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Cell Biology International (2003) 27, 785–789 (Printed in Great Britain)
Vacuolar accumulation and extracellular extrusion of electrophilic compounds by wild-type and glutathione-deficient mutants of the methylotrophic yeast Hansenula polymorpha
Vira M. Ubiyvovk1, Janusz Maszewski2, Grzegorz Bartosz23 and Andrei A. Sibirny13*
1Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street, 14/16, Lviv 79005, Ukraine
2Department of Molecular Biophysics, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland
3Rzeszów University, Cegielniana 12, 35-310 Rzeszów, Poland


Abstract

The methylotrophic yeast Hansenula polymorpha CBS4732 leu2 detoxifies electrophilic xenobiotics by glutathione (GSH)-dependent accumulation in vacuoles, as shown by fluorescence microscopy. GSH-dependent and GSH-independent export of xenobiotic derivatives were also demonstrated by high-performance liquid chromatography (HPLC). Conjugates of GSH and N-acetylcysteine with monobromobimane and N-[1-pyrene]maleimide were observed among the HPLC fractions, along with unidentified derivatives.


Keywords: Glutathione, Methylotrophic yeast, Hansenula polymorpha, Xenobiotics, Detoxification, Accumulation, Export.

*Corresponding author. Tel.: +380-322-740-363; fax: +380-322-721-648


1 Introduction

The tripeptide glutathione (GSH) is present in most living cells, from microorganisms to humans. It fulfils a variety of metabolic functions, participating in different stress responses, including detoxification of electrophiles (Penninckx, 2000). Proteins that pump glutathione-S-conjugates in mammalian, plant and yeast cells (MRP1, cMOAT, AtMRP, Ycf1) are structural and functional homologues and are responsible for extracellular elimination (mammals) or vacuolar sequestration (plants and yeasts) of several pharmacologically and agriculturally important compounds (Ishikawa et al., 1997).

It is well established that electrophilic xenobiotics in Saccharomyces cerevisiae are sequestered by GSH-dependent Ycf1p-mediated transport into the vacuole (Li et al., 1996). There is also some evidence for GSH-dependent extracellular extrusion of xenobiotics(St-Pierre et al., 1994; Z̊ądziński et al., 1996). Methylotrophic yeasts are evolutionarily adapted to metabolism and detoxification of the electrophilic metabolite formaldehyde, excess of which is excreted from cells during methylotrophic growth (Sahm, 1976). It is plausible that systems for excreting electrophilic compounds in the form of their GSH-conjugates have developed in methylotropic yeasts. Exogenous GSH is taken up by GSH-deficient mutants, using specific energy-dependent transport systems (Ubiyvovk et al., 1999).

We have mutants of the methylotrophic yeast Hansenula polymorpha that are defective in GSH synthesis and are unable to grow on GSH-deficient media with methanol or other carbon substrates. These mutants belong to two complementation groups, gsh1 and gsh2. They are devoid of γ-glutamylcysteine synthetase (γGCS) activity, but have GSH synthetase activity similar to that in the wild-type strain (Ubiyvovk et al., 1999). The mutant gsh2 is defective in the gene GSH2, which encodes γGCS (Ubiyvovk et al., 2002). Such a mutant enables the intracellular content of GSH to be manipulated and the role of this tripeptide in the metabolism of electrophilic xenobiotics to be elucidated.

The aim of this paper was to study the role of GSH in the detoxification of electrophilic compounds by intracellular accumulation and excretion from methylotrophic cells. The data could have implications for the modulation 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

The H. polymorpha strains used were CBS4732 leu2 (obtained from Centraalbureau voor Schimmelcultures, Delft, Netherlands) and the gsh mutant, gsh2-1 leu2, isolated as resistant to N-methyl-N′-nitro-N-nitrozoguanidine and sensitive to both cadmium ions and methanol (Ubiyvovk et al., 1999). The strains were grown in YPD medium (2% glucose, 2% bactopeptone, 1% yeast extract with or without 2% agar), and on a semi-synthetic medium with 0.05% yeast extract and leucine (20 mg/ml), or were incubated in the synthetic medium supplemented with biotin (5 μg/l), thiamine chloride (0.5 mg/l), trace elements and leucine (Sibirnyet al., 1990). The latter two types of media contained 1% glucose as a source of carbon and energy.

2.2 Glutathione assay

The total intracellular concentration of GSH (GSH+GSSG) (Brehe and Burch, 1976) was analysed in cell-free extracts as described by Sibirny et al. (1990). The protein concentration was determined by the Lowry method (Lowry et al., 1951).

2.3 Growth sensitivity of H. polymorpha yeast cells to electrophilic xenobiotics

Yeast cells of H. polymorpha CBS4732 leu2 (wild type) and gsh2-1 leu2 mutant were grown in YPD medium for 24 h at 30 °C to an O.D. of approximately 2.0 at 540 nm. Aliquots (5 μl) of the suspensions were transferred to the plates with solid YPD medium containing different concentrations of N-[1-pyrene]maleimide (5–20 μM) and monobromobimane (10–100 μM). Growth of wild type and gsh mutant cells was insensitive to the concentrations of xenobiotics used (tested on the fourth day).

2.4 Preparation of H. polymorpha cells with different GSH levels (dGSH- and rGSH-cells)

gsh2-1 leu2 mutant cells pre-grown in the semi-synthetic medium with glucose until mid-exponential phase were pelleted and incubated in fresh glucose-containing synthetic medium (1 mg cells/ml) for 16 h at 30 °C with shaking (for intracellular GSH exhaustion). These were referred to as dGSH-cells (GSH-deficient). gsh2-1 leu2 mutant cells prepared as dGSH-cells were further incubated in the synthetic medium with 0.1 mM GSH (1 mg cells/ml) for 40 min at 30 °C (for intracellular GSH enrichment); these were referred to as rGSH-cells (GSH-rich). Wild type cells of H. polymorpha CBS4732 leu2 strain were prepared by the same manner as dGSH- and rGSH-cells without significant changes in the intracellular levels of total GSH.

2.5 Transport of electrophiles out of yeast cells

Wild type cells of H. polymorpha CBS4732 leu2 strain, and gsh2-1 leu2 mutants prepared as dGSH- and rGSH-cells, were washed with distilled water and suspended to cell concentrations of 5% (v/v) in 6 ml of 1% glucose, 0.1 M sodium phosphate buffer, pH 7.5. The suspensions were preincubated for 10 min at 30 °C on a shaker and then incubated for 40 min with different electrophiles: 10 μM N-[1-pyrene]maleimide or 30 μM monobromobimane. At chosen time intervals, aliquots were centrifuged (5000×g, 1 min at 4 °C) and the supernatants were frozen at −20 °C and then applied to a C18column.

2.6 HPLC

The derivatives were eluted from the C18column in a Waters HPLC apparatus with a linear gradient ofacetonitrile (0%–100%) containing 0.05% trifluoroacetic acid, and were detected by measuring the absorbance at 340 nm for N-[1-pyrene]maleimide derivatives, and at 400 nm for monobromobimane derivatives. GSH-conjugated derivatives as well as N-acetylcysteine-conjugated derivatives of N-[1-pyrene]maleimide and monobromobimane were identified using authentic compounds synthesized non-enzymatically as follows: mixtures of 0.1 mM GSH with 0.1 mM N-[1-pyrene]maleimide or 0.1 mM monobromobimane and mixtures of 0.1 mM N-acetylcysteine with 0.1 mM N-[1-pyrene]maleimide or 0.1 mM monobromobimane were incubated in 0.1 M Na-phosphate buffer, pH 7.1, for 3–4 h at 30 °C. Retention times of the derivatives on the HPLC C18column during linear water-acetonitrile elution were determined as 21.4, 23.4 and 26.7 min for GS-N-[1-pyrene]maleimide, ACS-N-[1-pyrene]maleimide and N-[1-pyrene]maleimide, respectively; and 16.0, 16.7 and 19.2 min for GS-monobromobimane, ACS-monobromobimane and monobromobimane, respectively.

2.7 Fluorescence microscopy

This was performed according to Li et al. (1996), with some modifications. Wild type CBS4732 leu2 and gsh2-1 leu2 mutant cells of H. polymorpha were grown in YPD medium for 24 h at 30 °C to an O.D. of approximately 2.0 at 540 nm and 1 ml aliquots of the suspensions were transferred to 10 ml volumes of fresh synthetic medium containing 1% glucose and 100 μM monobromobimane (synthetic medium was used for intracellular GSH exhaustion in gsh mutant cells). After incubation for 6 h at 30 °C, the cells were pelleted by centrifugation, washed twice with 0.1 M Na-phosphate buffer, pH 7.1, and viewed without fixation under a Nikon fluorescence microscope with phase-contrast attachment.

3 Results and discussion

It is known that electrophilic compounds readily enter cells and conjugate with GSH, spontaneously or with the help of GSH-transferases. In the present investigation we used non-toxic concentrations of xenobiotics, determined by growth sensitivity studies (Materials and methods). The intracellular distribution of a fluorescent conjugate of GSH with monobromobimane, a non-fluorescent electrophile, was determined in H. polymorpha wild type cells CBS4732 leu2 (Fig. 1A, GSH2) and the GSH-deficient mutant gsh2-1 leu2 (Fig. 1A, gsh2). Phase-contrast microscopy (Fig. 1B), which allowed visualization of the vacuoles, confirmed the overlap of the fluorescence signal from GS-monobromobimane with the vacuoles in the wild type strain (GSH2). In contrast, the GSH deficient strain (gsh2) showed a significantly lower intensity of GS-monobromobimane fluorescence.


Fig. 1

Fluorescence (A) and phase-contrast (B) microscopy of methylotrophic yeast H. polymorpha of wild type CBS4732 leu2 (GSH2) and GSH-deficient mutant gsh2-1 leu2 (gsh2) after 6 h incubation in synthetic medium with 100 μM monobromobimane.


Using HPLC analysis, we showed that, duringincubation of H. polymorpha cells with electrophilic compounds, N-[1-pyrene]maleimide (Fig. 2a) or monobromobimane (Fig. 2b), several (5–7) of their derivatives were exported to the extracellular medium. Comparing the HPLC profiles of derivatives extruded by dGSH-cells (containing less than 1.0 nmol of total GSH/mg protein) and rGSH-cells (containing more than 200 nmol of total GSH/mg protein in both wild type and gsh mutant cells), we have identified GSH-dependent and GSH-independent extruded fractions. The former were almost totally absent in dGSH-cells (Fig. 2A), but appeared during incubation of rGSH-cells (Fig. 2B). The latter were observed in the extracellular media of both dGSH- and rGSH-cells, independently of intracellular GSH levels (Fig. 2A, B). The GSH-dependent fractions of N-[1-pyrene]maleimide derivatives were composed of peak 21.4, corresponding to GS-N-[1-pyrene]maleimide (identified as described in Materials and Methods), and another unidentified heterogeneous peak with weakly divided sub-peaks (21.9, 22.2 and 22.4) (Fig. 2a).


Fig. 2

HPLC profiles of extracellular derivatives of (a) N-[1-pyrene]maleimide and (b) monobromobimane exported by methylotrophic yeast H. polymorpha gsh2-1 leu2 mutant dGSH- and rGSH-cells with (A) negligible or (B) normal levels of intracellular GSH, respectively. Mutant cells, denoted dGSH- and rGSH-cells, were incubated with (a) 10 μM N-[1-pyrene]maleimide or (b) 30 μM monobromobimane for 40 min, and extruded derivatives were analyzed by HPLC as described in Materials and methods.


We observed only traces of the ACS-N-[1-pyrene]maleimide fraction, however, this was elevated when cells were grown on YPD instead of semi-synthetic medium (data not shown). Peak 16.7, which corresponded to ACS-monobromobimane, unidentified peak 15.2 and a negligible amount of fraction 16.0 (GS-monobromobimane) were exported only by GSH-enriched cells during incubation with monobromobimane (Fig. 2b). The main exported GSH-independent (unidentified) fractions were: 26.2 (major) and 22.9 (minor) for N-[1-pyrene]maleimide; 17.1 (major) and 15.8 (minor) for monobromobimane (Fig. 2a, b). Most of the exported derivatives of N-[1-pyrene]maleimide and monobromobimane accumulated linearly over time (for 40 min of incubation) in the extracellular medium, except for GSH conjugated derivatives. The export kinetics for the GS-N-[1-pyrene]maleimide conjugate were hyperbolic; moreover, the GS-monobromobimane conjugate was onlyobserved in small amounts at the beginning of the 40 min incubation and had disappeared by the end (data not shown).

Resorption of GS-N-[1-pyrene]maleimide and GS-monobromobimane, or their intracellular transformation with extrusion of other derivative intermediates, was assumed. This does not exclude the possible transformation of GS-conjugated derivatives of N-[1-pyrene]maleimide and monobromobimane through intermediates into the corresponding ACS-conjugates, as in the mercapturic acid pathway of higher eukaryotes (Tsuchida, 1997).

Future studies will include investigation of the chemical nature of unidentified extruded GSH-dependent HPLC fractions, as well as GSH-dependent fluorescent conjugate(s) that enter the vacuole. We plan to do this using isolated mutants of H. polymorpha that lack γ-glutamyltransferase activity, the key enzyme of the mercapturic acid pathway.

Acknowledgements

This study was supported by the Committee for Scientific Research (Poland). Dr V. Ubiyvovk was a recipient of the European Fellowship Fund (Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland). We are indebted to Dr M. Soszynski, Dr M. Maidan and O. Krasovska for friendly discussion and help.

References

Brehe J, Burch, HB. Enzymatic assay of glutathione. Anal Biochem 1976:74:189-97
Crossref   Medline   

Ishikawa T, Li, Z-S, Lu, Y-P, Rea, PA. The GS-X pump in plant, yeast and animal cells: structure, function and gene expression. Bioscience Reports 1997:17:189-207
Crossref   Medline   

Li ZS, Szczypka, M, Lu, YP, Thiele, DJ, Rea, PA. The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump. J Biol Chem 1996:271:6509-17
Crossref   Medline   

Lowry OH, Rosebrough, NJ, Farr, AL, Randall, RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951:193:265-75
Medline   

Penninckx M. A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses. Enzyme Microb Technol 2000:26:737-42
Crossref   Medline   

Sahm H. Metabolism of methanol by yeast. Adv Biochem Eng 1976:6:103

Sibirny AA, Ubiyvovk, VM, Gonchar, MV, Titorenko, VI, Voronovsky, AY, Kapultsevich, YG. Reaction of direct formaldehyde oxidation to CO2are non-essential for energy supply of yeast methylotrophic growth. Arch Microbiol 1990:154:566-75

St-Pierre MV, Ruetz, S, Epstein, LF, Gros, P, Arias, IM. ATP-dependent transport of organic anions in secretory vesicles of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 1994:91:9476-9
Crossref   Medline   

Tsuchida S. . Encyclopedia of Cancer 1997:1:733-43

Ubiyvovk VM, Telegus, YV, Sibirny, AA. Isolation and characterization of glutathione-deficient mutants of the methylotrophic yeast Hansenula polymorpha. Microbiology (Moscow) 1999:68:26-31

Ubiyvovk VM, Nazarko, TY, Stasyk, OG, Sohn, MJ, Kang, HA, Sibirny, AA. GSH2, a gene encoding γ-glutamylcysteine synthetase in the methylotrophic yeast Hansenula polymorpha. FEMS Yeast Research 2002:2:327-32
Medline   

Zadzinski R, Maszewski, J, Bartosz, G. Transport of glutathione S-conjugates in the yeasts Saccharomyces cerevisiae. Cell Biol Int 1996:20:325-30
Crossref   Medline   


Received 11 February 2003/27 March 2003; accepted 13 May 2003

doi:10.1016/S1065-6995(03)00127-6


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)