzes the transfer of the γ-glutamyl moiety of GSH and γ-glutamyl compounds to amino acids and also the hydrolytic release of
Effect of sulfur (ammonium-containing medium) and nitrogen (sulfate-containing medium) on the activity of γGT (μmol h−1 mg protein−1) of the H. polymorpha wild type strain CBS4732 leu2-2 ura3-20 (WT) and Δggt1LEU2 ura3-20 (Δggt1) mutant ± SEM
In our study we demonstrated that γGT activity did not initiate transformation of GSH to supply H. polymorpha cells with cysteine/sulfur when the GSH was added as the sole sulfur source (Fig. 2A, C). Yeast cells of null ggt1 mutant as well as the wild type strain were able to utilize exogenous GSH as the sole sulfur source and grow in two media with different nitrogen sources (ammonium or glutamate) (Fig. 2A, C data shown only for ammonium). It seemed that γGT of H. polymorpha was not implicated in supplying the cells with nitrogen from GSH, because both strains (wild type and mutant) demonstrated similarly weak growth in media with GSH as the sole nitrogen source compared to glutamate or ammonium-containing media (Fig. 2B, D). Weak growth of both strains on indicated concentrations of GSH could be explained by significantly higher yeast requirements in nitrogen than in sulfur sources and/or by GSH growth inhibitory effects in concentrations above 0.5
Growth of H. polymorpha wild type strain (A, B) and Δggt1 mutant (C, D) depending on sulfur and nitrogen source: (A, C) sulfur-deficient NH4+-containing medium (circles) supplied with 0.1
It was assumed that yeast H. polymorpha, similar to S. cerevisiae (Kumar et al., 2003), possesses an alternative γGT-independent GSH degradation pathway to supply it with sulfur and nitrogen from GSH. The second H. polymorpha gene GGT2 may be responsible for GSH breakdown. We plan to elucidate this in our further study with null ggt2 and double ggt1
H. polymorpha wild type strain and null ggt1 mutant were tested for growth in the media containing stress generating agents. It was shown that null ggt1 mutant was more sensitive to the cell surface polymer perturbing agent, calcofluor white, and tert-butyl hydroperoxide and more resistant to CdSO
Growth sensitivity of H. polymorpha wild type strain CBS4732 leu2-2∷LEU2 ura3-20 (WT) and Δggt1∷LEU2 ura3-20 (Δggt1) mutant was determined by spotting the cells on synthetic medium 2 containing one of the following toxic compounds: 10
3.2 Fluorescence microscopy of H. polymorpha and S. cerevisiae fluorogenic xenobiotic metabolism
Yeasts H. polymorpha and S. cerevisiae eliminate electrophilic xenobiotics (monobromobimane) from the cytosol after their spontaneous or GSH-transferase-mediated conjugation with GSH (GS-bimane) and transport them to vacuoles (Ubiyvovk et al., 2003; Zadzinski et al., 1996). Further fate of vacuolar GSH conjugates and possible detoxification pathways in yeast cells remains unknown (see, however, Elskens and Penninckx, 1997). It is well known that bimanes are almost nonfluorescent until they react with thiols. The fluorescence microscopy study of vacuolar accumulation of fluorescent GS-bimane conjugate (this thiol conjugate was identified earlier, Zadzinski et al., 1996; Ubiyvovk et al., 2003) and kinetics of the disappearance of vacuolar fluorescence by cells of wild type strains and mutants with null activity of γGT, null ggt, of H. polymorpha and S. cerevisiae were conducted. Initial GS-bimane vacuolar fluorescence was observed in both H. polymorpha and S. cerevisiae wild type strains and null ggt mutants after 3
Fluorescence (A) and phase-contrast (B) microscopy of the yeast H. polymorpha and S. cerevisiae wild type strains (HpWT, ScWT) and null ggt mutants (HpΔggt1, ScΔcis2) after 3
3.3 Study of electrophile derivative transport out of H. polymorpha and S. cerevisiae cells 3.3.1 Extrusion of monobromobimane derivatives HPLC study of extracellular extrusion of monobromobimane derivatives of the wild type and null ggt mutant cells of H. polymorpha and S. cerevisiae was performed with extracellular medium of yeast cells incubated for 1 Fig. 5 HPLC profiles of extracellular derivatives of monobromobimane (mBB) (A) and N-[1-pyrene]maleimide (N-Pyr) (B) exported by yeast H. polymorpha and S. cerevisiae wild type strains (WT) and null ggt (Δggt1,Δcis2) mutant cells. Cells were incubated with 100
3.3.1 Extrusion of monobromobimane derivatives
HPLC study of extracellular extrusion of monobromobimane derivatives of the wild type and null ggt mutant cells of H. polymorpha and S. cerevisiae was performed with extracellular medium of yeast cells incubated for 1
HPLC profiles of extracellular derivatives of monobromobimane (mBB) (A) and N-[1-pyrene]maleimide (N-Pyr) (B) exported by yeast H. polymorpha and S. cerevisiae wild type strains (WT) and null ggt (Δggt1,Δcis2) mutant cells. Cells were incubated with 100
3.3.2 Extrusion of N-[1-pyrene]maleimide derivatives
We have also studied the γGT-dependent extrusion of another electrophilic compound N-[1-pyrene]maleimide that could form conjugates with GSH and be extruded from cells as different xenobiotic derivatives (Ubiyvovk et al., 2003). Extracellular medium of cells of the null ggt mutants of H. polymorpha and S. cerevisiae incubated for 1
In conclusion, we have found for the first time that γGT is involved in detoxification of electrophilic xenobiotics in yeasts. In methylotrophic yeast, this enzyme can also fulfil some specific functions connected with methanol metabolism. In favor of this conclusion is the fact that methanol strongly induces γGT in gsh2 mutants of H. polymorpha defective in the first reaction of GSH synthesis (Ubiyvovk et al., 2002a,b). The specific role of γGT in methanol metabolism remains to be elucidated.
This study was supported by the Collaborative NATO Linkage Grant LST.CLG 979872. Access to H. polymorpha genome database provided by Rhein Biotech GmbH (Duesseldorf, Germany) is gratefully acknowledged. We are indebted to Dr. O.V. Stasyk for friendly discussion and help.
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Received 15 December 2005/24 March 2006; accepted 26 April 2006doi:10.1016/j.cellbi.2006.04.006