JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Oct. 1999, p. 6456–6462 Vol. 181, No. 20

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Biochemical and Genetic Analyses of the Role of YeastCasein Kinase 2 in Salt Tolerance

EULALIA DE NADAL,1 FERNANDO CALERO,2 JOSE RAMOS,2

AND JOAQUIN ARINO1*

Departamento Bioquımica i Biologia Molecular, Universitat Autonoma de Barcelona, Bellaterra 08193,Barcelona,1 and Departamento de Microbiologıa, Escuela Tecnica Superior de Ingenieros

Agronomos y Montes, 14080 Cordoba,2 Spain

Received 27 May 1999/Accepted 6 August 1999

Saccharomyces cerevisiae cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the geneCKB1, display a phenotype of hypersensitivity to Na1 and Li1 cations. The sensitivity of a strain lacking ckb1is higher than that of a calcineurin mutant and similar to that of a strain lacking HAL3, the regulatory subunitof the Ppz1 protein phosphatase. Genetic analysis indicated that Ckb1 participates in regulatory pathwaysdifferent from that of Ppz1 or calcineurin. Deletion of CKB1 increased the salt sensitivity of a strain lackingEna1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is notmediated by Ena1. Consistently, ckb1 mutants did not show an altered cation efflux. The function of Ckb1 wasindependent of the TRK system, which is responsible for discrimination of potassium and sodium entry, andin the absence of the kinase regulatory subunit, the influx of sodium was essentially normal. Therefore, the saltsensitivity of a ckb1 mutant cannot be attributed to defects in the fluxes of sodium. In fact, in these cells, boththe intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-typecells. The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the lightof these findings.

As for many cell types, sodium cations are rather toxic foryeast cells, and consequently, the maintenance of suitable in-tracellular concentrations of Na1 is a strong requirement forsurvival (see reference 42 for a review). Intracellular sodiumlevels are the result of influx and efflux processes that aresubjected to regulation. Saccharomyces cerevisiae actively ex-trudes sodium through the Na1-ATPase encoded by the geneENA1, the first member of the ENA (also called PMR2) locus(13, 18, 40, 47). ENA1 is barely expressed under normal growthconditions, but its expression is sharply increased by osmoticand saline (sodium or lithium) stresses, as well as by alkalinepH (13, 23, 25). As a consequence, cells lacking ENA1 arehighly sensitive to sodium and lithium. Several components ofthe regulatory network that controls ENA1 expression havebeen identified in the last few years. Interestingly, this regula-tion involves phospho-dephosphorylation mechanisms. For in-stance, the Ser/Thr protein phosphatase PP2B (calcineurin) isneeded for full response to sodium stress (25, 28). On the otherhand, the Ppz1 protein phosphatase represses ENA1 expres-sion through a mechanism that is independent from that ofcalcineurin. This repression of ENA1 results in phosphatasemutants that are hypertolerant to sodium (32). Recent workhas shown that HAL3, initially identified as a halotolerantdeterminant that influences ENA1 expression (11), is a nega-tive regulatory subunit of Ppz1 and thus defines a novel regu-latory pathway (8). Hal1, a conserved salt-induced protein(14), has been defined as an effector of ENA1 expression (39).Recently, HAL8 and HAL9 have been determined to be genesencoding putative transcriptional activators of the ENA1 re-sponse to salt stress (24).

In S. cerevisiae the uptake of K1 and Na1 is mediated by theTrk1-Trk2 transport system, being the Trk1 function predom-inant under normal growth conditions (12, 19, 20, 36). TheTRK system discriminates between Na1 and K1, thus prevent-ing the entry of an excess of Na1 when the levels of the cationin the medium are too high. Therefore, a proper functioning ofthis cation uptake system ought to be important for salt toler-ance, as demonstrated by the observation that trk1 trk2 mutantsare hypersensitive to sodium ions (17, 19). In addition, intra-cellular sequestration of sodium can also be an efficientmethod of improving salt tolerance, and confinement of Na1

in the vacuole has been proposed as a mechanism that reducesthe cytosolic levels of this cation (19). It has been documentedthat the putative Na1-H1 exchanger encoded by the geneNHX1 is involved in the vacuolar compartmentalization ofsodium ions (29, 30).

Therefore, sodium homeostasis in yeast appears to be acomplex process, still poorly understood at the molecular level.Casein kinase 2 (CK-2) has been proposed as an additionalcomponent of this regulatory system. CK-2 is a highly con-served Ser/Thr protein kinase that has also been related to cellpolarity and cell cycle progression (for a recent review, seereference 16). In yeast, CK-2 is an oligomer composed of tworelated catalytic subunits (a and a9), encoded by the genesCKA1 and CKA2 (6, 31), and two regulatory polypeptides (band b9), encoded by the genes CKB1 and CKB2 (5, 37), re-spectively. In order to survive, yeast cells require at least one ofthe catalytic subunits (31). On the contrary, the regulatorysubunits do not appear to be necessary for growth under nor-mal conditions. Interestingly, deletion of either CKB1 or CKB2results in the same phenotype of hypersensitivity to Na1 andLi1 (5). The effect of the mutations is not additive and doesnot affect the tolerance to potassium cations (5).

Our laboratories are interested in the analysis of the role ofprotein phosphorylation in the regulation of salt tolerance in

* Corresponding author. Mailing address: Dept. Bioquımica i Bio-logia Molecular, Facultat de Veterinaria, Ed. V, Universitat Au-tonoma de Barcelona, Bellaterra 08193, Barcelona, Spain. Phone: 34-93-5812182. Fax: 34-93-5812006. E-mail: J.Arino@cc.uab.es.

6456

on April 10, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

yeast cells. Therefore, to gain insight into the mechanism re-sponsible for the role of CK-2 in yeast biology, we undertooka genetic and biochemical study of the effects of the absence ofCkb1 on the different cell processes that affect the sensitivity toNa1 and Li1. Our results indicate that CK-2, in contrast withrecently reported data, does not regulate the influx or the effluxof sodium, thus suggesting that this kinase might be involved inthe regulation of a putative target for sodium toxicity.

MATERIALS AND METHODS

Strains and growth conditions. Escherichia coli NM522 and DH5a were usedas hosts for DNA cloning. Bacterial cells were grown at 37°C in Luria-Bertanimedium containing 50 mg of ampicillin per ml, when needed, for plasmid selec-tion. Yeast cells were grown at 28°C in yeast extract-peptone-dextrose (YPD)medium or, when indicated, in synthetic minimal (SD) or complete minimalmedium (43). The relevant genotypes of the strains described in this work can befound in Table 1.

Recombinant DNA techniques, gene disruptions, and plasmids. E. coli and S.cerevisiae cells were transformed by standard techniques as previously described(8). Restriction reactions, DNA ligations, and other standard recombinant DNAtechniques were carried out as described previously (41). Gene disruptions wereperformed as follows. Disruption of PPZ1 and CNB1 was as described in refer-ence 32. Disruption of CKB1 with the HIS3 marker was made by integration ofplasmid pAPB17 linearized by digestion with EcoRI (5). To disrupt CKB1 withthe marker TRP1, plasmid pAPB17 was digested with XhoI and SacI and theinsert (about 1.0 kbp) was cloned into plasmid pRS304. This plasmid was lin-earized as described above and used to transform yeast cells. Disruption of thegenes HAL1 and HAL3 with the LEU2 marker was performed in manners similarto those described in references 14 and 11, respectively.

To achieve high levels of expression of Hal1 and Hal2, the corresponding openreading frames were cloned into high-copy-number vectors carrying the PMA1promoter, as previously described (26, 39).

b-Galactosidase measurements. To evaluate the effect of the ckb1 mutationon ENA1 expression, wild-type DBY746 and EDN1 (ckb1D) cells were trans-formed with the multicopy plasmid pKC201 (1, 7), which contains ENA1 se-quences from 21385 to 135 (relative to the starting initiating Met), fused tolacZ. Cells (5 ml) were grown to an optical density at 660 nm of 0.5 to 1.0, solidNaCl was added to achieve a final concentration of 0.75 M, and growth wasresumed for 60 min. Cells were then centrifuged, and b-galactosidase activity wasmeasured as described in reference 38.

Determination of cation influx and efflux. For influx experiments, cells grownin SD medium were potassium starved by incubation in the minimal ammonium-phosphate medium (35). After 5 h, cells were harvested and resuspended inbuffer containing RbCl or LiCl (50 mM). Samples were taken at regular timeintervals, filtered immediately, and treated for determination of intracellular ioncontent.

For determination of efflux rate, cells were grown in SD medium up to opticaldensity at 660 nm of 0.3 to 0.4 and then LiCl or NaCl (100 mM) was added.Growth was resumed for 3 h in order to load the cells with the cation. After thistime, cells were harvested and resuspended in buffer {10 mM MES [2-(N-morpholino)ethanesulfonic acid] brought to pH 5.8 with Ca(OH)2 and contain-ing 0.1 mM MgCl2 and 2% glucose}, supplemented with 10 mM KCl to trigger

the efflux process. Samples were taken at regular time intervals, filtered, andtreated for determination of intracellular ion content.

The intracellular ion content of the cells was determined as previously de-scribed (34, 36). Briefly, samples of cells were filtered, washed with 20 mMMgCl2, and treated with acid and the cations were analyzed by atomic absorptionspectrophotometry.

Other methods. Salt sensitivity assays were performed with freshly preparedYPD plates containing different concentrations of the compound (drop tests) orwith liquid cultures as described in reference 32. Measurement of proton fluxeswere performed as described previously (2), except that cells were grown in YPDmedium. Differential extraction of potassium and sodium ions from the cyto-plasm and vacuole was essentially achieved as previously described (10), withminor modifications, including a treatment of the cells with 0.1 mg of digitoninper ml for 5 min.

RESULTS

CK-2 regulates sodium tolerance by a mechanism indepen-dent from that of calcineurin and Ppz1. It has been reportedthat deletion of CKB1 results in a phenotype of sensitivity tosodium and lithium ions (5). To evaluate the potency of thisphenotype, we deleted the CKB1 gene in the DBY746 back-ground and compared the sensitivities to sodium and lithium ofthe ckb1D mutant with those of cells lacking other genes knownto be involved in salt sensitivity, such as the regulatory subunitof calcineurin (CNB1), HAL1, and HAL3. Strains with muta-tions in the HAL1 gene displayed a very weak salt sensitivityphenotype. Deletion of CKB1 resulted in a phenotype that wasstronger than that of calcineurin mutants and almost as strongas that of cells lacking HAL3 (not shown). Dose-responseexperiments performed with SD liquid cultures showed thatthe tolerance to lithium ions of a ckb1D mutant was reduced byabout 30% compared to the tolerance of the wild-type strain(50% inhibitory concentration 18 mM versus 26 mM).

Because CKB1 encodes a regulatory subunit of a proteinkinase, we considered it interesting to test the possibility ofgenetic interaction between this gene and the pathways definedby the calcineurin and Ppz1 phosphatase genes. To this end, wedisrupted the CKB1 gene in cells lacking CNB1, the geneencoding the regulatory subunit of calcineurin, and tested thesensitivity of these cells to Li1. As shown in Fig. 1, lack of Ckb1resulted in an additional increase in sensitivity to lithium cat-ions, indicating that the kinase and the phosphatase do notshare a common regulatory pathway.

Disruption of the protein phosphatase Ppz1 resulted in in-creased salt tolerance. As shown in Fig. 2, the absence of Ckb1decreased the tolerance of a ppzlD strain, as would be expected

TABLE 1. Yeast strains used in this work

Strain Relevant genotype Source or reference

DBY746 MATa ura3-52 leu2-3,112 his3-D1 trp1-D239 A. Rodrıguez-NavarroJA30 DBY746 ppz1::ura3 32JA40 DBY746 cnb1::HIS3 32RH16.6 DBY746 ena1–ena4::LEU2 18EDN1 DBY746 ckb1::TRP1 This workEDN3 DBY746 ckb1::HIS3 This workEDN4 DBY746 hal3::LEU2 This workEDN6 DBY746 hal1::LEU2 This workEDN11 DBY746 ppz1::ura3 ckb1::TRP1 This workEDN21 DBY746 hal3::LEU2 ckb1::TRP1 This workEDN22 DBY746 cnb1::HIS3 ckb1::TRP1 This workEDN25 RH16.6 ckb1::HIS3 This workW303.1A MATa ade-2-2 his3-11,15 leu2-3,112 ura3-1 trp1-1W59 W301.1A trk1::LEU2 TRK2 22WD3 W301.1A trk1::LEU2 trk2::HIS3 22EDN42 W301.1A ckb1::TRP1 This workEDN44 WD3 ckb1::TRP1 This work

VOL. 181, 1999 ROLE OF CASEIN KINASE 2 IN SALT TOLERANCE 6457

on April 10, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

if Ckb1 and Ppz1 regulate independent pathways. Hal3 hasbeen defined as a regulatory subunit of Ppz1, thus placing Ppz1and Hal3 in the same regulatory pathway. To confirm ourobservation, we generated a ckb1 hal3 double mutant andanalyzed its sensitivity to lithium ions. As shown in Fig. 2, theckb1 hal3 double mutant was more sensitive than a single hal3or ckb1 deletion mutant.

Both calcineurin and Ppz1 are known to affect sodium tol-erance by regulating the expression of ENA1, a gene encodingthe Na1-ATPase which represents the major mechanism forNa1 efflux in budding yeast. HAL1 had been defined as a genethat, when it is expressed in multicopy numbers, was able toincrease ENA1 expression. We considered that if Ckb1 wasplaced downstream of Hal1, high levels of Hal1 would notconfer sodium tolerance to the mutant. However, as shown inFig. 3, high-copy-number expression of HAL1 clearly increasedthe tolerance of a ckb1D strain, indicating that the effect ofHal1 is independent of the presence of Ckb1.

Analysis of the sodium efflux mechanisms in a ckb1 mutant.Because of the relatively strong phenotype of the ckb1 muta-tion, we decided to explore in a systematic way the possibleeffect of Ckb1 on the expression of ENA1 and, therefore, onsodium efflux. To this end, we disrupted the CKB1 gene in anRH16.6 strain that lacks the ENA1-ENA4 gene cluster. Thisstrain has a very reduced sodium efflux, and therefore it ishighly sensitive to Li1 and Na1. Interestingly, the deletion ofCKB1 further increased the sensitivity to Li1 of the ena1-ena4mutant (Fig. 4A). This result was somewhat unexpected be-cause it indicated that, in contrast to preliminary publisheddata (16), the function of CK-2 does not involve the Ena1ATPase. To confirm this possibility, we transformed wild-typeand ckb1 strains with plasmid pKC201, which bears the entireENA1 promoter fused to b-galactosidase. The cells werestressed with 0.75 M NaCl for 1 h, and the b-galactosidaseactivity was measured. As shown in Fig. 4B, cells lacking Ckb1showed a response essentially identical to that of wild-type

FIG. 1. Additive effects of the calcineurin and ckb1 mutations. StrainsDBY746 (wild type [wt]), JA40 (cnb1), EDN1 (ckb1), and EDN22 (cnb1 ckb1)were plated on YPD plates containing the indicated concentrations of LiCl.Plates were incubated at 28°C, and growth was scored after 2 days.

FIG. 2. The effect of CK-2 on salt tolerance is not mediated by the Hal3/Ppz1 pathway. (A) YPD medium containing the indicated concentrations of LiCl wasinoculated (initial A660, 0.007) with wild-type strain DBY746 (F) or its derivatives EDN1 (ckb1) (ƒ), EDN4 (hal3) (■), and EDN22 (ckb1 hal3) ({). Cultures weregrown for 18 h, and the densities of the cultures were then measured. Relative growth was calculated as the ratio between growth in the presence and growth in theabsence of added salts and expressed as a percentage. (B) Cultures of DBY746 (F), JA30 (ppz1) (�), and EDN11 (ppz1 ckb1) (■) cells were grown as indicated above.Data are means 6 standard errors of the means of results from four independent experiments.

FIG. 3. High-copy-number expression of HAL1 increases salt tolerance in ackb1 background. Strains DBY746 (CKB1) and EDN1 (ckb1) were transformedwith the high-copy-number plasmid pRS699-HAL1 (denoted YEpHAL1) (1) orthe empty plasmid YEplac195 (2). Positive cultures were plated on YPD platescontaining the indicated concentrations of LiCl, and growth was monitored asdescribed in the legend to Fig. 1.

6458 DE NADAL ET AL. J. BACTERIOL.

on April 10, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

cells whereas, under the same conditions, hal3 and ppz1 mu-tants displayed decreased (hal3) and increased (ppz1) re-sponses, respectively, as previously described (11, 32). There-fore, our data did not support the notion that CK-2 is aneffector of ENA1 transcription and suggested that cation effluxmight not be affected in Cbk1-deficient yeast cells. This possi-bility was directly tested by loading wild-type, cnb1D, andckb1D cells with lithium and measuring the efflux of this cation.As shown in Fig. 5, whereas the cation efflux of calcineurin

mutants was reduced (as previously described), the efflux of theckb1D strain was essentially identical to that of wild-type cells.Therefore, from our data it can be concluded that the in-creased sensitivity of the ckb1 mutant to sodium and lithiumcannot be attributed to a reduced efflux of these cations.

Mutation of CKB1 does not alter sodium or potassium in-flux. Changes in the influx of sodium and potassium, mediatedby the TRK system, can be responsible for salt sensitivity phe-notypes. To test the possibility that CK-2 affects the TRKsystem, we introduced the ckb1 deletion in a strain lackingTRK1 and TRK2. When this strain was tested for sodium sen-sitivity, we observed that it was more sensitive than the trkdouble mutant (Fig. 6). This result supported the notion that

FIG. 4. The ckb1 mutation is additive to those of ENA1 to ENA4 and does not alter the expression of the ATPase. (A) Strains DBY746 (F), RH16.6 (ena1 to ena4)(E), and EDN25 (ena1 to ena4 ckb1) (�) were tested for LiCl sensitivity in liquid cultures as described for Fig. 2. Data are means 6 standard errors of the means ofresults from four independent experiments. (B) DBY746 (wild type [wt]), EDN1 (ckb1), EDN4 (hal3), and JA30 (ppz1) were transformed with the multicopy plasmidpKC201, which allows expression of the b-galactosidase protein from the ENA1 promoter. Cells were grown as described in Materials and Methods, and b-galactosidaseactivity was measured in permeabilized cells treated with (1) or without (2) 0.75 M NaCl for 60 min. Data are means 6 standard errors of the means of results from16 to 18 independent experiments performed with five independent clones (DBY746 and EDN1) or eight independent experiments performed with four independentclones (EDN4 and JA30).

FIG. 5. Measurement of the efflux of lithium cations in ckb1 cells. Wild-typeDBY746 (F), as well as EDN1 (ckb1) (E) and JA40 (cnb1) (�), cells were loadedwith LiCl for 3 h and washed, and the efflux of Li1 was monitored as describedin Materials and Methods. Data are means 6 standard errors of the means ofresults from three independent experiments.

FIG. 6. The deletion of CKB1 increases the salt sensitivity of a trk strain. TheCKB1 gene was disrupted in the wild-type (wt) strain W303.1A and in its isogenicstrain WD3 (trk1 trk2) to yield EDN42 and EDN44, respectively. The sensitivitiesto NaCl of these strains were tested on plates as described in the legend to Fig.1.

VOL. 181, 1999 ROLE OF CASEIN KINASE 2 IN SALT TOLERANCE 6459

on April 10, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

CK-2 is required for normal salt resistance in trk1 trk2 cells andsuggests that the TRK system is not regulated by CK-2. In fact,we have measured Li1 and Rb1 influx (the latter being used asan analog of K1 for transport experiments) in wild-type andckb1 cells (Fig. 7). The time course of the uptake of thesecations showed that the initial velocities of influx were virtuallyidentical in both strains but that, as expected, it was dramati-cally reduced in a trk1 mutant, which is defective in high-affinity potassium transport. Consequently, the salt sensitivityphenotype of the ckb1 mutant cannot be attributed to changesin the influx of these cations. Because changes in proton effluxcan affect salt tolerance, we determined this parameter in wild-type and ckb1 cells, obtaining values of 15 6 2 and 13 6 1.5nmol of H1/mg (dry weight) of cells. Therefore, our resultsindicate that the ckb1 mutation does not modify proton pump-ing.

The data presented so far indicate that the mutation ofCKB1 does not alter the normal influx and efflux of Na1 andK1. Consistently with this evidence, we have observed that,after the cells were challenged with a range of NaCl concen-trations (from 0.25 to 1 M), the intracellular contents of Na1

and K1, as well as the Na1/K1 ratio, were virtually identical inwild-type cells and ckb1 mutants (data not shown). Finally, wehave examined the possibility that CK-2 is somehow involvedin the process of sequestration of sodium into the vacuole. Tothis end, we measured the cytoplasmic and vacuolar contentsfor Na1 and K1, before and after 6 h of incubation of the cellswith 1 M NaCl. As shown in Fig. 8, the intracellular distribu-tions of both cations were very much alike in wild-type andckb1 cells.

The fact that mutation of Ckb1 affects Na1 and Li1 toler-ance in the absence of increased levels of these cations drewour attention to the HAL2 (also called MET22) gene, which

codes for an Na1- and Li1-sensitive phosphohydrolase identi-fied as a putative target for the toxicities of these cations.Overexpression of HAL2 in a wild-type background resulted ina relatively weak increase in salt tolerance. As shown in Fig. 9,high levels of Hal2 also increase the tolerance of a ckb1 strain,indicating that the regulatory subunit of CK-2 does not medi-ate Hal2 function.

DISCUSSION

The finding that the mutation of the regulatory subunits ofCK-2 (CKB1 and CKB2) results in a phenotype of sensitivity tosodium and lithium ions (5) raised the key question of whattype of cellular process, relevant for cation tolerance, involvesthis kinase. Because of the equivalence in potency and the lackof an additive phenotype, the disruption of only one gene,CKB1, was chosen as a working model. Our data indicated thatthe potency of the mutation is relatively strong, thus suggestingthat this cellular process is highly relevant for salt tolerance. InS. cerevisiae, a key factor for salt tolerance is the proper func-tion of the major determinant for sodium efflux, the Ena1Na1-ATPase (13, 19, 47). In addition, the expression of theENA1 gene is regulated by mechanisms involving phospho-dephosphorylation reactions. Therefore, it was reasonable toassume that the function of CK-2 is related to that of the Hal3

FIG. 8. Cytoplasmic and vacuolar distributions of sodium and potassium ionsin wild-type and ckb1 yeast cells. Wild-type (filled bars) and EDN1 (ckb1) (openbars) cells were grown on YPD medium and incubated for 6 h with or without 1M NaCl. The contents of Na1 and K1 in the cytoplasm (Cit.) and the vacuole(Vac.) were determined as described in Materials and Methods. Data aremeans 6 standard errors of the means of results from three experiments.

FIG. 7. Influx of Li1 and Rb1 in a ckb1 strain. Potassium-starved wild-typeW303.1A (F) and EDN1 (ckb1) (E) were incubated with RbCl (upper panel) orLiCl (lower panel), and the influxes of these cations were determined as de-scribed in Materials and Methods. Data from strain W59 (trk1) (�), which isknown to have a decreased potassium transport, is included for comparison.Data are means 6 standard errors of the means of results from four independentexperiments.

6460 DE NADAL ET AL. J. BACTERIOL.

on April 10, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

and Ppz1 (8) or the calcineurin (25) regulatory system. How-ever, our data clearly show that CK-2 participates in a mech-anism that is independent from the mentioned phosphatases.In addition, we demonstrate that the function of Hal1, which,when expressed at high levels, results in increased ENA1 ex-pression (39), does not require Ckb1.

While the above-mentioned results are still compatible withthe notion that CK-2 regulates a novel Ena1-regulatory path-way, we provide here biochemical and genetic data demon-strating that the function of Ckb1 in salt tolerance does notinvolve Ena1 and that the expression of the ENA1 ATPasegene is not altered by the absence of Ckb1. These conclusionsare in sharp contrast with recently published data suggestingthat the kinase regulates the transcription of the ENA1 gene(45). Tenny and Glover (45) derived their conclusions essen-tially from experiments with a b-galactosidase reporter system(similar to what is shown in our Fig. 4B). Although, at thismoment, we cannot account for these contradictory results, itis worth noting that the experimental conditions differed in anumber of circumstances, including strain background andconcentration (0.4 instead 0.75 M) and time of exposure toNaCl (30 min instead 60 min). However, we performed b-ga-lactosidase experiments after stressing the cells for differenttimes with 0.4 M NaCl and still could not find differencesbetween wild-type and ckb1 strains. As an additional proof forthe involvement of Ena1 in the mechanism of action of thekinase, Tenny and Glover invoked the fact that the overexpres-sion of ENA1 suppresses the salt sensitivity of the ckb1 mu-tants. However, it seems evident that overexpression of Ena1would result in active extrusion of sodium and lithium cationsand, probably, sequestration in intracellular compartments (4).This overexpression would also alleviate the salt sensitivityphenotype of an Ena1-independent mutation, simply by reduc-ing the cytosolic amount of the cations. Furthermore, our anal-ysis of cation efflux clearly shows that the output of sodium orlithium ions is not modified by the absence of Ckb1. In con-trast, and consistently with reported data (25), a cnb1 mutant(which has a weaker salt sensitivity phenotype), shows a clear-cut decrease in efflux rate. This decrease can be considered

further evidence against an involvement of ENA1 in CK-2function. We feel that our conclusions are further strength-ened by the fact that they are sustained by a combination ofboth genetic and biochemical evidence. A consequence of theindependence of Ckb1 and Ena1 is that the function of CK-2would also be independent from that of the SOP1 and SOP2gene products, which have been shown to require ENA1 forfunction (21). In addition, from our efflux data, one mightexpect that the absence of Ckb1 would not affect the functionof the Nha1 antiporter, a protein that under specific circum-stances also plays a role in sodium efflux (3, 33, 44).

A very relevant finding regarding the role of CK-2 in salttolerance is that the absence of Ckb1 does not increase theintracellular sodium content or alter the intracellular Na1/K1

ratio. These facts are in agreement with our findings that thelack of Ckb1 has very little effect on Li1 and Rb1 uptake andthat the ckb1 mutation shows, as far as sodium tolerance isconcerned, an additive effect on the trk1 trk2 mutation. Ourdata also rule out the possibility that the absence of Ckb1 altersthe ability of the cell to reduce the cytoplasmic levels of sodiumcations through vacuolar sequestration.

Therefore, our findings define a scenario in which ckb1 cellsare substantially more sensitive to sodium and lithium thanwild-type cells in the absence of an increased intracellularcation content. A reasonable hypothesis would be that theabsence of Ckb1 results in increased sensitivity to sodium andlithium cations of an important component of the cellularmachinery. This component would be a direct or an indirecttarget for CK-2 phosphorylation, being the dephosphorylatedsalt-sensitive protein and, therefore, a cellular target for salttoxicity.

So far, only Hal2/Met22 has been characterized throughgenetic and biochemical methods as a target for lithium toxic-ity (15, 26, 27). Hal2 degrades adenosine 39, 59-bisphosphate(pAp) and 39-phosphoadenosine, 59-phosphosulphate (pApS).These compounds are intermediates of the sulfate assimilationpathway, which is needed mainly for the synthesis of sulfur-containing amino acids (see references 46 for a review). Over-expression of HAL2 increases Li1 tolerance because this en-zyme is inhibited by Li1, and pApS is highly toxic for yeast.While at this moment we cannot rule out the possibility thatthe salt sensitivity phenotype of ckb1 mutants is related toalterations in the sulfate uptake pathway, several lines of rea-soning suggest that this does not occur through the regulationof Hal2. For instance, Hal2 does not appear to contain con-sensus sequences for CK-2 phosphorylation. We show herethat overexpression of HAL2 still increases Li1 tolerance evenin the absence of Ckb1 and that the ckb1 mutant has a ratherstrong phenotype but that the hal2 deletion has almost noeffect on salt tolerance (15). Finally, hal2D mutants display anauxotrophy for methionine (15), presumably because Met sup-plementation greatly reduces the need for sulfate intake and,hence, pAp and pApS formation, whereas we have found thatckb1 mutants grow well in the absence of Met (data notshown). It has been recently suggested that RNA processingmight be a target function for lithium toxicity and that thiswould be the result of the existence of several Li1-sensitivecomponents displaying synergistic toxicity (9). Certain compo-nents would be inhibited by an excess of pAp or pApS (as aresult of Hal2 inhibition), whereas others, such as the RNaseMRP ribonucleoprotein, would be directly inhibited by lithiumions. A conceivable hypothesis would be that one of the lattercomponents is phosphorylated by CK-2 and that the absence ofCkb1 would yield a dephosphorylated protein, hypersensitiveto Li1 and Na1 ions.

FIG. 9. Overexpression of HAL2 increases the Li1 tolerance of a ckb1 strain.Wild-type DBY746 (circles) and EDN1 (ckb1) (triangles) cells were transformedwith plasmid pRS699-HAL2 (open symbols) or the empty plasmid YEplac185(filled symbols), and their sensitivities to LiCl were measured in liquid culturesas described for Fig. 2. Data are means 6 standard errors of the means of resultsfrom four independent experiments.

VOL. 181, 1999 ROLE OF CASEIN KINASE 2 IN SALT TOLERANCE 6461

on April 10, 2021 by guest

http://jb.asm.org/

Dow

nloaded from

ACKNOWLEDGMENTS

We thank C. V. Glover for the CKB1 disruption cassettes, A. Ro-drıguez-Navarro for the RH16.6 strain, R. Haro for the W59 and WD3strains, and R. Serrano for the HAL1 and HAL2 plasmids. The skillfultechnical help of Anna Vilalta and Mireia Zaguirre is acknowledged.

This work was supported by grants PB95-0663 and PB95-0976 fromthe Direccion General de Investigacion Cientıfica y Tecnica, Spain, toJ.A. and J.R., respectively; by an Ajut de Suport als Grups de Recercade Catalunya (SGR97-127) from the Generalitat de Catalunya to J.A.;and by grant BIO4-CT97-2210 from the European Union toJ.R. E.d.N. is the recipient of a predoctoral fellowship from the Min-isterio de Educacion y Cultura, Spain.

REFERENCES1. Alepuz, P. M., K. W. Cunningham, and F. Estruch. 1997. Glucose repression

affects ion homeostasis in yeast through the regulation of the stress-activatedENA1 gene. Mol. Microbiol. 26:91–98.

2. Balcells, L., R. Martın, M. C. Ruiz, N. Gomez, J. Ramos, and J. Arino. 1998.The Pzh1 protein phosphatase and the Spm1 protein kinase are involved inthe regulation of the plasma membrane H1-ATPase in fission yeast. FEBSLett. 435:241–244.

3. Banuelos, M. A., H. Sychrova, C. Bleykasten-Grosshans, J. L. Souciet, andS. Potier. 1998. The Nha1 antiporter of Saccharomyces cerevisiae mediatessodium and potassium efflux. Microbiology 144:2749–2758.

4. Benito, B., F. J. Quintero, and A. Rodrıguez-Navarro. 1997. Overexpressionof the sodium ATPase of Saccharomyces cerevisiae: conditions for phosphor-ylation from ATP and Pi. Biochim. Biophys. Acta 1328:214–226.

5. Bidwai, A. P., J. C. Reed, and C. V. Glover. 1995. Cloning and disruption ofCKB1, the gene encoding the 38-kDa beta subunit of Saccharomyces cerevi-siae casein kinase II (CKII). Deletion of CKII regulatory subunits elicits asalt-sensitive phenotype. J. Biol. Chem. 270:10395–10404.

6. Chen-Wu, J. L., R. Padmanabha, and C. V. Glover. 1988. Isolation, sequenc-ing, and disruption of the CKA1 gene encoding the alpha subunit of yeastcasein kinase II. Mol. Cell. Biol. 8:4981–4990.

7. Cunningham, K. W., and G. R. Fink. 1996. Calcineurin inhibits VCX1-dependent H1/Ca21 exchange and induces Ca21 ATPases in Saccharomycescerevisiae. Mol. Cell. Biol. 16:2226–2237.

8. de Nadal, E., J. Clotet, F. Posas, R. Serrano, N. Gomez, and J. Arino. 1998.The yeast halotolerance determinant Hal3p is an inhibitory subunit of thePpzlp Ser/Thr protein phosphatase. Proc. Natl. Acad. Sci. USA 95:7357–7362.

9. Dichtl, B., A. Stevens, and D. Tollervey. 1997. Lithium toxicity in yeast is dueto the inhibition of RNA processing enzymes. EMBO J. 16:7184–7195.

10. Dunn, T., K. Gable, and T. Beeler. 1994. Regulation of cellular calcium byyeast vacuoles. J. Biol. Chem. 269:7273–7278.

11. Ferrando, A., S. J. Kron, G. Rios, G. R. Fink, and R. Serrano. 1995. Regu-lation of cation transport in Saccharomyces cerevisiae by the salt tolerancegene HAL3. Mol. Cell. Biol. 15:5470–5481.

12. Gaber, R. F., C. A. Styles, and G. R. Fink. 1988. TRK1 encodes a plasmamembrane protein required for high-affinity potassium transport in Saccha-romyces cerevisiae. Mol. Cell. Biol. 8:2848–2859.

13. Garciadeblas, B., F. Rubio, F. J. Quintero, M. A. Banuelos, R. Haro, and A.Rodrıguez-Navarro. 1993. Differential expression of two genes encodingisoforms of the ATPase involved in sodium efflux in Saccharomyces cerevi-siae. Mol. Gen. Genet. 236:363–368.

14. Gaxiola, R., I. F. de Larrinoa, J. M. Villalba, and R. Serrano. 1992. A noveland conserved salt-induced protein is an important determinant of salt tol-erance in yeast. EMBO J. 11:3157–3164.

15. Glaser, H. U., D. Thomas, R. Gaxiola, F. Montrichard, Y. Surdin-Kerjan,and R. Serrano. 1993. Salt tolerance and methionine biosynthesis in Saccha-romyces cerevisiae involve a putative phosphatase gene. EMBO J. 12:3105–3110.

16. Glover, C. V. 1998. On the physiological role of casein kinase II in Saccha-romyces cerevisiae. Prog. Nucleic Acid Res. Mol. Biol. 59:95–133.

17. Gomez, M. J., K. Luyten, and J. Ramos. 1996. The capacity to transportpotassium influences sodium tolerance in Saccharomyces cerevisiae. FEMSMicrobiol. Lett 135:157–160.

18. Haro, R., B. Garciadeblas, and A. Rodrıguez-Navarro. 1991. A novel P-typeATPase from yeast involved in sodium transport. FEBS Lett. 291:189–191.

19. Haro, R., M. A. Banuelos, F. J. Quintero, F. Rubio, and A. Rodrıguez-Navarro. 1993. Genetic basis of sodium exclusion and sodium tolerance inyeast. A model for plants. Physiol. Plant. 89:868–874.

20. Ko, C. H., and R. F. Gaber. 1991. TRK1 and TRK2 encode structurallyrelated K1 transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 11:4266–4273.

21. Larsson, K., F. Bohl, I. Sjostrom, N. Akhtar, D. Strand, B. M. Mechler, R.Grabowski, and L. Adler. 1998. The Saccharomyces cerevisiae SOP1 andSOP2 genes, which act in cation homeostasis, can be functionally substitutedby the Drosophila lethal(2)giant larvae tumor suppressor gene. J. Biol. Chem.273:33610–33618.

22. Madrid, R., M. J. Gomez, J. Ramos, and A. Rodrıguez-Navarro. 1998. Ec-topic potassium uptake in trk1 trk2 mutants of Saccharomyces cerevisiaecorrelates with a highly hyperpolarized membrane potential. J. Biol. Chem.273:14838–14844.

23. Marquez, J. A., and R. Serrano. 1996. Multiple transduction pathways reg-ulate the sodium-extrusion gene PMR2/ENA1 during salt stress in yeast.FEBS Lett. 382:89–92.

24. Mendizabal, I., G. Rıos, J. M. Mulet, R. Serrano, and I. F. de Larrinoa. 1998.Yeast putative transcription factors involved in salt tolerance. FEBS Lett.425:323–328.

25. Mendoza, I., F. Rubio, A. Rodrıguez-Navarro, and J. M. Pardo. 1994. Theprotein phosphatase calcineurin is essential for NaCl tolerance of Saccha-romyces cerevisiae. J. Biol. Chem. 269:8792–8796.

26. Murguıa, J. R., J. M. Belles, and R. Serrano. 1995. A salt-sensitive 39(29),59-bisphosphate nucleotidase involved in sulfate activation. Science 267:232–234.

27. Murguıa, J. R., J. M. Belles, and R. Serrano. 1996. The yeast HAL2 nucle-otidase is an in vivo target of salt toxicity. J. Biol. Chem. 271:29029–29033.

28. Nakamura, T., Y. Liu, D. Hirata, H. Namba, S. Harada, T. Hirokawa, and T.Miyakawa. 1993. Protein phosphatase type 2B (calcineurin)-mediated,FK506-sensitive regulation of intracellular ions in yeast is an importantdeterminant for adaptation to high salt stress conditions. EMBO J. 12:4063–4071.

29. Nass, R., K. W. Cunningham, and R. Rao. 1997. Intracellular sequestrationof sodium by a novel Na1/H exchanger in yeast is enhanced by mutations inthe plasma membrane H1-ATPase. Insights into mechanisms of sodiumtolerance. J. Biol. Chem. 272:26145–26152.

30. Nass, R., and R. Rao. 1998. Novel localization of a Na1/H1 exchanger in alate endosomal compartment of yeast. Implications for vacuole biogenesis.J. Biol. Chem. 273:21054–21060.

31. Padmanabha, R., J. L. Chen-Wu, D. E. Hanna, and C. V. Glover. 1990.Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinaseII is essential for viability in Saccharomyces cerevisiae. Mol. Cell. Biol. 10:4089–4099.

32. Posas, F., M. Camps, and J. Arino. 1995. The PPZ protein phosphatases areimportant determinants of salt tolerance in yeast cells. J. Biol. Chem. 270:13036–13041.

33. Prior, C., S. Potier, J. L. Souciet, and H. Sychrova. 1996. Characterization ofthe NHA1 gene encoding a Na1/H1-antiporter of the yeast Saccharomycescerevisiae. FEBS Lett. 387:89–93.

34. Prista, C., A. Almagro, M. C. Loureiro-Dias, and J. Ramos. 1997. Physio-logical basis for the high salt tolerance of Debaryomyces hansenii. Appl.Environ. Microbiol. 63:4005–4009.

35. Ramos, J., P. Contreras, and A. Rodrıguez-Navarro. 1985. A potassiumtransport mutant of Saccharomyces cerevisiae. Arch. Microbiol. 143:88–93.

36. Ramos, J., R. Alijo, R. Haro, and A. Rodrıguez-Navarro. 1994. TRK2 is nota low-affinity potassium transporter in Saccharomyces cerevisiae. J. Bacteriol.176:249–252.

37. Reed, J. C., A. P. Bidwai, and C. V. Glover. 1994. Cloning and disruption ofCKB2, the gene encoding the 32-kDa regulatory beta9-subunit of Saccharo-myces cerevisiae casein kinase II. J. Biol. Chem. 269:18192–18200.

38. Reynolds, A., V. Lundblad, D. Dorris, and M. Keaveney. 1997. Yeast vectorsand assays for expression of cloned genes, p. 13.6.1–13.6.6. In F. M. Ausubel,R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K.Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons,New York, N.Y.

39. Rıos, G., A. Ferrando, and R. Serrano. 1997. Mechanisms of salt toleranceconferred by overexpression of the HAL1 gene in Saccharomyces cerevisiae.Yeast 13:515–528.

40. Rudolph, H. K., A. Antebi, G. R. Fink, C. M. Buckley, T. E. Dorman, J.LeVitre, L. S. Davidow, J. I. Mao, and D. T. Moir. 1989. The yeast secretorypathway is perturbed by mutations in PMR1, a member of a Ca21 ATPasefamily. Cell 58:133–145.

41. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.

42. Serrano, R. 1996. Salt tolerance in plants and microorganisms: toxicity tar-gets and defense responses. Int. Rev. Cytol. 165:1–52.

43. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Laboratory course manualfor methods in yeast genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

44. Sychrova, H., J. Ramırez, and A. Pena. 1999. Involvement of Nha1 antiporterin regulation of intracellular pH in Saccharomyces cerevisiae. FEMS Micro-biol. Lett. 171:167–172.

45. Tenney, K. A., and C. V. Glover. 1999. Transcriptional regulation of the S.cerevisiae ENA1 gene by casein kinase II. Mol. Cell. Biochem. 191:161–167.

46. Thomas, D., and Y. Surdin-Kerjan. 1997. Metabolism of sulfur amino acidsin S. cerevisiae. Microbiol. Mol. Biol. Rev. 61:503–532.

47. Wieland, J., A. M. Nitsche, J. Strayle, H. Steiner, and H. K. Rudolph. 1995.The PMR2 gene cluster encodes functionally distinct isoforms of a putativeNa1 pump in the yeast plasma membrane. EMBO J. 14:3870–3882.

6462 DE NADAL ET AL. J. BACTERIOL.

on April 10, 2021 by guest

http://jb.asm.org/

Dow

nloaded from