YeastYeast 2010; 27: 817–835.Published online 28 June 2010 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/yea.1780

Research Article

Genome-wide analysis of caesium and strontiumaccumulation in Saccharomyces cerevisiae

Sabine Heuck,1 Udo C. Gerstmann1, Bernhard Michalke2 and Ulrike Kanter1*1Helmholtz Zentrum Munchen, Institut fur Strahlenschutz, Neuherberg, Germany2Helmholtz Zentrum Munchen, Institut fur okologische Chemie, Neuherberg, Germany

*Correspondence to:Ulrike Kanter, HelmholtzZentrum Munchen, Institut furStrahlenschutz, IngolstadterLandstrasse 1, 85764Neuherberg, Germany.E-mail: ulrike.kanter@helmholtz-muenchen.de

Received: 4 February 2010Accepted: 29 March 2010

Abstract137Cs and 90Sr contribute to significant and long-lasting contamination of theenvironment with radionuclides. Due to their relatively high biological availability,they are transferred rapidly into biotic systems and may enter the food chain. In thisstudy, we analysed 4862 haploid yeast knockout strains of Saccharomyces cerevisiaeto identify genes involved in caesium (Cs+) and/or strontium (Sr2+) accumulation.According to this analysis, 212 mutant strains were associated with reproduciblealtered Cs+ and/or Sr2+ accumulation. These mutants were deficient for a wide rangeof cellular processes. Among those, the vacuolar function and biogenesis turned out tobe crucial for both Cs+ and Sr2+ accumulation. Disruption of the vacuole diminishedCs+ accumulation, whereas Sr2+ enrichment was enhanced. Further analysis with asubset of the identified candidates were undertaken comparing the accumulation ofCs+ and Sr2+ with their essential counterparts potassium (K+) and calcium (Ca2+).Sr2+ and Ca2+ accumulation was highly correlated in yeast excluding the possibilityof a differential regulation or uptake mechanisms. In direct contrast, the respectiveresults suggest that Cs+ uptake is at least partially dependent on mechanisms distinctfrom K+ uptake. Single candidates (e.g. KHA1) are presented which might bespecifically responsible for Cs+ homeostasis. Copyright 2010 John Wiley & Sons,Ltd.

Keywords: caesium; strontium; ion homeostasis; comparison of essential and non-essential ions

Introduction

The radionuclides 137Cs and 90Sr are produced bynuclear fission with high yields. Released in largeamounts by nuclear weapon tests and accidents, anddue to their long half-life (30.17 and 28.64 years,respectively) they are of high radiological concern.Due to their transfer from soil to the edible parts ofplants, the use of highly contaminated landscapesfor nutrition production is limited in certain areas,e.g. in the vicinity of the Chernobyl nuclear powerplant (White and Broadley, 2000). Among others,breeding of crops with low radionuclide uptakeis one option for possible future use of theseareas. However, breeding or genetic engineering

of such plants requires a profound knowledge ofthe genetic and mechanistic principles underlyingcaesium (Cs+) and strontium (Sr2+) uptake andaccumulation by plants. Thus, it is crucial to knowthe central pathways involved in the regulation ofCs+ and Sr2+ homeostasis.

Due to their chemical similarity, the biologi-cal behaviours of caesium and strontium resem-ble those of potassium and calcium, respectively.The uptake of Cs+ is well characterized in plants,whereas only few data are available for the uptakeof Sr2+. Cs+ uptake by plants is supposed to befacilitated via K+ transport pathways. Classicaluptake studies proposed that Cs+ enters the cellvia K+ transport pathways, since the addition of

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K+ to the growth medium or soil clearly affectsCs+ uptake by the plants (Epstein and Hagen, 1952;Menzel and Heald, 1955). It was therefore assumedthat K+ and Cs+ compete for the same transporters.More recent publications favour the idea that anup- or downregulation of different K+ transportproteins with variable Cs+ selectivity with respectto the external K+ concentration expose the majoreffect of changed Cs+ uptake, rather than sim-ple competition (Wang, et al., 1998). However, itis not definitely known which of the plant K+uptake pathways provide the explicit route for Cs+and whether there are additional factors that areunknown to date. Theoretical models and pharma-cological studies implied the main influx of Cs+via voltage-insensitive cation channels (VICs) ofthe low-affinity transport system (Hampton et al.,2005; White and Broadley, 2000). Furthermore, itwas found that the high-affinity K+ uptake systemof the KUP/HAK transporter family is involved inCs+ uptake at least under K+ depleted conditions(Qi et al., 2008; White and Broadley, 2000).

In contrast to Cs+, only little information isavailable concerning the uptake of Sr2+ into plants.Early trials with a variety of plant species impli-cated a close relationship of Sr2+ and Ca2+.This was elicited by the fact that high Ca2+-accumulating species also tends to retain higheramounts of Sr2+ (Bollard and Butler, 1966). Recentdecades have yielded in much progress concerningthe identification of Ca2+ transport mechanisms.Within roots, the uptake of Ca2+ occurs mainlyvia Ca2+ channels, which exhibit permeability to avariety of ions, also including Sr2+ (White et al.,2002).

However, the plant system is complex and theidentification of single factors concerning Cs+ andSr2+ accumulation might often be hampered by thepresence of gene families with redundant functions(White and Broadley, 2000). Therefore, an investi-gation of the principal cellular mechanisms under-lying Cs+ and Sr2+ accumulation is more suitablein the model system Saccharomyces cerevisiae. Thebudding yeast S. cerevisiae represents an acceptedcellular model for ion homeostasis in plants, due tothe conservation of several fundamental pathwaysand proteins involved (Rodriguez-Navarro, 2000).

In budding yeast, the main mechanism concern-ing K+ and Ca2+ homeostasis are well charac-terized and also some data concerning the Cs+and Sr2+ uptake are available (Avery, 1995; Avery

et al., 1999). Like plants, yeast cells possess ahigh- and a low-affinity K+ transport system(RodrigueZ -Navarro, 2000). The high-affinity K+transport is facilitated by the extensively stud-ied Trk1p/Trk2p transporters (Liang et al., 1998;Mulet et al., 2004), whereas the low-affinity uptakeoccurs via transporters and channels that arenot thoroughly characterized at the molecularlevel. However, electrophysiological experimentsrevealed the presence of a non-selective cationchannel, termed NSC1, which facilitates the entryof a variety of ions, among them Cs+ (Bihler et al.,1998, 2002). Furthermore, there are hypothesesthat a rather non-specific and low-affinity uptakeof monovalent cations occurs via transporters forsugars and other organic compounds (Liang et al.,1998; Madrid et al., 1998; Wright et al., 1997).Cs+ uptake is supposed to occur via both pathways,while the main influx is facilitated by the relativelyunspecific low-affinity transport mechanisms.

The influx of Ca2+ into yeast cells is primarilyenabled by a Ca2+-permeable channel composed ofMid1p/Cch1p (Paidhungat and Garrett, 1997; Peiteret al., 2005), while two Ca2+-ATPases, Pmc1pand Pmr1p, participate in the cellular sequestrationand homeostasis of Ca2+ (Cui and Kaandorp,2006; Cunningham and Fink, 1994). ComparingCa2+ and Sr2+, classical kinetic studies in S.cerevisiae revealed a highly similar uptake patternwith comparable affinities for both ions (Borst-Pauwels and Theuvenet, 1984).

The overall goal of this study was the identifica-tion of gene products without restriction to a certainphysiological function, viz. K+ and Ca2+ home-ostasis, as previous studies have done. Therefore,an unbiased genome-wide screen in the unicellu-lar model system S. cerevisiae was performed. Intotal, 4862 haploid knockout strains were investi-gated in respect to their Cs+ and Sr2+ accumulationand compared to the wild-type BY4741, which wasthoroughly characterized as well. To conduct thislarge-scale survey, we used a combination of stableand radioactive isotopes of Cs+ and Sr2+ to esti-mate the concentrations within the yeast cells, sinceno discrimination of different isotopes are reportedfor Cs+ and Sr2+. On the basis of this genome-widescreen, we conducted functional classification andprotein network analyses to elucidate cellular pro-cesses and functions with a strong impact on Cs+and Sr2+ accumulation. The results of the genome-wide screen gave insights into the cellular systems

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involved in Cs+ and Sr2+ homeostasis and newpathways, which influenced the accumulation ofboth non-essential cations, are described. Further-more, we investigated the accumulation of charac-teristics for corresponding essential elements (K+,Ca2+) of the candidate mutant strains. Thereby, dif-ferences in regulation or uptake of the ions wererevealed for specific mutant strains, at least withrespect to Cs+.

Materials and methods

Yeast strains and growth conditions

The whole collection of haploid knockout strains ofS. cerevisiae was purchased from EUROSCARF(Frankfurt, Germany). The genotype of the wild-type strain is MAT a his3∆1 leu2∆0 met15∆0ura3∆0 (BY4741). The cells were cultivated rou-tinely in deep 96-well plates in YPD (1% yeastextract, 2% peptone, 2% glucose) or on YPD agar(YPD medium plus 2% bacto agar) in 96-wellmicrotitre plates.

Spot assay

Cells from cultures grown to mid-logarithmic phasewere washed twice with distilled water and thecell titre was determined. Starting from OD600 0.1,a five-fold dilution series was set up in distilledwater and 2.5 µl of each dilution was spotted onan agar plate composed of YPD with the respectivesalt additives. The cells were incubated for 48 h at30 ◦C prior to scanning the colonies.

Measurement of Cs+ and Sr2+ accumulationin yeast cells

Routinely conducted screens were undertaken inbatches of 96 samples. The cells were preculturedin YPD for 48 h at 30 ◦C with agitation. For exper-iments, a 100-fold dilution was set up in YPD andincubated for 8 h under the same conditions, withagitation. Next, the optical density of the cell cul-tures was determined using a Tecan 96-multiwellreader at 595 nm (Tecan Genios, Crailsheim, Ger-many). The cells were harvested by centrifugationat 2000 × g for 5 min and the medium was aspi-rated. The cells were washed twice with assaybuffer (50 mM Hepes, pH 7.0) and resuspended in100 µl assay buffer with 2% glucose. After 30 min

regeneration at 30 ◦C and 200 rpm, equal volumesof cell suspension and assay buffer were mixed in96-well filter plates (HTS plates; Millipore) to afinal composition of 2–4 × 108 cells/ml in 50 mM

Hepes, pH 7.0, 2% glucose with each 50 µM CsCland SrCl2, as well as 4 kBq/ml 134Cs and 85Sr.The cells were incubated for 13 h at 30 ◦C and200 rpm. Afterwards, the liquid was aspirated byvacuum filtration and the filters were washed twicewith assay buffer prior to transferring them to vialsfor γ -detection. The quantification of 134Cs and85Sr activity was processed with an automatic γ -counter (Wallac Wizard 1480, Perkin-Elmer, USA).The measured activity was used to calculate Cs+and Sr2+ molarity (nmol) which was normalized tothe determined cell count (nmol/1 ×107 cells).

Accumulation studies were performed with ris-ing concentration: 50 µM, 0.5 mM, 1 mM, 5 mM,10 mM, 20 mM, 50 mM and 100 mM of CsCl orSrCl2, respectively. Competition experiments werealways performed with 50 µM CsCl or SrCl2; theconcentrations of added K+ and Ca2+ are indicated.Kinetic experiments were performed individuallyfor Cs+ or Sr2+ in larger volumes and aliquots ofthe cell suspension were withdrawn and filtered, aspreviously described, at the indicated time points.

Measurement of Ca2+ and Rb+ accumulationin yeast cells

The uptake assays with Ca2+ and Rb+ were per-formed as described for Cs+ and Sr2+, with slightmodifications: Experiments with 86Rb were donewith an activity of 50 kBq/ml to meet the mea-surement sensitivity of the automatic γ -counter.The quantification of 45Ca, a pure β-emitter, hadto be done by liquid scintillation counting, requir-ing a different type of sample processing. In brief,the filters with the cell material were digested inconcentrated nitric acid (20 min at 50 ◦C) after thewashing steps. After complete digestion, the sam-ples were diluted with distilled water. Scintillationfluid (Ultima Gold, Perkin-Elmer) was added andmixed carefully prior to quantification of the 45Caactivity with a liquid scintillation counter (WallacQuantulus 1220, Perkin-Elmer).

Determination of K+ and Ca2+ contentin whole-cell extracts

Cells were precultured in YPD as described aboveand a 100-fold dilution was incubated for another

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20 h at 30 ◦C and 200 rpm in fresh YPD medium.Next, the cell titre was determined. For eachmeasurement, approximately 3 × 109 cells wereharvested by filtration and washed three timeseach with 10 µM EDTA, pH 8.0, and distilledwater. The filters and cell material were digestedovernight in 30% nitric acid at 65 ◦C. The solu-tion was diluted with distilled water to a finalconcentration of 10% nitric acid and centrifuged.The clear supernatant was subsequently usedfor Inductively Coupled Plasma-Atomic EmissionSpectrometry (ICP–AES) measurements with anICP–AES Spectro Ciros Vision system (SpectroAnalytical Instruments GmbH & Co. KG, Kleve,Germany). Sample introduction was carried outusing a peristaltic pump equipped with an ‘anti-pulse head’ (SPETEC, Erding, Germany) con-nected to a Meinhard nebulizer with a cyclon spraychamber.

Sample normalization and scaling

To employ a normalization to the great number ofmeasurements undertaken, the differences betweenmutant strains and wild-type were expressed as Z -scores, using the following equation:

Z -score = (cko − cwt)/(σwt)

where cko and cwt are the ion content of theknockout strain (ko) or the wild-type (wt) and σwt

is the standard deviation (SD) of the wild-typedata. By using Z -scores, the SD of the wild-typeis used as a unit of measurement and thereforeallows comparison of the multitude of samples pro-cessed. The selection of interesting mutant strainswas carried out on the basis of the calculatedZ -scores. In accumulation experiments (Cs+, Sr2+,Rb+ and Ca2+), candidates were selected using athreshold of ±2.5 (= a divergence of 30–40%from the wild-type). In the initial screen of Cs+and Sr2+ accumulation, the batch mean (of 96 ran-dom samples) was used as reference instead of thewild-type, since only one wild-type was includedper batch and the batch mean was generally in goodagreement with the wild-type data. In all follow-upanalyses, the wild-type (n > 8/measurement) wasused as reference. On account of the very low SD ofthe wild-type within measurements of K+ and Ca2+content, a cut-off of four SD units was employed(=deviation of ∼15% from the wild-type).

Statistical and bioinformatic analyses

Correlation studies were routinely performed withthe STATISTICA 7 software package (StatSoft;Tulsa, USA), using the programs Multiple Regres-sion and ANOVA with default parameters.

Gene ontology (GO) analyses were conductedwith the GO Slim Mapper from SGD (http://www.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl).

Identification of overrepresented gene clusterswas performed with Cytoscape 2.6 (Shannon et al.,2003), using the Bingo 2.3 plug-in (Maere et al.,2005). Bingo 2.3 applies a hypergeometric distri-bution for quantitative determination of function-ally overrepresented GO categories, based on agiven input list of genes. The analysis was per-formed with a p value cut-off of 0.01, usingthe Benjamin and Hochberg False Discovery Ratecorrection for multiple comparisons, since theGO categories were not independent from eachother throughout. The input lists of genes forthe Cs+ and Sr2+ phenotypes were tested vs.the whole annotation (based on 5819 annotatedgenes).

Protein network analyses were also performedwith Cytoscape 2.6, using the jActive plugin 2.2(Ideker, et al., 2002). Prior to analysis, deviatingCs+ and Sr2+ accumulation of the selected mutantstrains was validated statistically, using a two-sided t-test for pairwise comparison with the wild-type (p = 0.05), employing at least three measure-ments/mutant. These statistical data were mappedonto the extended Wi-Phi interactome (Kiemeret al., 2007), which consists of >10 000 pro-tein–protein interactions. Significant subnetworksof activity were identified using the jActive module2.2 plug-in with default parameters. The identi-fied subnetworks corresponded to regions in theinteractome that affect Cs+ or Sr2+ accumulation.The resulting subnetworks were visualized withCytoscape.

Results

Cs+ and Sr2+ accumulate in wild-type yeastcells

We examined Cs+ and Sr2+ accumulation in latelogarithmic stage yeast cells exposed to low, non-toxic concentrations (50 µM) of Cs+ and Sr2+chloride salts, which were conducted from initial

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growth inhibition experiments performed with thewild-type strain BY4741 (Figure 1A).

Both ions were rapidly incorporated into yeastcells and their accumulation reached saturationover a time course of 8–13 h (Figure 1B).

Figure 1

Consecutive investigations tested the accumulationcapacity of yeast cells exposed to rising externalCs+ or Sr2+ concentrations ([Cs+ ]ex or [Sr2+]ex).An increase of [Cs+]ex to 10 mM led to a maximalaccumulation of approximately 100 nmol Cs+/107

cells, which equals an internal concentration of180 mM if a homogeneous intracellular distributionis assumed (Figure 1C). The obtained high Cs+concentration reflects the efficient accumulation ofthe monovalent ion Cs+, which is comparable tothat of the essential K+ (Rodriguez-Navarro, 2000).In the case of Sr2+, a steady increase in cellu-lar concentration is measurable up to a [Sr2+]ex ofapproximately 50 mM. The investigated yeast cellswere capable of accumulating Sr2+ to a final cel-lular concentration of 23 nmol Sr2+/107 cells (or40 mM), indicating that at a certain external con-centration (∼40 mM) no further Sr2+ enrichmentoccurs.

Since the uptake of Cs+ and Sr2+ is supposedto be facilitated by transport systems concern-ing K+ or Ca2+, respectively, we also investi-gated the inhibitory effects of these essential ions(Figure 1D). The simultaneous presence of K+ andCs+ in the assay buffer led to an only moder-ate reduction of the Cs+ accumulation, which wascomparable to the effect of an equal concentrationof Ca2+ (10 mM KCl or CaCl2; Figure 1D). Unlike

Figure 1. Characterization of Cs+ and Sr2+ accumula-tion in wild-type strain BY4741. (A) Elucidation of toxicCs+ and Sr2+ concentrations to the wild-type using spotassays on YPD agar with varying concentrations of Cs+and Sr2+ chloride salts. Starting from cell suspensions of0.1 OD a five-fold dilution series was spotted onto agarplates. The wild-type showed first growth sensitivity toYPD supplemented with 75 mM CsCl and 500 mM SrCl2,respectively. (B) Representative time curve describing theaccumulation of Cs+ and Sr2+ in the wild-type BY4741under standard conditions (uptake assay with 50 µM CsClor SrCl2). Accumulation is expressed in nmol/1 × 107 cells.(C) Representation of Cs+ and Sr2+ accumulation after13 h uptake, depending on the external Cs+ and Sr2+concentration. Cs+ accumulation reaches a maximum of100 nmol/1 × 107 cells when 10 mM external CsCl isreached or exceeded. Sr2+ accumulation approaches amaximum around 23 nmol/1 × 107 cells. (D) Influence ofK+ ( ) and Ca2+ (- - - - ) on Cs+ and Sr2+ accu-mulation. The uptake assay was routinely performed witheither 50 µM Cs+ and Sr2+ and accumulation under stan-dard conditions without K+ and Ca2+ Supplementationwas defined as 100%. Changes in Cs+ and Sr2+ accumu-lation were expressed as a percentage of the standardconditions

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822 S. Heuck et al.

Figure 2. Genome-wide screen of yeast knockout strains for Cs+ and Sr2+ accumulation. (A) Exemplary representationof the initial screen for Cs+ accumulation. 4862 mutant strains were analysed and the respective Z-scores were calculated,where a Z-score of 0 equals wild-type characteristics. The threshold of ±2.5, which refers to a variation of 2.5 standarddeviations (SDs) from the wild-type, was used for the selection of interesting mutant strains (indicated by a grey barbeneath the data points). (B) Venn diagram indicating the distribution of the 212 verified mutant strains to their respectivephenotypes and the overlap between the two groups according to the results of the rescreen. (C) Number of mutantsshowing increases (open bars) and decreases (filled bars) in Cs+ and Sr2+ accumulation

Cs+ accumulation, we observed a dramatic reduc-tion of Sr2+ accumulation upon the addition ofdivalent cations, such as Ca2+ (Figure 1D) or Mg2+(not shown).

Analysis of yeast deletion mutant strainsfor Cs+ and Sr2+ accumulation

To identify genes contributing to the homeostasisof Cs+ and Sr2+ in yeast cells, a total of 4862strains were subjected to a first-pass analysis, wherefor every strain the cellular Cs+ and Sr2+ contentwas measured twice (see Supporting information,Table S1). The evaluation of the mutants wasdone by calculating Z -scores, using a threshold of±2.5 (Figure 2A), which resulted in 557 putativecandidates (for 64 candidates, only one dataset wasavailable). These candidates were rescreened usingquadruple measurements in a randomized design,to avoid positional effects. 212 mutant strains (4%of the screened mutants) could be verified to showa reproducible increase or decrease in Cs+ and/orSr2+ accumulation. This collection also excludesall putative candidates that failed to grow in twoor more measurements of the rescreen procedure(n = 92).

Within the 212 selected mutants, three groupscould be assigned according to their phenotypes(Figure 2B): (a) strains exhibiting a Cs+ phenotype(n = 64 or 30%); (b) a Sr2+ phenotype (n = 76or 36%); or (c) a phenotype for both ions (n =

72 or 34%). Among all strains with Cs+ pheno-type, roughly the same proportions assigned toan increase or a decrease in ion accumulation(Figure 2C). Thereby, the mutants with the low-est and the highest accumulation capacities variedby a factor of 125 (0.02–2.5-fold of the wild-type).Regarding mutant strains revealing a Sr2+ pheno-type, only very few candidates exhibited a reducedaccumulation (n = 8) and nearly 95% (n = 140)of the identified candidates were characterized byan increase in Sr2+ accumulation (Figure 2C). Toexclude an unspecific attachment of Sr2+ to thecell wall, which might have caused this dispro-portionality, we performed separate measurementson soluble cell components and cell debris withrandomly chosen mutant strains and the wild-type(not shown). The cells were disrupted after theuptake phase and the subsequent washing steps.In all cases, <5% of the Sr2+ was found to beassociated with the cell wall. Although this highdiscrepancy exists, the variance among the iden-tified high- and low-accumulating strains equals afactor of 14 (0.22–3.0-fold of the wild-type). Alto-gether, the yeast mutants exhibited a greater varietyin Cs+ than in Sr2+ accumulation.

The last group of candidates showed deviationsin Cs+ and Sr2+ accumulation (34% of all identi-fied strains). This great overlap might indicate anunexpected relationship between the homeostasisof the two ions, which was tested by statisticalmeans. Among the referred candidates (n = 72),

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Figure 3. (A) and (B) Manually curated classification of the identified genes into 10 broad categories based on results ofGO ontology analyses. The percentage of genes in each functional class is represented. (C) and (D) Representation of GObiological process terms overrepresented within the identified gene clusters (Cs+ or Sr2+ phenotype; for a detailed list ofthe specific genes or GO terms, see Supporting information, Tables S2 and S3)

no correlation was clearly detectable, indicated bya correlation coefficient of r = 0.08 (p value notsignificant; tested by ANOVA and linear correlationanalysis). Consequently, these mutant strains mustexert deletions interfering with general aspects ofcellular ion homeostasis.

A variety of cellular processes influence Cs+and/or Sr2+ accumulation

The genes altered in the identified mutant strainswere grouped into 10 manually curated, broad cat-egories, based on the results of GO analyses con-ducted with the GO Slim mapper tool from theSGD database to give a general overview of all bio-logical processes concerned (Figure 3A, B). Fur-thermore, GO enrichment analyses were performed

to search for significantly overrepresented func-tional gene clusters within the two candidate groupsreferring to Cs+ or Sr2+ phenotype (Figure 3C, D;see also Supporting information, Table S3).

For both phenotypes, one large group of candi-dates showed defects in vacuolar biogenesis andfunction (Figure 3A, B: Cs, 17.6%; Sr, 20.9% ofall candidates). This broad category corresponds tomost of the highly overrepresented genes whosedisruption led to a Cs+ as well as a Sr2+ pheno-type. Mutants with Cs+ phenotype show enrich-ments in biological processes concerning vacuolaracidification (GO, 0007035; p < 2.0 × 10−12), cel-lular pH control (GO, 0030641; p < 2.4 × 10−12)or vesicle-mediated transport (GO, 0016192; p <

1.1 × 10−11) (Figure 3C). Regarding mutants with

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Table 1. Genes involved in K+ and Ca2+ homeostasis

Gene ORF Cs Sr Description

K+ homeostasis TRK1 YJL129C 4.69 14.89 K+ transporter (plasma membrane)NHA1 YLR138W 2.00 −3.25 Na+/K+ transporter (plasma membrane)NHX1 YDR456W −2.41 4.79 Na+/K+ transporter (intracellular)KHA1 YJL094C −2.78 1.31 K+ transporter (intracellular)TRK2 YKR050W 0.26 0.26 K+ transporter (plasma membrane)TOK1 YJL093C −1.83 −2.05 K+ channel (plasma membrane)SAT4 YCR008W 0.09 −0.76 Kinases, regulation TRK1HAL5 YJL165C −0.13 0.33 Kinases, regulation TRK1PPZ1 YML016C 0.38 4.07 Phosphatases, regulation TRK1PPZ2 YDR436W 0.11 0.48 Phosphatases, regulation TRK1HOG1 YLR113W −0.84 1.50 Kinase, regulate osmotolerance and TOK1/NHA1VNX1 YNL321W −0.61 −0.92 Monovalent cation/H+ antiporter (vacuole)

Ca2+ homeostasis PMC1 YGL006W 0.18 −5.03 Ca2+ ATPase (vacuole)SPF1 YEL031W −0.96 8.12 P-type ATPase (ER)PMR1 YGL167C −1.83 0.54 Ca2+ ATPase (Golgi)CCH1 YGR217W −0.63 −1.43 Ca2+ channel (plasma membrane)MID1 YNL291C 0.98 0.21 Ca2+ channel (plasma membrane)VCX1 YDL128W −1.20 −1.46 Ca2+ transporter (vacuole)YVC1 YOR087W 0.62 0.96 Ca2+ channel (vacuole)CNA1 YLR433C 0.87 0.96 Calcineurin UE A isoformCNA2 YML057W −0.72 −1.05 Calcineurin UE A isoformCNB1 YKL190W −1.29 −0.50 Calcineurin UE B

Representation of known key players in K+ and Ca2+ homeostasis with their respective Cs+ and Sr2+ accumulation given in Z-scores(phenotype is defined as ‘low’ if the Z-score is ≤−2.5, and as ‘high’ if the Z-score is ≥2.5). Candidates showing a phenotype regarding Cs+or Sr2+ accumulation are shown in bold.

Sr2+ phenotype, mainly gene deletions are overrep-resented, which results in disturbed vacuolar bio-genesis, expressed by the following categories: vac-uolar transport (GO, 0007034; p < 1.8 × 10−11),endosomal transport (GO, 0016197; p < 1.5 ×10−6) or vesicle-mediated transport (GO, 0016192;p < 3.7 × 10−4) (Figure 3D; for complete list ofenriched GO biological processes, see Supportinginformation, Table S3).

Beside this highly overrepresented GO cate-gory, a variety of other biological processes wereshown to have an impact on Cs+ and/or Sr2+accumulation, as shown in Figure 3A, B. Amongthem, translational and transcriptional processesand metabolic pathways have been detected. Wecould also identify 12 genes associated with a Cs+or Sr2+ phenotype, which are not yet fully char-acterized and may be new players in ion home-ostasis: ENT4, IRC6, TMA22, UBP11, YCR062W,YDR089W, YDR090C, YGR125W, YJL123C,YLR414C, YOL162W and YOR205C (see also Sup-porting information, Table S2). To elucidate theirspecific function, further investigations will benecessary.

Key players in K+ and Ca2+ homeostasis showlittle impact on Cs+/Sr2+ accumulationA smaller fraction of the mutant strains with Cs+and/or Sr2+ phenotype are defective for genesinvolved in ion homeostasis. In association withthis functional category, a overrepresentation ofgenes concerning cation homeostasis (Cs+ pheno-type: GO, 0055080; p < 1.4 × 10−6) or cellularion homeostasis (Sr2+ phenotype: GO, 0006873;p < 7.8 × 10−3) could be shown (Figure 3C, D).Most of the overrepresented genes interfere withthe activity of the V-ATPase but a few of thegenes are also involved in K+ or Ca2+ homeosta-sis, as highlighted in Table 1. Trk1� showed anincreased Cs+ and also Sr2+ accumulation, whichargues against a specific correlation between K+and Cs+. On the contrary, kha1� displayed solelya decreased Cs+ accumulation, which may indicatea regulation of Cs+ enrichment via this K+-specificpathway. Furthermore, disruption of several trans-porter and regulatory proteins important for K+homeostasis primarily show unbalanced Sr2+ accu-mulation: NHA1, NHX1 or PPZ1. Furthermore,deletions of only two genes involved in Ca2+

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homeostasis, PMC1 and SPF1, could be associatedwith a deviating Sr2+ enrichment under our experi-mental conditions. Both genes express ATPases thatare proposed to transport divalent cations as wellas exerting the Ca2+ signalling function in the cell(Cronin et al., 2002; Cunningham and Fink, 1996).

Comparison of Cs+ and Sr2+ with essentialelements

As has been pointed out, only a small fraction of thefactors concerning K+ and Ca2+ homeostasis couldbe identified within the genome wide screen. Thecurrent literature links Cs+ and Sr2+ uptake to thatof K+ and Ca2+, respectively (Avery, 1995; Borst-Pauwels, 1981; Rodriguez-Navarro, 2000). To meetthis discrepancy between observations and theliterature, further uptake assays with the essentialelement Ca2+ (and 45Ca) and with Rubidium (Rb+and 86Rb) as a substituent of K+ were performed(Rodriguez-Navarro, 2000). The experiments wereconducted with a subset of 153 mutant strains(excluding strains with defects in translational and

transcriptional processes) and the wild-type strain.In a second step, the K+ and Ca2+ content of alarge part of these strains was analysed. For thispurpose, the yeast cells were cultivated in YPDuntil diauxic shift. The ion content was measuredby ICP–AES.

The accumulation of Cs+ is not tightlyconnected to K+ homeostasisTo elucidate differences between the accumula-tions of Cs+ and Rb+ ions, correlation analyseswere performed which showed a moderate posi-tive correlation expressed by an correlation coef-ficient of r = 0.65 (p < 0.001) for all 153 testedmutant strains (Figure 4A). In particular, mutantstrains with increased Cs+ accumulation showedno comparable tendency in their Rb+ enrichment(for a summary of all data, see Supporting infor-mation, Table S2). Otherwise, a larger fraction ofthe mutants with decreased Cs+ accumulation alsoshowed a slightly diminished Rb+ accumulation,which rarely exceeded the threshold of 2.5, indicat-ing a phenotype. Altogether, the initially identified

Figure 4. Comparison of the accumulation of Cs+ and Sr2+ with that of essential elements. Represented are plots of(A) Cs+ against Rb+ and (B) Ca2+ vs. Sr2+ accumulation, respectively. Light grey filled symbols represent mutant strainswith reduced accumulation, while a dark grey filling indicates an increased accumulation of the corresponding mutants.Correlation analyses were performed by linear regression and the correlation coefficients are indicated in the respectiveplots. Between Ca2+ and Sr2+ a very strong correlation (r = 0.97) is measured, whereas Cs+ and Rb+ are only moderatelystrongly correlated (r = 0.65). (C, D) Investigation of the K+ and Ca2+ content of mutant strains with deviating Cs+ orSr2+ accumulation. For each group (Cs+ or Sr2+ phenotype) the respective fractions of mutants are indicated, which alsoshowed deviations in K+ and/or Ca2+ content (�), respectively

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826 S. Heuck et al.

mutant strains with a Cs+ phenotype do not gen-erally exert analogous and moreover such largedeviations in their Rb+ accumulation.

Further studies concerning the K+ and Ca2+content revealed that approximately 75% of theidentified strains with Cs+ phenotype showed nochanges in the K+ content when grown understandard conditions (Figure 4C). Only 5% of themutants exert exclusive deviations in their K+ con-tents, while another 18% are disturbed in K+ aswell as Ca2+ homeostasis. For the small fractionof mutants that showed alteration for both Cs+and K+, most often a negative correlation wasdetected, meaning that elevated K+ levels were

accompanied by a reduced Cs+ accumulation, ascould be observed for mutants of V-ATPase sub-units. Interdependencies between an altered Cs+accumulation and a disturbed Ca2+ homeostasiswere much more pronounced. Approximately 50%of all mutants with a Cs+ phenotype showed irreg-ular Ca2+ levels. Most often, a positive correlationunderlined this association (63%). Altogether, ourresults showed a far less pronounced dependencybetween K+ or Rb+ and Cs+ homeostasis, based,as is often implied, on chemical similarities of theions. Table 2 summarizes all genes that show cleardifferences between Cs+ and Rb+ accumulation.Mutants showing solely a Cs+ phenotype but no

Table 2. Genes with solely deviations in Cs+ accumulation

Gene ORF Cs+∗ Rb+∗ Sr2+∗ K+∗∗ Ca2+∗∗ GO biological process/notes

TFP1 YDL185W −6.51 −0.72 2.36 −4.13 1.13 Vacuolar H+-ATPase subunitVPS35 YJL154C −3.86 −0.27 −0.63 −1.21 −2.45 Vacuolar protein sorting (A), retromer complexUBP3 YER151C −3.77 −0.24 2.15 8.09 6.80 Ubiquitin-specific proteaseAPL4 YPR029C −3.66 −0.72 2.36 0.42 1.45 γ -Adaptin, large subunit of the AP-1 complexAPS3 YJL024C −3.32 −0.77 −0.77 1.74 −0.31 Small subunit of the AP-3 complexVPS17 YOR132W −2.94 0.08 3.03 0.54 7.80 Vacuolar protein sorting (A), retromer complexNPR1 YNL183C −2.88 −0.40 0.75 2.66 2.45 Protein kinase, stabilize amino acid transportersRMD11 YHL023C −2.81 −0.80 5.35 −0.37 −1.56 Deletion confers delays in meiotic DNA replication/nuclear divisionsKHA1 YJL094C −2.78 −0.31 1.31 −0.25 5.20 Putative K+/H+ antiporterFCY2 YER056C −2.71 −0.85 −1.39 −3.10 1.88 Purine-cytosine permease (purine, cytosine)VAM10 YOR068C −2.71 1.00 −0.55 2.96 5.72 Vacuole fusionVAM6 YDL077C −2.64 −0.06 14.45 0.02 1.77 Vacuolar protein sorting (B), membrane fusionVPH1 YOR270C −2.57 −0.31 4.22 2.39 −8.12 Vacuolar H+-ATPase subunitTPO1 YLL028W 2.57 0.34 −2.59 −1.17 5.95 Polyamine transporterRHO4 YKR055W 2.83 −0.56 9.48 7.36 14.12 GTPase involved in establishment of cell polarityATG2 YNL242W 3.15 0.93 −0.66 0.27 −1.01 Vesicle formation during autophagyMDM34 YGL219C 3.18 0.63 4.51 −4.66 −2.45 Required for mitochondrial morphology/inheritanceCYK3 YDL117W 3.88 −0.31 1.10 3.98 5.24 SH3-domain protein in the mother-bud neck/cytokinetic actin ringFPS1 YLL043W 3.97 0.33 −0.11 0.05 1.25 Channel (efflux of glycerol, uptake of acetic acid/trivalent metalloids)CSF1 YLR087C 4.00 0.79 1.25 −1.74 7.83 Fermentation at low temperaturesPMP3 YDR276C 4.18 −1.00 7.08 −0.25 13.38 Related to plant polypeptides, overexpressed under high salt

concentrationEDE1 YBL047C 4.20 0.84 −0.26 −1.99 3.07 Key endocytic proteinTRK1 YJL129C 4.69 0.09 14.89 −2.72 3.10 Component of the high affinity K+-transport systemRVS161 YCR009C 5.11 0.69 14.53 4.11 9.36 Amphiphysin-like lipid raft protein, regulates polarization of the actin

cytoskeletonYPK1 YKL126W 5.34 0.31 2.01 6.04 16.55 Serine/threonine protein kinase required for receptor-mediated

endocytosisYDR090C YDR090C 5.36 0.37 −0.58 1.61 3.68 Unknown function, homologue to cystinosinMGM101 YJR144W 5.77 −0.06 7.60 −3.13 7.89 Required for normal mitochondrial morphologyLST4 YKL176C 5.81 0.35 −0.40 2.82 1.67 Nitrogen metabolism, vesicle-mediated transportSUR4 YLR372W 6.93 0.92 4.27 −1.54 8.22 Elongase, sphingolipid biosynthesis

Summary of all genes whose deletion led to a Cs+ phenotype but no deviation in Rb+ accumulation in the respective mutant strains. All dataare given in Z-scores, meaning that values between −2.5 and 2.5 classify wild-type character. Genes showing no further anomalies (e.g. in K+or Ca2+ content) are highlighted in bold.∗ Measurement of ion accumulation (according the described uptake assay).∗∗ Measurement of ion content after cultivation in YPD.

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Cs and Sr accumulation in yeast 827

dramatic deviations in either Rb+, Sr2+ Ca2+ accu-mulation or K+ and Ca2+ content are highlightedin bold. The respective genes are classified withrespect to different biological processes, such asGolgi-to-vacuole transport (VPS35, APL4, APS3 ),autophagy (ATG2 ), endocytosis (EDE1 ) and trans-port (FCY2, FPS1 and YDR090C ) or metabolism(NPR1 ) (for a summary of the complete data for allinvestigated mutant strains, see Supporting infor-mation, Table S2).

Sr2+ and Ca2+ accumulation is very similar

The accumulation of Sr2+ and Ca2+ was measuredfor equal concentrations in triplicates for all 153mutant strains. The following correlation analysisrevealed that all tested yeast strains did not discrim-inate between these two ions (r = 0.97; p = 0.000;Figure 4B). Furthermore, it could be clearly shownthat approximately 50% of all investigated strainswith Sr2+ phenotype also exhibited a disturbedCa2+ content when grown in YPD. In 72% of allcases, a positive correlation was found betweendeviating Sr2+ accumulation and irregular Ca2+contents (Figure 4D).

Discussion

Cs+ and Sr2+ accumulate in yeast althoughthey are not essential

We analysed the accumulation of the non-essentialCs+ and Sr2+ in S. cerevisiae because both ionshave an impact on the environment in terms ofradioactive pollution, in the form of 137Cs and90Sr. To elucidate the mechanism contributing tothe uptake of the Cs+ and Sr2+, we performed agenome-wide analysis investigating the uptake ofboth ions in the low micromolar range (50 µM), asit occurs in the environment (White and Broadley,2000). Therefore, secondary effects provoked byhigh osmolarity or by toxicity could be largelyexcluded (for toxic concentrations of Cs+ andSr2+, see Figure 1A). Under these conditions, Cs+and Sr2+ become highly enriched in the yeastcells against a step gradient, which indicates thatactive or secondary active mechanism underlies themechanism of uptake. This is highlighted by theenrichment of 1.25 nmol Cs+/1 × 107 cells or 1.82nmol Sr2+/1 × 107 cells, which equal a 40- and

60-fold enrichment, respectively, compared to themedium.

212 mutant strains show large variations in Cs+and Sr2+ accumulation

The analysis of the 4862 individual deletion strainsrevealed that a variety of processes participatein balancing the accumulation of Cs+ and Sr2+.After a two-stage screening process, 212 mutantswere associated with reproducible variations in Cs+and/or Sr2+ accumulation. The high discrepancybetween the first screen, which revealed 553 poten-tial candidates, and the rescreen (212 confirmedcandidates) was largely caused by positional effectscaused by the use of microtitre plates. Therefore,we changed in the rescreen to a randomized designto create a compensation for this effect, excludinglarge parts of false-positive hits.

One intriguing point is the large variation inthe accumulation capacity, which is depicted bya factor of 125 for Cs+ and a factor of 14for Sr2+. These variation factors are a multiplehigher than comparable data of investigations withplant varieties (wheat and maize), which onlyencompassed a two- to three-fold range (Gerst-mann and Schimmack, 2006; Schimmack et al.,2004; Schneider et al., 2008). This result clearlyreflects the advantages in investigating single dele-tion strains, where gene disruptions show strong,and moreover assignable, effects. The large dif-ference in the variety of uptake capacity betweenCs+ and Sr2+ is caused by an unequal decrease inion accumulation. Several gene disruptions nearlyextinguish Cs+ accumulation. This is not measur-able for Sr2+, so that it could be assumed that thecells are leakier for divalent cations.

A direct comparison with other studies concern-ing cellular responses to monovalent cations pro-vides only little overlap, since all of them were con-ducted with higher concentrations, eliciting eitherosmotic or toxic stress, which was not our goal.The most recent study concerning the effects ofCs+ on yeast cells was conducted by Del Vescovoet al. (2008) and clearly highlighted the responseof cells to Cs+ stress by reconstituting the cell wallin a HOG-dependent and also -independent fashion.In contrast to our study, which was conducted inthe low micromolar range, Del Vescovo et al. usedat least 100 mM CsCl, which was toxic to the yeastcells in our experimental set-up (Figure 1A) rather

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828 S. Heuck et al.

than only provoking a growth delay. Nevertheless,our results also imply that cell wall biogenesis con-tributes to the extent of Cs+ accumulation by yeastcells (see Supporting information, Table S2) but theidentified factors are not identical to those identi-fied by Del Vescovo et al. under stress conditions.

Another study that presents data comparableto our investigation dealt with the genome-wideionome profiling of a collection of knockout strainsof non-essential genes (Eide et al., 2005). In thatstudy, the vacuolar function was also identifiedas one central aspect in ion accumulation andhomeostasis.

The vacuole plays a central role in Cs+and Sr2+ accumulation

GO enrichment analysis proved a distinct overrep-resentation of genes concerning vacuolar functionand biogenesis (see Figure 3C, D; see also Support-ing information, Table S3). Thereby, a restrictionof vacuolar functions tends to result in differentphenotypes: while the accumulation of Cs+ is dras-tically reduced, the Sr2+ accumulation is elevated.

The vacuole is a central player in cellular ionhomeostasis, due to regulation of cellular pH, ionfluxes and adjustment of the cellular turgor (Nel-son, 2003), and it is also very important for detox-ification processes, due to the removal of toxiccomponents out of the cytoplasm (Eide et al.,1993). Consequently, the miscellaneous function-ality of the vacuole has effects not only on Cs+and Sr2+ accumulation but also on the regulationof a broad variety of other ions, as was shown byother genome-wide analyses presented by severalstudies (Eide et al., 2005; Jo et al., 2008; Lesu-isse et al., 2005). For a better understanding, addi-tional protein network analyses were performed,which uncovered three significant subnetworks ofactivity within the yeast interactome. The threesubnetworks clearly link disturbed Cs+ and Sr2+accumulation to vacuole function and biogenesis(Figure 5).

V-ATPase function is crucial for Cs+ accumulation

The first cluster comprising nearly all of the V-ATPase subunits (Kane, 2006) and several ofits assembly factors (Figure 5A, summarized inTable 3). The V-ATPase is an intracellular local-ized H+-ATPase that acidifies vacuole, Golgi and

endosome (Kane, 2006; Nelson, 2003). Muta-tions disrupting the V-ATPase exhibit a drasti-cally reduced Cs+ accumulation, while Sr2+ levelsranged generally within the limits of the wild-typethreshold. Cs+ is supposed to enter the vacuolevia transporters, which depend on electrochemi-cal gradient build-up by the V-ATPase (Avery,1995; Nelson, 2003). A decreased V-ATPase activ-ity would reduce the transport of Cs+ ions into thevacuole and its subsequent cellular accumulation.Other organelles, such as the Golgi or endoplas-mic reticulum, apparently do not compensate forthe missing vacuolar Cs+ storage capacity. Fur-thermore, the cytosolic Cs+ concentration appearsto be relatively low in these mutants, which mightbe achieved either by an efficient efflux of Cs+ ordue to a downregulation of uptake. This assump-tion is, furthermore, in strict accordance with theobservation that most V-ATPase mutants are alsoless sensitive to high Cs+ concentrations in themedium (Figure 5B). The uptake of Sr2+ into thevacuole is thought to occur predominantly via theCa2+-permeable ATPase Pmc1p (Cunningham andFink, 1994). In this case, the presence of a func-tional V-ATPase is not necessary for sequestrationof divalent ions, at least under non-stressed condi-tions, which is visible in the wild-type, like Sr2+accumulation of these mutants.

Mutations concerning vacuolar protein sortingprimarily affect Sr2+ accumulation

The second cluster represents a large group ofvacuolar protein sorting (vps) mutants (Figure 5C,Table 3). Previous investigations on vacuolar pro-tein sorting defects revealed six different classesof vps mutants (termed A–F), depending on theirparticular vacuolar morphologies. All mutants dis-play defects in protein transport from the Golgito the vacuole (Bowers and Stevens, 2005). Thescreen carried out identified members of classesA, B, D, E and F, whereas mutants of vps classC grew very poorly and therefore could not beinvestigated here. Among all vps mutants, membersof class E exclusively showed an increased Sr2+accumulation, whereas members of the remain-ing classes revealed a high Sr2+ accumulationin combination with frequently low Cs+ levels(Figure 5D).

The vacuole is the primary storage site for Ca2+,where it is bound to polyphosphates to reduceits cytoplasmic concentration, and it is assumed

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Cs and Sr accumulation in yeast 829

Figure 5. Protein network analysis identifying subnetworks of activity in Cs+ and Sr2+ accumulation. Differences betweenmutant and wild-type strains were validated by a two-sided t-test (pairwise comparison) with a significance level ofp = 0.05. The resulting data were used for protein network analysis, using the jActive module 2.2 of Cytoscape. Twosubnetworks were identified, based on Cs+ accumulation data. (A) Subnetwork representing subunits and assembly factorsof the VATPase. Almost all subunits of the V-ATPase were associated in our screen with a diminished Cs+ accumulation(nodes coloured in dark grey). The whole complex is essential for ion homeostasis in yeast. (B) Comparison of toxiceffects to high medium concentrations of Cs+ and K+ towards the wild-type and mutants of the V-ATPase subunits(subunit labels are given in brackets, for V0 complex unit A–E and V1 complex unit a–c). High Cs+ concentration ofthe medium led indeed to a delayed growth of the mutants but clearly not in the magnitude displayed by the wild-type.Concerning high K+ concentrations, the effect was, conversely, that mutant strains with the strongest growth on highCs+ displayed the most poorly growth on high K+. (C) This subnetwork included two interconnected AP complexesinvolved in protein delivery to the vacuole (AP-1, APS1–APL2–APL4–APM1; AP-3, APS3–APL5–APL6). Furthermore,the subnetwork revealed additional players in the protein trafficking system of the yeast associated to the AP-3 complex.These genes/proteins are predominantly involved in membrane fusion processes (VAM3, VAM6, VAM7, YPT7, PEP12).(D) A third subnetwork was found based on Sr2+ accumulation data (nodes coloured in light grey). All represented genesbelong to the vps class E and are involved in protein sorting within the prevacuolar compartment, with the exceptionof DOA4, although the ubiquitin isopeptidase Doa4p is functional and locally strongly connected with the vps classE machinery

that Sr2+ is also enriched in the vacuole (Dunnet al., 1994). Since the vacuole is impaired in vpsmutant strains, the observed increase in Sr2+ accu-mulation, instead of a reduction, was not antici-pated. However, a former study of Miseta et al.(1999) demonstrated a comparable, elevated Ca2+level in vps33� cells. VPS33 is a class C vac-uolar protein-sorting gene (Bowers and Stevens,2005). Miseta et al. (1999) imply that the increasedCa2+ content in �vps33 is caused by the compen-satory action of Pmr1p, a Ca2+/Mn2+-transportingATPase of the Golgi apparatus, which leads to a

hyperaccumulation of Ca2+ in this compartment.The similar observation of Sr2+ levels in vpsmutants implies that Pmr1p also transports Sr2+and causes a hyperaccumulation.

AP-1 and AP-3 dependent protein sorting influenceCs+ accumulation

The third cluster of genes involved in vacuolarbiogenesis comprises subunits of AP-1 and AP-3 complexes (Figure 5C) that are involved in theformation and liberation of clathrin-coated trans-port vesicles at the late Golgi (Spang, 2008). The

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830 S. Heuck et al.

Table 3. Genes affecting vacuolar function and biogenesis

Gene ORF Cs+ Sr2+ GO cellular component GO biological process/notes

V-ATPase functionCUP5 YEL027W −6.27 0.32 Vacuole Vacuolar H+-ATPase subunitPPA1 YHR026W −5.36 3.32 Vacuole Vacuolar H+-ATPase subunitSTV1 YMR054W −5.00 1.42 Golgi/vacuole Vacuolar H+-ATPase subunitTFP1 YDL185W −6.51 2.36 Vacuole Vacuolar H+-ATPase subunitTFP3 YPL234C −6.41 1.80 Vacuole Vacuolar H+-ATPase subunitVMA2 YBR127C −6.54 1.32 Vacuole Vacuolar H+-ATPase subunitVMA4 YOR332W −6.49 2.08 Vacuole Vacuolar H+-ATPase subunitVMA5 YKL080W −6.45 1.47 Vacuole Vacuolar H+-ATPase subunitVMA8 YEL051W −6.53 2.16 Vacuole Vacuolar H+-ATPase subunitVPH1 YOR270C −2.57 4.22 Vacuole Vacuolar H+-ATPase subunitRAV1 YJR033C −3.61 17.87 Cytoplasm Vacuolar H+-ATPase assemblyRAV2 YDR202C −1.83 4.77 Cytoplasm Vacuolar H+-ATPase assemblyVMA21 YGR105W −4.97 3.64 Endomembrane system Vacuolar H+-ATPase assemblyVMA22 YHR060W −6.53 2.07 Endomembrane system/ER Vacuolar H+-ATPase assemblyVPH2 YKL119C −6.52 1.95 Endomembrane system/ER Vacuolar H+-ATPase assemblyVacuolar protein sortingVPS8 YAL002W −2.06 9.63 Cytoplasm Vacuolar protein sorting (A)VPS17 YOR132W −2.94 3.03 Endosome Vacuolar protein sorting (A), retromer complexVPS29 YHR012W −3.97 −0.37 Endomembrane system/ER Vacuolar protein sorting (A), retromer complexVPS35 YJL154C −3.86 −0.63 Endomembrane system Vacuolar protein sorting (A), retromer complexVPS38 YLR360W −1.82 10.06 Unknown Vacuolar protein sorting (A)VAM6 YDL077C −2.64 14.45 Vacuole Vacuolar protein sorting (B), membrane fusionVAM7 YGL212W −4.28 8.48 Vacuole Vacuolar protein sorting (B), membrane fusionVPS41 YDR080W −2.34 15.59 Vacuole/endosome Vacuolar protein sorting (B), HOPS complexVPS54 YDR027C −3.84 6.65 Golgi/mitochondrion Vacuolar protein sorting (B), GARP complexVPS72 YDR485C 0.92 3.20 Cytoplasm/nucleus Vacuolar protein sorting (B)PEP12 YOR036W −3.24 15.80 Golgi/endosome Vacuolar protein sorting (D)VPS3 YDR495C −4.24 11.43 Cytoplasm Vacuolar protein sorting (D)DID2 YKR035W-A −0.70 8.85 Cytoplasm Vacuolar protein sorting (E), ESCRT III complexDID4 YKL002W −1.38 6.89 Cytoplasm/membrane Vacuolar protein sorting (E), ESCRT III complexSNF7 YLR025W −1.74 5.06 Cytoplasm Vacuolar protein sorting (E), ESCRT III complexSRN2 YLR119W 0.99 12.07 Cytoplasm Vacuolar protein sorting (E), ESCRT I complexSTP22 YCL008C 0.84 8.35 Cytoplasm Vacuolar protein sorting (E), ESCRT I complexVPS4 YPR173C 1.95 9.50 Cytoplasm/ER Vacuolar protein sorting (E), ESCRT III complexVPS20 YMR077C 1.81 11.67 Cytoplasm/membrane Vacuolar protein sorting (E), ESCRT III complexVPS24 YKL041W −0.03 11.78 Cytoplasm/membrane Vacuolar protein sorting (E), ESCRT III complexVPS27 YNR006W 0.85 12.53 Cytoplasm Vacuolar protein sorting (E)VPS28 YPL065W 1.69 9.92 Cytoplasm/membrane Vacuolar protein sorting (E), ESCRT I complexVPS36 YLR417W 0.34 12.67 Cytoplasm/membrane Vacuolar protein sorting (E), ESCRT II complexVPS1 YKR001C −4.21 8.75 Mitochondrion/peroxisome Vacuolar protein sorting (F)GMH1 YKR030W 0.14 2.72 Golgi Protein–vacuolar targetingVAM3 YOR106W −2.14 14.19 Vacuole Vesicle fusionVAM10 YOR068C −2.71 −0.55 Vacuole Vacuole fusionVTC3 YPL019C 1.11 8.35 Vacuole Vacuolar transportYPT7 YML001W −1.77 12.07 Vacuole/mitochondrion Vacuole fusionIntracellular traffickingAPS1 YLR170C −3.74 1.59 Golgi Small subunit of the AP-1 complexAPM1 YPL259C −4.81 0.26 Golgi µ 1-Like medium subunit of the AP-1 complexAPL2 YKL135C −4.73 3.25 Golgi β-Adaptin, large subunit of the AP-1 complexAPL4 YPR029C −3.66 2.36 Golgi γ -Adaptin, large subunit of the AP-1 complexAPS3 YJL024C −3.32 −0.77 Membrane Small subunit of the AP-3 complexAPL5 YPL195W −3.44 −1.67 Membrane δ-Adaptin-like subunit of the AP-3 complexAPL6 YGR261C −3.03 −0.79 Membrane β 3-like subunit of the AP-3 complex

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Table 3. Continued

Gene ORF Cs+ Sr2+ GO cellular component GO biological process/notes

ARF1 YDL192W −3.58 6.10 Cytoplasm/Golgi GTPase of the Ras superfamilyGCS1 YDL226C −4.77 12.51 Cytoplasm/Golgi GTPase activating protein (ARF-GAP)GOS1 YHL031C −2.44 2.70 Membrane v-SNARE protein involved in Golgi transportSEC28 YIL076W −2.28 10.70 Golgi/endosome ε-COP subunit, retrograde Golgi-to-ER traffic

Mutant Cs+ and Sr2+ accumulation is defined as ‘low’ if the Z-score is ≤−2.5, and as ‘high’ if the Z-score is ≥2.5 (data in bold). Valuesbetween −2.5 and 2.5 represent accumulation data that were not considered to be changed from the wild-type (data in italic). The letters inparentheses indicate the assigned class of vacuolar protein sorting defect, which are described in the text.

AP-1 complex is necessary for vacuolar proteintransport following the classical CPY route via theendosome (Phelan et al., 2006), in which also vpsproteins participate, while the AP-3 complex fulfilssimilar functions in the context of an alternativepathway bypassing the late endosome (ALP route)(Cowles et al., 1997). Both complexes are involvedin vacuolar function by enabling the transport ofdifferent subsets of proteins to the vacuolar mem-brane or lumen. The mutants are characterized bya reduced Cs+ accumulation but an overall normalSr2+ phenotype.

Generally, the low Cs+ accumulation in AP-1/-3mutants and also some vps mutants is most likelycaused by the loss of the vacuole as Cs+ storagecompartment, which has already been discussed inaccordance to the V-ATPase (Avery, 1995). Intrigu-ingly, we observed a normal Cs+ accumulation inmutants of vps class E. Those mutants exhibit animpaired vacuole but also possess an enlarged pre-vacuolar compartment (PVC) (Bowers and Stevens,2005). So it is possible that an efficient Cs+ uptakeoccurs into the PVC, which would be supported bytwo aspects: (a) the PVC contains a huge variety ofproteins originally destinated for the vacuole; and(b) it was shown that V-ATPase is functional inthis compartment, enabling secondary active trans-port events (Kane, 2006). So the accumulation intointracellular compartments is a key event for theenrichment of Cs+ in yeast cells. Consequently,it would be helpful to identify the specific trans-port proteins involved in this Cs+ sequestration,to understand and potentially manipulate the Cs+accumulation.

Cs+ accumulation in the context of K+and Ca2+ homeostasis

The literature gives opposing views on the uptakeof Cs+ into yeast cells. It is largely assumed

that Cs+ enters the cells via K+ pathways andthat there is a clear competition between the twoions, which we could not back up (Figure 1D),while others clearly exclude the main K+ uptakesystem (Trk1/2p) as a Cs+ entry route (Bertl et al.,2003). The competition experiments presented hereshowed only a moderate, and moreover saturable,inhibitory effect of K+ ions on Cs+ uptake underlow (50 µM) external Cs+ concentration. It seemsthat a large quantity of Cs+ (approximately 50%;Figure 1D) is taken up by yeast independently ofthe K+ concentration in the medium or the K+status of the cell itself. This observation led to thehypothesis that Cs+ uptake could partially occurvia different uptake mechanisms than describedfor K+.

Special attention in the genome-wide screen ofCs+ accumulation was laid upon already knownfactors participating in K+ homeostasis. As shownin Table 1, only a minor fraction of the respec-tive mutant strains showed deviations in Cs+ accu-mulation, the two transporter mutants �trk1 and�kha1. The loss of TRK1 led to an increased Cs+accumulation, but this was rather provoked by ahyperpolarization of the plasma membrane in thismutant strain, which caused increased and unspe-cific uptake of mono- and divalent ions, since anincreased Sr2+ accumulation was also measured(Madrid et al., 1998). In accordance with this,a recent electrophysiological study with isogenicyeast strains lacking either the main transport fac-tors TRK1, TRK2 and TOK1 or combinations ofthem indicated that the high-affinity K+ uptake sys-tem consisting of Trk1/2p does not facilitate Cs+entry into the cell on a large scale. The authorsrather propose that Tok1p is in some ways involvedin the permeability of yeast cells to Cs+, as wellas a so-far only electrophysiologically describednon-selective channel, NSC1 (Bertl, 2003). Never-theless, we could neither observe a deviating Cs+

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832 S. Heuck et al.

accumulation in mutants of TOK1 (or TRK2) norcould we identify a gene corresponding to NSC1.Here, the use of double or triple mutants would benecessary to uncover pathways that might be notvisible, due to a certain redundancy.

The loss of KHA1 led to a decrease in Cs+ accu-mulation. Kha1p is a putative K+/H+ antiporterresiding in intracellular membranes (Flis et al.,2005) and could potentially be one entry path-way for Cs+ ions into the vacuole or endosome.The loss of KHA1 was described to provokean increased cellular K+ content (Ramirez et al.,1998), which might reduce the Cs+ accumulationdue to a displacement by K+ ions. But in ourstudy no increased K+ content could be measured,so that it can also be speculated that in �kha1cells inhibited transport of Cs+ ions into the vac-uole could be the reason for the overall loweredaccumulation. A comparable but slightly weakerphenotype could be observed for �nhx1, a mutantstrain disrupted for another intracellular localizedNa+/K+/H+ antiporter (see Table 1; deviations inCs+ accumulation are a little below the threshold),potentially providing a second transporter involvedin Cs+ sequestration within the cell (Kinclova-Zimmermannova et al., 2006; Nass and Rao, 1998).

Although our study showed only moderate over-lap between K+ homeostasis and Cs+ accumu-lation, pronounced interactions between Cs+ andCa2+ could be revealed. A first hint was thatCs+ accumulation was inhibited by Ca2+ in aneven more efficient way than by the competi-tive ion K+ (Figure 1D). Several studies provedthat Ca2+ signalling is crucial for the salt stressresponses of yeast cells. In response to sodium(Na+) stress, two major responses are known.Osmotic stress is mediated via the HOG path-way, leading to an activation of the P-type ATPasePMR2 (ENA1), which facilitates increased Na+efflux (Posas et al., 1996). The second signallingpathway is related to the action of calcineurinand may be an answer to the toxicity of Na+.This protein phosphatase is activated due a burstof cytosolic Ca2+ concentration, which provokesthe action of Ca2+/calmodulin. As a consequence,calcineurin dephosphorylates the transcription fac-tor Crz1p, leading to the transcriptional regulationof up to 150 genes. Among others, PMR2 alsobecomes upregulated, contributing to a clearanceof the toxic Na+ (Stathopoulos and Cyert, 1997).The inhibition of Cs+ accumulation by Ca2+ might

be a result of the described mechanism, since Cs+is even more toxic than Na+. Otherwise, it is alsopossible that the electrophysiologically-describednon-selective channel (NSC1) is involved in thisobservation, since it was also reported to be reg-ulated via Ca2+ (Bihler et al., 2002). The authorsshowed that higher external Ca2+ concentrationssuppressed the ion permeability of this channel,which would lead to a reduced Cs+ influx (if NSC1is the main uptake route in yeast).

For a regulation of Cs+ accumulation via Ca2+/calmodulin- and calcineurin-dependent mechanismfurthermore argues that approximately 50% of allmutants with Cs+ phenotype showed disturbancesin their Ca2+ content when grown under standardconditions (Figure 4B). A deregulation in Ca2+homeostasis also implies disturbed Ca2+ signallingand would finally result in an impaired reaction ofthe cell towards Cs+.

Sr2+ accumulation in the context of Ca2+homeostasis

In the case of Sr2+ uptake, only very few dataexist and it is assumed that it occurs via the samepathways that account for Ca2+. The active uptakeof divalent cations is thereby mainly driven viachannels due to the extremely low cytoplasmicconcentration, which is kept up by the action ofintracellular ATPases transferring ions into intracel-lular compartments (Cunningham and Fink, 1994).All investigations carried out on Sr2+ and Ca2+led to the conclusion that yeast cells do not dis-criminate between the two ions, at least under theexperimental conditions chosen.

Comparison of Cs+ accumulation in yeastand plant

Cs+ uptake in plants has been investigated exten-sively over recent decades, and also the distri-bution of Cs+ within plant cells, among othertopics. The vacuole was identified as one of theprimary compartments involved in Cs+ retentionslacking a defined saturation level (Le Lay et al.,2006), which is in good agreement with the yeastmodel (Avery, 1995). Furthermore, most of the fac-tors involved in vacuolar function and biogenesis(Table 3) are conserved throughout both kingdoms,which offer the possibility of inferring the effectsof gene deletions from the yeast model.

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Cs and Sr accumulation in yeast 833

Table 4. Homologues in plants

Saccharomycescerevisiae

Homologues in Arabidopsisthaliana

Gene name ORF Gene name Locus

KHA1 YJL094C AtCHX15 At2g13620AtCHX16 At1g64170AtCHX17 At4g23700AtCHX18 At5g41610AtCHX19 At3g17630AtCHX20 At3g53720

NHX1 YDR456W AtNHX1 At5g27150AtNHX2 At3g05030AtNHX3 At5g55470AtNHX4 At3g06370AtNHX5 At1g54370AtNHX6 At1g79610

Representation of plant homologues of KHA1 and NHX1 in themodel plant Arabidopsis thaliana. Indicated are the gene names andloci of the plant genes.

Manipulations of the plant vacuole, either byenhancing V-ATPase function or manipulating asingle cation transporter, were already shown tocontribute to improved salt and drought stressresponses (Gaxiola et al., 2002). In this context, thetwo putative Cs+-sequestering transporters, Kha1pand Nhx1p, would also be good candidates formanipulations in plants. Table 4 lists all homo-logues of KHA1 and NHX1 in the model plantArabidopsis thaliana, giving an impression of thecomplexity of the endeavour to apply changes inthe plant system.

General conclusion

This study was performed to identify genetic fac-tors contributing to a regulation of Cs+ or Sr2

accumulation in the model system S. cerevisiae,in an unbiased approach. According to the candi-dates identified, strong evidence is given that thevacuole plays an important role in normal accumu-lation of Cs+ and Sr2+ in the cell. Based on ourscreen, we suggest that the vacuole is the decisiveentity for Cs+ accumulation in yeast, since almostall mutations affecting vacuolar function lead toa significantly reduced Cs+ content in the yeastcells. In a broader sense it is therefore reasonableto hypothesize that also in plant cells the vacuoleis very important for an efficient accumulation ofCs+. Therefore, it would be reasonable to take

more efforts in the characterization of ion trans-porter of intracellular compartments with respect toCs+. Beside the vacuolar function, other promisingcandidates pointed towards a connection betweenCs+ accumulation and biological processes suchas endocytosis and autophagy, lipid metabolism orcell wall biogenesis (see Supporting information,Table S2). Also in this context, continued inves-tigation could deepen our understanding of Cs+homeostasis.

Otherwise, the work presented here gave thestrong impact that a distinction between the non-essential Sr2+ and the essential Ca2+ does notoccur in yeast cells and, concerning the literature,in plants either. Nevertheless, the results of thegenome-wide screen presented new genes whichwere not associated with Ca2+ homeostasis beforenow (see Supporting information, Table S2). Fur-ther characterization of these genes may refine ourunderstanding of how Ca2+ fluxes and content areregulated in yeast cells.

Acknowledgements

We gratefully thank Professor Dr Durner and Dr AntonSchaffner from the Institute of Biochemical Plant Pathol-ogy, Helmholtz Zentrum Munchen, for their helpful advice.

Supporting information on the internet

The following supporting information may befound in the online version of this article:

Table S1. Cs+ and Sr2+ accumulation of alltested haploid knock-out strains

Table S2. Summary of ion accumulation, ioncontent and toxicity

Table S3. Cs+ and Sr2+ overrepresented genes

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