Arch Microbiol (2010) 192:909–918

DOI 10.1007/s00203-010-0614-4

ORIGINAL PAPER

Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic

Tatina T. Todorova · Anna V. Kujumdzieva · Stéphane Vuilleumier

Received: 3 January 2010 / Revised: 30 June 2010 / Accepted: 2 August 2010 / Published online: 26 August 2010© Springer-Verlag 2010

Abstract The response of Saccharomyces cerevisiae toarsenic involves a large ensemble of genes, many of whichare associated with glutathione-related metabolism. Therole of the glutathione S-transferase (GST) product of theURE2 gene involved in resistance of S. cerevisiae to abroad range of heavy metals was investigated. Glutathioneperoxidase activity, previously reported for the Ure2p pro-tein, was unaVected in cell-free extracts of an ure2� mutantof S. cerevisiae. Glutathione levels in the ure2� mutantwere lowered about threefold compared to the isogenicwild-type strain but, as in the wild-type strain, increased2–2.5-fold upon addition of either arsenate (AsV) or arse-nite (AsIII). However, lack of URE2 speciWcally causedsensitivity to arsenite but not to arsenate. The protectiverole of URE2 against arsenite depended solely on the GST-encoding 3�-end portion of the gene. The nitrogen sourceused for growth was suggested to be an important determi-nant of arsenite toxicity, in keeping with non-enzymaticroles of the URE2 gene product in GATA-type regulation.

Keywords Ure2 · Glutathione S-transferase · GATA regulation · Arsenic detoxiWcation

AbbreviationsGSTs Glutathione S-transferasesNCR Nitrogen catabolite repressionGPx Glutathione peroxidaseGdh1 NADPH-dependent glutamate dehydrogenaseGln1 Glutamine synthetaseGdh2 NAD+-dependent glutamate dehydrogenase

Introduction

The protein Ure2p of Saccharomyces cerevisiae has beenstudied in three diVerent aspects: as a precursor of [URE3],the protease-resistant prion-like altered form of Ure2p(Wickner et al. 2000); as a negative regulator of GATA-dependent transcription (Cooper 2002); and as a glutathioneS-transferase (GST) (Bousset et al. 2001; Choi et al. 1998).

The Ure2 protein sequence consists of two distinctregions (see Lian et al. 2006 for a review). Its N-terminalpart, from residue 1 to residue 93, constitutes the prion-inducing domain responsible for self-propagation of[URE3] aggregates (Masison and Wickner 1995). TheC-terminal part of the URE2 gene product (residues 94–354) is similar in sequence and three-dimensional structure(Bousset et al. 2001; Coschigano and Magasanik 1991) toglutathione S-transferases (GSTs), a family of enzymes cat-alysing the conjugation of toxic electrophilic compounds toglutathione (Allocati et al. 2009; Frova 2006; McGoldricket al. 2005; Vuilleumier and Pagni 2002). Ure2p binds glu-tathione and has glutathione peroxidase (GPx) activity withinorganic and organic peroxides (Bai et al. 2004), as well asglutaredoxin activity (Zhang and Perrett 2009). However,

Communicated by Axel Brakhage.

Electronic supplementary material The online version of this article (doi:10.1007/s00203-010-0614-4) contains supplementary material, which is available to authorized users.

T. T. Todorova · A. V. KujumdzievaFaculty of Biology, Department of General and Applied Microbiology, SoWa University “St. Kliment Ohridski”, 1164 SoWa, Bulgaria

T. T. Todorova · S. Vuilleumier (&)Université de Strasbourg, UMR 7156 CNRS, 28 rue Goethe, 67083 Strasbourg Cédex, Francee-mail: vuilleumier@unistra.fr

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attempts to demonstrate activity of wild-type Ure2p withtypical GST model substrates such as 1-chloro-2,4-dinitro-benzene have been unsuccessful (Choi et al. 1998),although single residue variants of the protein wereobtained which displayed this activity (Zhang et al. 2008).Thus, an enzymatic role of physiological importanceremains to be identiWed for the Ure2p protein.

It was demonstrated that URE2 disruption mutants wereparticularly sensitive to a broad series of toxic agents (Basuet al. 2004; Rai et al. 2003; Rai and Cooper 2005; Todorovaet al. 2009). The sensitivity of S. cerevisiae to arsenic hasbeen the focus of much interest (Wysocki and Tamás 2010)given the importance of this metalloid as a contaminant ofdrinking water in many parts of the world (Aposhian et al.2004). Arsenic exists in two major oxidation states—As(III) (arsenite) and As(V) (arsenate). DetoxiWcation ofAs(III) and As(V) involves diVerent transport and transfor-mation mechanisms in S. cerevisiae.

Arsenite is imported into the cell by aquaglyceroporinssuch as Fps1p (Wysocki et al. 2001), whereas arsenate entersby way of carriers of inorganic phosphate (Bun-ya et al.1996; Yompakdee et al. 1996). Somewhat paradoxicallygiven the greater toxicity of As(III), As(V) is reduced to themore toxic form As(III) after entering the cell (Rosen 2002).

A mammalian GST enzyme capable of arsenate reduc-tion in vitro, GSTO1, has been identiWed (Aposhian andAposhian 2006; Zakharyan and Aposhian 1999), but lacksclose homologues in microorganisms. In S. cerevisiae,reduction of arsenate to arsenite occurs through the actionof arsenate reductase Acr2p, which requires glutathione asa donor of reduction equivalents (Mukhopadhyay et al.2000). Following this reduction step, As(III) may be conju-gated to glutathione and pumped to the vacuole by Ycf1p(Ghosh et al. 1999), or extruded by the membrane proteinAcr3p (Ghosh et al. 1999; Wysocki et al. 1997) and, asrecently suggested, by Fps1p, which may therefore repre-sent a bidirectional arsenite transporter (Maciaszczyk-Dziubinska et al. 2010). Taken together, this suggests thatglutathione-dependent reactions, including those poten-tially catalysed by the Ure2p glutathione S-transferase, arecrucial in arsenic detoxiWcation.

The response of S. cerevisiae to arsenic exposure is alsocomplex, because of the involvement of several proteins withmultiple regulatory roles (e.g. Haugen et al. 2004; Hosineret al. 2009; Ilina et al. 2008; Menezes et al. 2008; Thorsenet al. 2006, 2007, 2009; Wysocki and Tamás 2010). In thiscontext, it is important to consider the implication of Ure2pin nitrogen catabolite repression (NCR) (see e.g. Cooper2002; Georis et al. 2009; Magasanik and Kaiser 2002; Wonget al. 2008). When S. cerevisiae is cultivated on preferrednitrogen sources such as ammonia or glutamine, the expres-sion of genes required for utilisation of alternative nitrogensources (such as glutamate or proline) is strongly repressed.

In the absence of a preferred nitrogen source, in contrast, thelatter genes are expressed at high levels to ensure the assimi-lation of other forms of nitrogen. When preferred nitrogensources are present in the growth medium, the URE2 geneproduct of S. cerevisiae was shown to form cytoplasm-localised complexes with zinc Wnger proteins Gln3p andGat1p (Crespo et al. 2001; Scherens et al. 2006; Springaeland Penninckx 2003). These proteins belong to the GATAfamily of transcription regulators, which recognise speciWcGATAA containing core sequences in the promoter regionsof genes involved in the assimilation of non-preferred nitro-gen sources (Cooper 2002), but also of genes associated withvarious aspects of the cell stress response in S. cerevisiae(Crespo et al. 2001; Tate and Cooper 2007), including homo-eostasis of glutathione (Springael and Penninckx 2003); alsosee Online Resource). The negative GATA-dependent regu-lation of genes for assimilation of non-preferred nitrogensources is lost in the ure2� mutant, in which nitrogen cata-bolic genes are expressed at high levels irrespectively of thenature of nitrogen source in the medium (Coschigano andMagasanik 1991).

The relation of this nitrogen-related characteristic of theure2� mutant to the sensitivity of ure2� mutant to toxiccompounds such as arsenic is not well understood. In thiswork, we address in more detail the role of the URE2 gluta-thione S-transferase for arsenic detoxiWcation in S. cerevi-siae, also taking this nitrogen dependence of arsenicsensitivity into consideration.

Materials and methods

Strains and media

S. cerevisiae strains BY4741 (Mat a; his3�1; leu2�0;met15�0; ura3�0), and its isogenic disruption mutanture2� (BY4741 YNL229c::kanMX4), were both obtainedfrom EUROSCARF (http://web.uni-frankfurt.de/fb15/mikro/euroscarf).

Strains were grown on rich YPD medium (1% yeastextract, 2% peptone, 2% glucose (Q-BioGene)) and onminimal medium (0.17% YNB without amino acids orammonia (Q-BioGene)). Minimal YNB medium was sup-plemented with 0.1% glutamate or 0.5% (NH4)2SO4 as thenitrogen source, and 2% glucose or 2% galactose as the car-bon source as indicated. Auxotrophic supplements wereadded at standard concentrations (Ura, His, Met at20 mg L¡1 and Leu at 60 mg L¡1), as required.

Arsenic sensitivity assays

Yeast strains were grown overnight at 30°C/140 rpm inYPD or YNB liquid medium. Cells were pelleted, washed

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twice with fresh medium and resuspended to OD600 » 1.Samples (5 �L) of successive tenfold serial dilutions withOD600 » 1; 0.1; 0.01; 0.001; 0.0001 were spotted onto YPDor YNB plates containing NaAsO2 (As(III)) or NaH-AsO4·7H2O (As(V)) (Sigma) in the 0.1–10 mM concentra-tion range, and plates were incubated at 30°C for 3–5 days.

Growth rate determination

Cultures of S. cerevisiae were grown overnight in YPD liq-uid medium, collected and washed twice with freshmedium. Two hundred microlitres of YPD with NaAsO2

(As(III)) or NaHAsO4.7H2O (As(V)) at the indicated con-centrations were inoculated with overnight cultures, result-ing in an initial OD600 of 0.05. Cultures were thenincubated in 96 MicroWell™ Xat-bottom plates (Nunclon)for 24 h at 25°C/160 rpm, and absorbance was measured at600 nm at regular intervals using a Bio-Tek KC4 platereader. Relative speciWc growth rates were determined fromthe early exponential phase of growth between 6 and 14 hafter inoculation.

Complementation of ure2� mutant phenotype

Genes URE2 and URE2�N (a truncated URE2 gene with a279-bp 5�-end deletion) were cloned from plasmidspET23b-URE2 and pET23b-URE2(�N) (Choi et al. 1998)by standard procedures. BrieXy, after digestion of theseplasmids with XbaI and XhoI, the resulting 1.0- and 0.7-kbfragments were ligated with XbaI/XhoI digested yeast/E. coli shuttle vector p413 (Funk et al. 2002). The resultingplasmids pME8530 (p413-URE2) and pME8531 (p413-URE2�N), and the p413 vector as a control, were trans-formed into S. cerevisiae by electroporation (BioRadMicropulser) using the standard protocol provided by themanufacturer. Transformants were selected on minimalmedium without histidine. Transformant strains were culti-vated in minimal medium with glucose as the carbon sourceto an OD600 » 1. Cultures were transferred to inductionmedium containing galactose at a concentration of 2% andspotted on YNB plates containing 2% galactose as the car-bon source and As(III) at various concentrations after 24 hof induction (OD600 » 1).

Biochemical assays

Wild-type BY4741 and ure2� strains were cultivated inYPD medium to an OD600 of approximately 0.8 and thentreated for 30 min with 1 mM NaAsO2 or NaHAsO4.7H2O.Cultures were collected, washed, resuspended in anequal volume of 0.05 M potassium phosphate buVer pH7.8 and disrupted with glass beads (0.5 mm, Sigma) onice (4 £ 10 min) using a vibration homogeniser VHG1

(Biotech International Melsungen). The obtained suspen-sions were centrifuged for 30 min at 14,000 rpm at 4°C,and the resulting supernatants were used as cell-freeextracts. Protein concentration was determined by themethod of Lowry with bovine serum albumin as a standard(Lowry et al. 1951).

Activities in cell-free extracts were determined as theaverage of 3 independent experiments. Glutathione peroxi-dase (GPx) activity was measured as described (Veal et al.2002), with H2O2 as the substrate. Initial rates were deter-mined from the maximal slope of product formation, usingan extinction coeYcient for NADPH of 6,220 M¡1 cm¡1.Activities of NADPH-dependent glutamate dehydrogenase(Gdh1) and NAD+-dependent glutamate dehydrogenase(Gdh2) were assayed as described (Doherty et al. 1970),with the following modiWcations. For Gdh1, 50 �L of ten-fold diluted cell-free extract were added to 450 �L reactionmixture in 50 mM potassium phosphate buVer pH 7.75 con-taining 50 mM NH4Cl, 10 mM �-ketoglutarate and 2 mMNADPH. The assay mixture for Gdh2 was 50 mM potassiumphosphate buVer pH 8, containing 0.1 mM �-mercaptoethanol,0.5 mM EDTA, 50 mM NH4Cl, 10 mM �-ketoglutarate and2 mM NADH. The oxidation of NADPH or NADH wasmonitored by recording the absorbance at 340 nm, asdescribed above.

Glutamine synthetase (Gln1) activity was determined asfollows: 50 �L of cell-free extract were incubated for10 min at 45°C with 50 mM imidazole–HCl buVer pH 7.15containing 100 mM glutamate, 50 mM NH4Cl, 50 mMMgCl2 and 1 mM ATP in a Wnal volume of 0.5 mL. Thereaction was stopped with 1 mL 10% TCA. Inorganic phos-phate produced from ATP degradation was determined inthe supernatant after centrifugation as described (Tausskyet al. 1953).

Cellular glutathione content was determined as the aver-age of three independent measurements by the method ofEllman (Zhang 2000). BrieXy, 1 mL 5% TCA was added to2 mL cell-free extract to precipitate the proteins, and theresulting suspension was centrifuged for 5 min at5,000 rpm. From this, 50 �L supernatant was added to400 �L 0.1 M potassium phosphate buVer pH 7.5. Derivati-sation was initiated by addition of 50 �L 10 mM5,5�-dithio-bis(2-nitro)benzoic acid. Concentration ofreduced glutathione was determined spectrophotometri-cally at 412 nm using an extinction coeYcient of13,600 M¡1 cm¡1. Total glutathione was determined aftertreatment of protein-free samples with glutathione reduc-tase and NADPH, in 1 mL 0.1 M potassium phosphatebuVer pH 7.5 containing 0.5 U enzyme (Sigma) and0.3 mM NADPH at 25°C for 30 min. The content of oxi-dised glutathione in the samples was obtained after subtrac-tion of the content in reduced glutathione determinedbefore reduction.

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RNA isolation and RT–PCR

Total RNA was isolated using TRIzol Reagent (Invitrogen).BrieXy, strains were grown in 30 ml cultures toOD600 » 0.8 at 30°C/140 rpm in YPD medium, and 1 mMarsenite or the same volume of YPD were added followedby further incubation for 30 min. Cultures were then har-vested and homogenised with 1 ml TRIzol and 0.2 mLchloroform, vortexed with glass beads (0.5 mm, Sigma),and RNA precipitated with isopropanol. After 10 min ofincubation at room temperature, RNA samples were centri-fuged, washed with ethanol, air-dried and dissolved inRNase-free water (50 �L). Isolated total RNA was DNAse1treated (RQ1, Promega; one unit of enzyme per 1 �g ofRNA) and conserved in aliquots at ¡80°C until use. RNApurity was determined spectrophotometrically, and prepara-tions with A260/A280 > 1.9 were used.

RT–PCRs were performed in duplicate on RNA isolatedfrom three independent cultures, as follows. TemplatemRNA (10 ng) was ampliWed in 10 �L Wnal volume usingthe OneStep RT–PCR Kit (Qiagen), with gene-speciWcprimers at a Wnal concentration of 0.6 �M. Primers of simi-lar length and annealing temperature were designed usingPrimer3 (http://frodo.wi.mit.edu), and ampliWed a 191-bpfragment of the genes of interest. Primer sequences were asfollows: FPS1 gene, 5�-AGGAATTTTTAGCCGAGTTT-3�

and 5�-GGATGAAACCAAGGATGTTA-3�; 18S rRNAgene, 5�-TTAACGAGGAACAATTGGAG-3� and 5�-CACAAGGACTCAAGGTTAGC-3�. Reactions were initiatedat 50°C for 30 min to synthesise cDNA, followed by inacti-vation of reverse transcriptase at 95°C for 15 min. AmpliW-cation of cDNA by PCR was then performed by 30 cyclesof 30 s at 95°C; 1 min at 50°C and 1 min at 70°C. Theseconditions were chosen so that reactions remained in theexponential phase of DNA ampliWcation, i.e. none of thetarget PCR products had reached a plateau before the end ofthe PCR. AmpliWed products were visualised on 1% (w/v)agarose gels and analysed by Quantity One Software (Bio-Rad Gel Documentation System). Signal intensities of theFPS1 product were normalised relative to the 18S RNAproduct obtained from the same RNA sample. ControlPCRs omitting the reverse transcriptase step demonstratedthe absence of contaminating DNA in the initial RNA prep-arations.

Measurements of �-galactosidase activity

Expression of the FPS1 gene was also followed by measur-ing �-galactosidase activity in wild-type and ure2� mutantstrains transformed with a derivative of plasmid YEp367Sc(Dang et al. 1994) carrying a FPS1-lacZ translationalreporter fusion (Tadi et al. 1999). This plasmid contains a1.5-kb insert with 254 bp of sequence upstream of the FPS1

ATG start codon, as determined by sequencing with prim-ers olPUC18 and olZ119 (Tadi et al. 1999). Transformantswere grown in selective minimal medium to OD600 » 0.8,induced for 30 min with 1 mM NaAsO2, cell-free extractswere obtained as described above, and �-galactosidaseactivity was measured as described (Reynolds et al. 2001).

Statistical analysis

The Sigma Stat 3.1 statistical package was used for dataanalysis. Growth and biochemical parameters were com-pared between the wild-type and ure2� mutant strainsusing Student’s t-test. Where the normality test failed, theMann–Whitney rank-sum test was used. Values of P < 0.05were considered statistically signiWcant.

Results

URE2-dependent sensitivity of S. cerevisiae to arsenic

The previously noted role of URE2 in arsenic resistance ofS. cerevisiae (Rai and Cooper 2005) was reinvestigated inmore detail on the wild-type strain BY4741 and its isogenicure2� mutant. Viability of the ure2� mutant was indistin-guishable from wild-type on solid YPD medium. However,the viability of the ure2� mutant was strongly aVected byeven low concentrations of arsenite (0.2 mM), while thewild type was able to grow in the presence of up to 1.3 mMAs(III) (data not shown, and Fig. 3, controls). In liquidmedium supplemented with As(III), the speciWc growth rateof the mutant was more strongly aVected than that of thewild-type strain (Fig. 1a, P < 0.05 for all As(III) concentra-tions tested).

In contrast, disruption of the URE2 gene did not aVectviability of S. cerevisiae in the presence of arsenate (seeFig. 3, controls), and arsenate aVected the growth rate inliquid culture of the ure2� mutant to a similar extent as thatof wild type (Fig. 1b). Indeed, the ure2� mutant grew at avery similar rate as wild type when cultivated in presenceof As(V) (Fig. 1b).

The GST-encoding domain of URE2 confers arsenite resistance to Saccharomyces cerevisiae

The ure2� mutant strain was transformed with centromericlow copy plasmids bearing all or part of the gene URE2under the control of the weak galactose-inducible regulatorGALS. Expression of both the full-length and truncatedforms of Ure2p from plasmids pME8530 and pME8531was conWrmed by Western blot analysis using anti-Ure2pantiserum following addition of galactose (data not shown).Plasmid pME8530 expressing the URE2 gene in its entirety

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fully restored growth of the ure2� mutant in the presenceof As(III) (Fig. 2), conWrming that arsenite sensitivity ofure2� is the consequence of URE2 gene disruption in strainBY4741. Importantly, transformation of mutant ure2� withplasmid pME8531 containing a truncated version of theURE2 gene, encoding the C-terminal GST domains butlacking the N-terminal prion propagation region (Choi et al.1998), was suYcient to abolish arsenite sensitivity in theure2� mutant (Fig. 2). This demonstrates that the protec-tive role of URE2 against arsenite depended solely on theGST-encoding 3�-end portion of the gene.

Arsenic dependence of glutathione peroxidase activity

PuriWed recombinant Ure2p was previously reported topossess GPx activity with both H2O2 and organic peroxidesin vitro (Bai et al. 2004). Arsenic exposure resulted in a

minor overall increase of GPx activity in cell-free extractsin both wild-type and ure2� mutant (by approx. 15% forAs(V) and 20% for As(III)). However, extracts of mutanture2� showed no diVerence in GPx activity compared towild-type extracts.

Arsenic dependence of glutathione content

Decreased levels of intracellular glutathione in a ure2�mutant were previously reported (Perrone et al. 2005;Todorova et al. 2009). Here, levels of reduced and oxidisedforms of glutathione were measured in both wild-type andure2� mutant strains as a function of arsenic concentration(Table 1). As expected, the ure2� mutant showed signiW-cantly decreased glutathione levels, but exposure to eitherAs(III) or As(V) arsenic forms caused a similar 2–2.5-foldincrease in levels of total glutathione in both wild-type and

Fig. 1 EVect of arsenic on growth rate of liquid cultures of S. cerevi-siae. Relative maximal growth rates of YPD cultures, expressed as thepercentage of growth rates of YPD without addition arsenic, of wild-type (Wlled square) and ure2� mutant (square) as a function of theconcentration of arsenite (a) or arsenate (b) (measured rates of

0.173 § 0.014 h¡1 for wild type and 0.110 § 0.017 h¡1 for the ure2�mutant in the absence of arsenic under the chosen experimental condi-tions, see “Materials and methods”). Vertical bars represent the stan-dard deviation from three independent experiments

A B

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Fig. 2 Complementation of arsenite sensitivity of the ure2� mutantby diVerent URE2 constructs. Transformants of the ure2� mutant con-taining plasmids p413 (vector), pME8530 (p413-URE2) and pME8531(p413-URE2�N) were cultivated in histidine-free minimal medium

containing 2% glucose to OD600 » 1, and then induced with 2% galact-ose. After 24 h incubation, tenfold serial dilutions were spotted onminimal YNB plates containing galactose with or without 0.65 mMsodium arsenite, and plates were incubated at 30°C for 3–5 days

control

+ p413

BY4741

0.65 mM As(III)

ure2Δ

+ pME8530

+ pME8531

+ p413

+ pME8530

+ pME8531

Plasmid

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mutant strains, presumably as the result of oxidative stressinduced by arsenic (Haugen et al. 2004; Kitchin andAhmad 2003). The ratio of oxidised to reduced glutathionewas higher in the mutant strain and increased further uponexposure to either form of arsenic (Table 1).

URE2-mediated, nitrogen source–dependent arsenic sensitivity in S. cerevisiae

Repression of genes for assimilation of poor nitrogensources was shown previously to be alleviated in a ure2�mutant, irrespectively of the nitrogen source in the medium(Cooper 2002). In particular, the activities of NADPH-dependent glutamate dehydrogenase (Gdh1), NAD+-dependent glutamate dehydrogenase (Gdh2p) and glutaminesynthetase (Gln1) were strongly increased in a ure2�mutant (Coschigano and Magasanik 1991; ter Schure et al.2000), and this was conWrmed here (Table 1). In addition,our results showed that in itself, exposure to arsenic did notalter activities of Gdh1p, Gdh2p and Gln1p in both wild-typeand ure2� mutant backgrounds (Table 1).

A direct role of Ure2p in GATA-type nitrogen catabolicrepression, leading to the observed As(III) sensitive pheno-type in the ure2� mutants, was suggested from viabilityexperiments performed on solid minimal medium supple-mented either with ammonium (a preferred nitrogensource) or glutamate (a poor nitrogen source) as the solesource of nitrogen. Indeed, and unlike what was observed

with the ure2� mutant strain, exposure to 0.65 mM arsenitehad no eVect on wild-type S. cerevisiae growing withammonium as a preferred nitrogen source (Fig. 3), whilethe wild-type strain grown with glutamate as a non-preferred nitrogen source became sensitive to arsenite(Fig. 3). In the presence of As(V), in contrast, and asexpected from the known growth behaviour of wild-typeand ure2� mutant strains (Fig. 1), no signiWcant diVerenceswere observed between the wild-type strain and the ure2�mutant irrespectively of the source of nitrogen (Fig. 3).

URE2-dependent regulation of gene expression and arsenite sensitivity: the case of FPS1

To further evaluate the possibility of URE2-mediatedGATA-type regulatory eVects on arsenite sensitivity, theS. cerevisiae genome sequence was checked using the Pat-tern Matching tool of the Saccharomyces Genome DatabaseGATA-type regulator for potential binding sites. The analy-sis was performed for genes known to be subject to GATA-type regulation as well as for genes involved in arsenicmetabolism (Online resource). Six GATAA/TTATC motifswere identiWed as potential binding sites for GATA-typeregulators in the 1-kb upstream of FPS1 (Fig. 4a). Thisnumber is typical of that found for other genes known to beregulated by GATA factors (Springael and Penninckx2003; ter Schure et al. 2000), but larger than other genesknown to be involved in arsenic metabolism, such as the

Table 1 Activities in cell-free extracts (mU mg¡1 protein) and intracellular glutathione content (nmoles mg¡1 protein) for S. cerevisiae grownwith or without 1 mM arsenic

Units are deWned as �mol substrate transformed per minutea No signiWcant diVerence (P > 0.05) between enzymatic activities in cell-free extracts of the same strain under diVerent conditions and betweenthe enzymatic activities in cell-free extracts of the wild-type and ure2� mutant strains under the same conditionsb No signiWcant diVerence (P > 0.05) between the enzymatic activities in cell-free extracts of the same strain under diVerent conditions, but sig-niWcant diVerences (P < 0.05) between the enzymatic activities in cell-free extracts of the wild-type and the ure2� mutant strains under the sameconditionsc SigniWcant diVerences (P < 0.05) for the same strain under diVerent conditions and between the wild-type and ure2� mutant strains under thesame conditions

Wild-type BY4741 ure2� mutant

Control As(III) As(V) Control As(III) As(V)

Enzyme activities

Glutathione peroxidasea 24 § 3 29 § 2 28 § 1 24 § 1 28 § 3 27 § 4

Glutamate dehydrogenase (NADP+-dependent)b 1,261 § 70 1,402 § 98 1,433 § 227 2,246 § 407 2,494 § 211 2,191 § 133

Glutamate dehydrogenase (NAD+-dependent)b 111 § 20 92 § 9 105 § 13 4,012 § 97 4,723 § 184 4,105 § 246

Glutamine synthetasea 352 § 68 362 § 95 412 § 27 378 § 81 454 § 135 434 § 71

Glutathione content

Totalc 163 § 38 429 § 25 414 § 14 57 § 12 143 § 20 116 § 15

Reducedc 144 § 30 334 § 7 278 § 9 34 § 7 41 § 10 27 § 9

Oxidisedc 19 § 6 95 § 19 136 § 22 23 § 6 102 § 26 90 § 21

Oxidised/reduced ratio 0.1 0.3 0.5 0.7 2.5 3.3

Fold increase 2.6 2.5 2.5 2.0

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glutathione-dependent arsenate reductase ACR2, the arse-nite transporter ACR3, and the vacuolar glutathione S-con-jugate transporter YCF1 (Online resource). The FPS1 geneencodes an aquaglyceroporin involved in glycerol uptakeand in arsenite inXux (Liu et al. 2004; Wysocki et al. 2001),but was recently shown to have a role in arsenite eZux aswell (Maciaszczyk-Dziubinska et al. 2010). Additionally,two GATAA sequences, in the form of a tandem AGATAAGATAAAG site, and two other TTGGT and TTGTTsequences, previously proposed to deWne binding sites forauxiliary regulators (Magasanik and Kaiser 2002), werealso noted in the FPS1 upstream region. Transcripts ofFPS1 were increased by 50% in the ure2� mutant com-pared to wild type (Fig. 4b). Nevertheless, both strainsshowed a 20% decrease in transcripts of the FPS1 geneupon As(III) addition, as Wrst shown by Wysocki et al.(2001), and later conWrmed by Haugen et al. (2004) using amicroarray approach. The similar decrease in transcriptionin both wild-type and mutant backgrounds (Fig. 4) suggeststhat arsenite aVects transcription of the FPS1 gene in waysthat are at least partly independent of URE2. However, thiseVect of arsenite on transcription of the FPS1 gene is minorcompared to the eVect of the URE2 mutation itself (Fig. 4).

These Wndings were conWrmed in experiments on theexpression, in wild-type S. cerevisiae and its isogenicure2� mutant, of a plasmidic FPS1-lacZ reporter fusion(Tadi et al. 1999) containing the GATAA motif upstreamof the FPS1 gene closest to the ATG start codon (Fig. 4a).A higher level of �-galactosidase activity was observed inthe mutant background relative to wild type, both in thepresence and in absence of arsenite (Fig. 4c), while a

similar, approximately twofold decrease in �-galactosidaseactivity was observed in both wild-type and mutant back-grounds after treatment with As(III).

Discussion

Lack of URE2 has been known for some time to decreasetolerance of S. cerevisiae towards arsenic (Rai and Cooper2005). Here, we show that this eVect arises speciWcally fromthe arsenite form of the metalloid (Fig. 3), and that the gluta-thione S-transferase encoding domain of the URE2 gene issuYcient for full complementation of the arsenite sensitivityphenotype of the ure2� mutant. This demonstrated thatURE2 is indeed involved in arsenic resistance by way of aspeciWc glutathione S-transferase associated function(Fig. 2). Two possible explanations for the observed sensi-tivity of the ure2� mutant towards As(III) were investi-gated: Enzymatic, glutathione-dependent participation ofUre2p in the detoxiWcation of arsenite, and altered expres-sion of speciWc GATA-regulated genes mediated by URE2.

Our results do not support a direct enzymatic role ofUre2p in arsenic detoxiWcation. Although glutathione per-oxidase activity was previously reported for the puriWedrecombinant Ure2p protein (Bai et al. 2004), overall levelsof glutathione peroxidase activity were similar in wild-typeand ure2� mutant, and remained unaltered after exposureto As(III) or As(V) (Table 1). However, it is possible thatthe glutathione peroxidase activity of Ure2p makes only aminor contribution to the total glutathione peroxidase activ-ity of the cell.

Fig. 3 Sensitivity of ure2� to arsenic in medium containing diVerent forms of nitrogen. Wild-type BY4741 and mutant ure2� strains were cultivated overnight in either liquid YPD medium, and in YNB minimal medium with glucose as a car-bon source and either 0.5% ammonium or 0.1% glutamate as a nitrogen source. Serial decimal dilutions of each strain were spotted on solid agar minimal medium containing sodium arse-nite (As(III)) or sodium arsenate (As(V)) as indicated, and plates were incubated at 30°C for 3–5 days

BY4741 ure2Δ

YPD

6 mM As(V)

control

0.65 mM As(III)

2 mM As(V)

NH4+

control

0.65 mM As(III)

6 mM As(V)

glutamate

6 mM As(V)

control

0.65 mM As(III)

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916 Arch Microbiol (2010) 192:909–918

Total glutathione content was strongly decreased in theure2� mutant (Table 1). It had been noted previously thatexpression of �-glutamyl peptidase, the main enzyme indegradation of glutathione, was under GATA control(Springael and Penninckx 2003; Online resource), andmore highly expressed in the absence of the URE2 gene.However, it was also demonstrated independently by otherresearchers that exogenous addition of glutathione to themedium was insuYcient to alleviate sensitivity of an ure2�mutant to arsenic (Rai and Cooper 2005), so the implica-tions of the observed levels of glutathione in the ure2�mutant for sensitivity to arsenic are not yet fully under-stood. Also, several other experiments reported by Rai andCooper (2005) suggest that glutathione, or the lack of it, isnot a signiWcant factor in the sensitivity of the ure2�mutant to arsenite. In particular, the ure2� strain cultivatedin medium with glutathione as the sole nitrogen sourceremained sensitive to As(III). In our view, insuYcient lev-els of reduced glutathione in the ure2� mutant would beassociated with ineYcient reduction of arsenate within thecell, and thus lead to enhanced sensitivity to arsenate, sincedetoxiWcation of arsenate requires reduced glutathione byway of the glutathione-dependent reduction to arsenite byAcr2p (Mukhopadhyay et al. 2000; Rosen 2002; Zakharyanand Aposhian 1999)Accordingly, deletion of the glutathi-

one-dependent arsenate reductase Acr3p, and to a lesserdegree of the vacuolar transporter of glutathione conjugatesof As(III) Ycfp, was shown to confer pronounced arsenatesensitivity to the corresponding mutants (Wysocki et al.2001). However, sensitivity to arsenate was not observedhere (Figs. 1, 3), suggesting that the low levels of glutathi-one measured here for the ure2� mutant are suYcient forarsenic metabolism.

Therefore, basing on the available body of evidencefrom this and previous studies, sensitivity of the ure2�mutant to arsenic may originate at least in part in a yetuncharacterised aspect of arsenic detoxiWcation in S. cerevi-siae. Our data on increased expression of FPS1 in theure2� mutant (Fig. 4) suggest one possible element ofexplanation for the enhanced speciWc sensitivity of anure2� mutant to arsenite relative to wild type, i.e. thatFPS1, a gene taken here as an example for potential Ure2p-dependent GATAA-type regulation at the level of tran-scription in the context of arsenic metabolism, and codingfor an aquaglyceroporin speciWcally involved in As(III)transport (Wysocki et al. 2001; Maciaszczyk-Dziubinskaet al. 2010), is a target for GATA-type regulation by Ure2p.On this particular point as well as on others, however, therecent new data of Maciaszczyk-Dziubinska et al. (2010)clearly suggest that the physiological role of FPS1 is more

Fig. 4 URE2-dependent expression of the FPS1 gene (a) Schematicrepresentation of the 1-kb FPS1 upstream region with GATAA/TTATC motifs (open boxes) and TTGGT/TTGTT auxiliary motifs(shaded boxes). Numbers indicate the nucleotide position relative tothe ATG start codon of the FPS1 gene. The initial nucleotide of thegene upstream region in the used FPS1-lacZ fusion construct (Tadiet al. 1999) is highlighted by an asterisk. Levels of FPS1 transcript in

wild-type BY4741 (black bars) and ure2� mutant (white bars) wereestimated by (b) RT–PCR (relative to 18S RNA; see “Materials andmethods”) and (c) �-galactosidase activity of a FPS1-lacZ fusion (Tadiet al. 1999; expressed in U/mg protein). All diVerences between wild-type and ure2� mutant under the same conditions, and between thelevels of FPS1 transcript or �-galactosidase in the presence or absenceof 1 mM arsenite for the same strain, were signiWcant (P < 0.05)

AGATAAGATAAAG

-603

TTATCGATAA GATAATTATC

-706 -561 -159-967A

ATG

AGATAAGATAAAG TTATCGATAA GATAATTATC

*

-713

TTGTT

-676

TTGGT

CB

12

14

35

40

8

10

pt le

vels

(%

)

20

25

30

sida

se (

U/m

g)

2

4

6

tran

scrip

5

10

15

β-ga

lact

os0

no As(III) 1 mM As(III)

0

no As(III) 1 mM As(III)

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Arch Microbiol (2010) 192:909–918 917

complex than hitherto envisaged and needs to be investi-gated anew.

In conclusion, our Wndings hint at the importance, in theresponse of S. cerevisiae to arsenic, of regulatory rolesrelated to nitrogen catabolite repression of the URE2 geneproduct, a GST-like protein of still uncertain enzymaticfunction in the context of arsenic detoxiWcation. Regarding apossible physiological function of Ure2p as an enzyme, thepossibility remains that Ure2p protects cells against heavymetals by way of a glutathione transferase activity, whichyet has to be discovered. The involvement of this glutathi-one S-transferase-like protein in the resistance to arsenicexempliWes the complex and intriguing interrelationshipsthat exist between the enzymology of central metabolic pro-cesses, their regulation as a function of prevailing environ-mental conditions, and the detoxiWcation of pollutants.

Acknowledgments We are grateful to Ivan Tarassov for providingthe p413 vector, to Ales Vancura for pET23b-URE2 and pET23b-URE2(�N) plasmids, and to Emmanuelle Boy-Marcotte for theFPS1-lacZ fusion plasmid. Work in S.V.’s laboratory is supported byREALISE, the Alsace Research Network in Environmental Sciences.The present study was supported by CNRS ATIP to S.V., by theNational Science Fund of Bulgarian Ministry of Education and Science(Project No. <-BE-201/06) and by a visiting scientist grant of the Euro-pean Doctoral College of Strasbourg to A.K. T.T. was the recipient ofan Agence Universitaire de la Francophonie PhD grant and a memberof the European Doctoral College of Strasbourg.

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