The Protein Phosphatase Calcineurin Is Essential for NaCl Tolerance ...

THEJOLIRNAL UF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 12, Issue of March 25, pp. 8792-8796, 1994 Printed in U.S.A.

The Protein Phosphatase CalcineurinIs Essential for NaCl Tolerance of Saccharomyces cerevisiae* (Received for publication, October 13, 1993, and in revised form, January 3, 1994)

Imelda MendozaS, Francisco Rubioli, Alonso Rodriguez-Navarroli, and Jose M. PardoSn From the SInstituto de Recursos Naturales y Agrobiologia, Consejo Superior de Investigaciones Cientificas, Apdo 1052, Seuilla-41089, Spain and $Departamento de Microbiologia, Escuela Tecnica Superior de Ingenieros Agronomos, Madrid-28040, Spain

NaC1-sensitive yeast mutants were isolated to identify genes essential for NaCl tolerance. Complementationof a mutant highly sensitive to Na' and Li' led to the isolation of the CNBl gene. This gene encodesthe regulatorysubunit(CNB)of the Ca2'/calmodulin-dependent protein phosphatase calcineurin. Cells deficient in CNB accumulated Li' due to reduced expression of ENAl, a gene encoding a P-type ATPase involved inNa' and Li' efflux. In addition, the K+ transport system of c n b l A cells was not converted to the high affinity state that facilitates better discriminationof K' over Na'. Thusthe cnblA strain resembled at r k l mutant. Theseresults indicate that adaptationto NaCl stress in Saccharomyces cerevisiae requires a signal transduction pathway involving Ca2+ and protein phosphorylation-dephosphorylat.ion.In this pathway, calcineurin would coordinate gene expression and activity of ion transporters to facilitate ion homeostasis.

(CNA),' -60-68 kDa) and a regulatory subunit (calcineurin B (CNB), -19 kDa). The regulatory CNB subunit binds up to four molecules of free Ca2+, stimulating the protein phosphatase activity of t h e CNA.CNB complex that is inactive in the absence of Ca". CNA can also bind Ca2+/calmodulin complexes, thus yielding a fully active trimeric enzyme. The physiological function of calcineurin is becoming clearer in different organisms and cell types. The immunosuppressants cyclosporin A a n d FK506, when associated with the respective binding proteins, inhibit calcineurin in lymphocytes. This inhibition results in both impaired gene expression of interleukin-2 and failure of T-cell activation, indicating that calcineurin is an essential component of the T-cell receptor signal transduction pathway (8, 9). Calcineurin is required for inactivation of Ca2+ channels in mammalian brain cells and of S. cerevisiae cells K+ channels in plant guard cells (10, 11). deprived of CNA and/or CNB by null mutations fail to recover from GI arrest in the presence of a-mating factor (12, 13). However, no otherdefect could be discerned in generation time, carbon source utilization, heat- or cold-sensitivity, mating, and In the yeast Saccharomyces cereuisiae, Na+ homeostasis is sporulation. In this paper we report that calcineurin is required achieved by the coordinate regulation of plasma membrane for NaCl andLiCl tolerance in yeast cells(Li' is a toxic analog influx and efflux systems. Na' and other alkali cations enter of Na'). A mutant lackinga functional regulatory CNB subunit t h e cell through the K' uptake system anddown their respecaccumulated abnormally high levels ofLi'. This phenotype is tive electrochemical gradients (1).Under Na+ stress, the K' the resultof reduced expressionof t h e ENAl gene anda failure a high affinity mode of K+ of the K+ transport system to convert to the high affinity state. uptake system is converted into transport that results in higher K+/Na+ discrimination, thereby reducing the influx of Na' (2). The expression of this high MATERIALS AND METHODS affinity state depends onTRKl, a gene encodinga putative K' Genetic andRecombinantDNA Methods-S. cerevisiae GRF167 transporter (3). Na+ efflux is mediated by the P-type ATPase (MATa ura3 his3)and DBY746 (MATa ura3 leu2 his3t r p l ) were used (4).The as wild-type strains for NaCl tolerance and ion fluxes. YP5-lb (MATa encoded by ENAl, an essential gene for NaCl tolerance expression of ENAl is induced by Li', Na', or by alkaline pH. ura3 his3 leu2)and W3-5b (MATa ura3 leu2c n b l ) were derived from GRF167 and the mutant SS2 (MATa ura3 his3 cnbl), respectively. Little is known about the signal transduction pathway leading to the induction of ENAl a n d t h econversion tothe high affinity RH16.6 is a derivative of DBY746 that carries a disruption in the ENA tandem array (enalh::LEU2::ena4h).RHW2l (MATa ura3 leu2 trpl mode of K' transport. However, recent reports implicate prohis3 ude2 enalA::LEU2::ena4h) was obtained from a crossbetween tein phosphorylation in NaCl adaptation and osmotic adjustW303-1A (MATa uru3 leu2 trpl his3 ade2can1 and RH16.6. TE12 is ment in yeast cells(5, 6). a strain disrupted for TRKl and ENA genes (2). Standard procedures Among the four types of protein phosphatasesthat have been were followed for growth and genetic manipulations of yeast (14, 15). described, calcineurin (PP2B-type) is unique that in it requires The pH was adjusted to 7.0 with NaOH to allow agar solidification in Ca2+ and calmodulin for activity (7). Calcineurin holoenzyme is medium containing 0.7 M NaCl. EMS mutagenesis of GRF167 cells wasas described (16).There was a heterodimer consisting of a catalytic subunit (calcineurin A 60% survival after EMS treatment. The cnblhl::HlS3 disruption was achieved by replacement of the 544-base pair BstXI fragment in CNBl * This work was supported by grants PB89-0180 from Direccion Gen- with a 1.3-kb BamHI-XhoI fragment containing HIS3. A 2.1-kb fragment containing cnblhl::HIS3 was excised with EcoRV (sites flanking eral de Investigacion Cientifica y Tecnica (to A. R.-N.)andAGR91-0858 from Plan Nacional de InvestigacionAgraria (to J. M. P.), The costs of C N B l ) and electroporated (17) into the indicated strains to produce publication of this article were defrayedin part by the payment of page cnblAl::HIS3 null mutants. Accurate gene replacement was assessed charges. This article must therefore be hereby marked "advertisement" by Southern analysis (18), using as a probe the 3.25-kb BamHI fragment containing DPH2 and CNBl sequences (see Fig. 3). For Northern in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. blots of ENAl and CNBl transcripts, total RNA was isolated and elecThe nucleotide sequence(s1 reported in this paper hasbeen submitted to the GenBankTMIEMBL Data Bank with accession numbeds) 226521. trophoresed using formaldehyde-agarose gels (19). Probeswereob7l Towhom correspondence should be addressed: Center for Plant Environmental Stress Physiology, 1165 Horticulture Building, Purdue The abbreviations used are: CNA, calcineurin A , CNB, calcineurin University,West Lafayette, IN 47907. Tel.: 317-494-1323; Fax: 317-494B; EMS, ethylmethane sulfonate; kb, kilobase pairs. 039 1.

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Calcineurin and Na' Homeostasis tained from the 1.6-kb XbaI internal ENAl fragment and the 544-base pair BstXI internal CNBl fragment. Hybridization signalsfrom Northern blots were quantifiedby densitometric scanning of several autoradiograms of the same blot, using a Molecular Dynamics densitometer, model 300A. Relative signal intensities were normalizedt o the amount of RNA loaded onto the gels based on reprobing of the blots with theG EcoRI fragment from the radish 18 S rRNA gene (20). ForP-galactosidase analysis, an in-frame ENAl-lac2 fusion was obtained by cloning the SalI-EcoRI fragment from ENAl (-1384 to +40) intoYIp356R (4, 21). The resulting plasmid, pFR7Oi, was linearized with NcoI before electroporation t o direct integration into the ura3 locus. DNA sequences were obtained from double-stranded templates (22). Sequence analysis and comparisons were performed on a VAX computer with theUWGCG software package ( 2 3 ) . Biochemical Procedures-For measurement of ion fluxes, cells were grown in AP medium (8 mbf phosphoric acid, 10 mM L-arginine, 2 mhf MgSO,, 0.2 mM CaCl,, 2% glucose, plus vitamins and trace elements, brought to pH 6.5 with arginine) that was devoid of K' and other alkali cations (24). AP medium was supplemented with KC1 as required, and the concentrationwas as indicatedinparenthesis. Li* uptakewas measured in cells grown overnight in AP ( 3 mM K+), harvested, and resuspended in fresh AP (1mM K+) containing30 mM Li+.At the indicated intervals, samples were harvested by filtration and washed, and the Li' content was analyzed by atomic absorption spectrophotometry (25). For Lit efflux determination, cells were grown in AP ( 3 mM K+) and then transferredt o 20 ml of fresh AP (0.2 mM K*) medium supplemented with 50 mM LiCI. After 30 min of loading, 80 ml ofAP with 62.5mM KC1 were added, togive final concentrationsof 50 mMK' and 10 mM Li'. At the indicated intervals, samplesof cells were removed, and internal Lit contents were determined. Rb' influx into DBY746 or DBY746cnblA cells was calculatedfrom the initial ratesof Rb+ uptake in the presence of varying K' concentrations (24). Intracellular Rb' content was monitored by atomic absorption spectrophotometry. Na+-stressed cells were obtained as described under "Results." K+-starved cells were prepared as described (26). P-galactosidase activity was measured aftercell permeabilization with chloroform and SDS (27).

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RESULTS FIG.1. Growth inhibition of strains GRF167 and SS2 by NaCl, Isolation and Characterization of the NaCl-sensitive Mutant KCI, and LiC1. Liquid YPD medium included the indicated finalconcentrations of NaCl (open symbols, panel A),KC1 (closed symbols, panel SS2-To identify yeast genes required for cellular adaptation A), or LiCl (panel B ) , and the growthof the parental (GRF167, circles) to high salinity stress, a mutational analysis was initiated in and the mutant (SS2, squares) strains recorded were byARoo" ~Growth . the yeast S. cereuisiae. GRF167 cells weremutagenized by in e.xhmedium was normalized to the value obtained inYPD without EMS and screened for inability to grow in solid media supple- added salts.

mented with 0.7 M NaCl (a moderate NaCl shock for yeast; see Fig. lA). With this selection scheme, we identified 11 NaClsensitive mutants among 8100 EMS survivorsscreened. To quantify the sensitivity to NaCl, isolated mutants and theparental strain were compared in liquid YF'D supplemented with increasing amounts of NaCl (not shown). The concentration of NaCl that decreased the growth rate by 50% relative to medium without saltwas estimated, and it referred is hereafter as Is0. The mutant strain SS2 was selected for further characterization becauseof its high sensitivity to NaCI. I,, for strain SS2 was 0.1 M NaCl, about 5-fold lower than the value for the parental strain (I5o = 0.47 M NaCI) (Fig. lA). The NaC1-semitive phenotype of strain SS2 segregated consistently as a recessive mutation in a single nuclear locus (not shown). To further characterizeSS2, its growth wascompared in YF'D supplemented with increasing amounts of KCl, LiC1, or polyethylene glycol. Li' can be used as ananalog of Na' for transport studies as Na+ and Li' influx (l),and efflux (28) are mediated by thesametransportsystemsinyeast. However, because internal Li' is much more toxic than Na', effective growth inhibition is obtained at millimolar concentrations. SS2 had identical growth rates to the wild-type strain inKC1 (Fig. lA)or polyethylene glycol (not shown). On the contrary, SS2 was more sensitive toLiCl than the control strain; I,, were 7.7 these results and 29 mM, respectively (Fig. 1B). Taken together, indicated that SS2 was specifically Na+- andLi+-sensitive but not osmosensitive. This phenotype might be explained by defects in ion fluxes or by a mutation rendering a protein essential for growth more sensitive to those ions. To test these pos-

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Time (rnin) FIG. 2. Time-dependentlithiumaccumulation in strains GRL'167 and 552. GRF167 (circles) and SS2 (squares)cells were incubated inAP medium containing1mMK' and 30 mM Li'. Samples were removed at the indicated times, and the Li' content was determined. Values were normalized t o the dry weight of the sample.

sibilities, we measured the time course of Li' accumulation in the SS2 and parental strains after incubation in AP medium containing 30 mM Li' and 1 mMK' (Fig. 2). The initial rates of Li+ uptake in cells of both strains were similar, but SS2 cells clearly accumulated more Li' than control cells after 30 min. These data indicate a defect in the cellular mechanisms controlling the homeostasis of alkali cations.

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FIG.3. Physical map of the DPHZ and CNBl genes and subclone analysis. Upper part, location of DPH2 and CNBl coding regions (arrows) and relevant restriction sites (B, BgZII; H, BamHI; N , NcoI; S, SalI; X, BstXI). Solid bars indicate the DPH2 and CNBl nucleotide sequences in the data banks (see "Results"). Lower part, fragments subcloned to identify the functional region that complemented the NaClsensitivity of SS2. Bottom line, bracketsindicate aBamHI deletion. Right, phenotypes conferred t o SS2 in solidYNB medium containing 0.7 M NaCl by subcloned fragments. +, tolerant; -, sensitive.

Na' and Li' effluxes in yeast are mediated by the ATPase CNBl gene and that the regulatory subunit of calcineurin is encoded by ENAl(4). ENAl mutants were sensitive to these required for NaCl tolerance. ions and to high external pH (28). The growth of SS2 was also Effect of cnblA on Li' Efflux-Because the cnbl mutation reduced 2.6-fold when compared with the parental strain in mimicked the phenotype of an enal mutation(i.e. sensitivity to medium buffered to pH 8.0. However, a genetic cross between Na',Li', andalkaline pH), we addressedthe question of strains YF'3-5b (a derivative of SS2) and RHWBl(ena1A) re- whether calcineurin was controlling ENAl expression and/or sulted in a NaC1-tolerant diploid strain, indicating that both protein activity. RH16.6 and RH16.6cnblA cells, transformed mutations were not allelic. Furthermore, thegene ENAl cloned with plasmid pGB34 (which contains the ENAl gene cloned into the single-copy plasmid YCp50 (pGB34) (4) did not restore into the single copy plasmid YCpBOJ, were preloaded with Li', growth of SS2 in 0.7 M NaC1. Therefore, the SS2 strain was not and the net emux was recorded. Disruption of CNBl resulted an enal mutant despite the coincidental phenotypes. in a 2-fold reduction in net Li' efflux (0.53 versus 0.97 nmol Zsolation of the CNBl Gene-We undertook the molecular mg min l J(Fig.4). Cells without pGB34 lacked significant cloning of the gene restoring the capacity of SS2 to grow in 0.7 Li' eMux. M NaCl. SS2 cells were electroporated in the presence of a S. Because ENAl transcription is induced in S. cerevisiae by cerevisiae genomic DNA library constructed into the plasmid Li',Na', and, maximally, by alkaline pH (4) and calcineurin YEp24 (19). One NaC1-tolerant clone was isolated out of 7870 has been shown to modulate gene expression by dephosphoURA' transformants. When the plasmid (pST1) in this tolerantrylation of specific transcription factors in lymphocytes (9), we transformant was isolated and transformed into SS2 cells, all determined the effect of a cnblA mutation on ENAl gene exURA' clones were NaC1-tolerant also. The functional region pression. The plasmid pFR70i, containing a ENAl-lac2 fusion, was mapped to a 2.5-kb BamHI-BglII fragment (Fig. 3).When was integrated into the genome of strains GRF167, a 3.25-kb BamHI fragment containing this region was sub- GRF167cnblA, RH16.6, and RH16.6cnblA. As expected, cloned into the single-copy plasmid YCp50, the resulting plas- GRF167(pFR70i) and RH16.6(pFR70i) cells had low constitumid mediated the growth of SS2 cells in medium with 0.7 M tive P-galactosidase activity, which was induced substantially NaCl, indicating that pSTlwas not amulticopy suppressor for by NaCl and alkaline pH (Table I). However, the induced exstrain SS2. pression of the ENAl-lac2fusion was less in cnblA cells than The nucleotide sequence of the complementing 2.5-kb in control cells. BamHI-BglII fragment was obtained from both strands and To directly compare the expression of the ENAl-lac2fusion comparedwithsequences in the GenBank and EMBL data and Li' emux, P-galactosidase activitywasdetermined in bases. The sequence matched two entries in the data bases, RH16.6 and RH16.6cnblA after incubation under similar connamely the genes DPHZ (required for diphtamide biosynthe- ditions to those used for Li' loading. In the presence of 50 mM sis)2 and CNBl (encoding the regulatory CNB subunit of cal- Li', p-galactosidase activity in RH16.6cnblA was half of that cineurin) (13). According to our sequence analysis, DPHZ and detected in RH16.6, in accordance with the 2-fold reduction in CNBl, which were independently deposited in the data banks, Li' efflux (Fig. 4). Consistent withthe result of Garciadeblas et are separated by only 71 base pairs (see Fig. 3). Because the al. (4), incubation of RH16.6(pFR70i) cells in A p medium incomplementing 2.5-kb BamHI-BglII fragment contained only duced the expression of the ENAl-lac2 fusion. However, this 52% of the carboxy terminal DPHZ coding region, it is likely induction was also lower in cells lacking the CNBl gene. that the CNBl gene complemented SS2. A null allele (cnblDifferences in ENAl gene expression between CNBl and A 1 ::HZS3 ) was constructed by replacement of the 544-base pair cnbl strains were assessed by Northern blot (Fig. 5). DensitoBstXI internal CNBl fragment with the HIS3 gene. GRFcnblA metric scanning of autoradiographic signals indicated a %fold cells were sensitive to NaCl, but the I,, was higher (0.17 M reduction in ENAlmRNA abundance in cnblcells over control NaC1) than that of SS2 (0.1 M NaC1). A genetic cross between cells a t pH 8.5,in agreementwith P-galactosidase activity measstrains YP5cnblA and SS2 rendered a NaC1-sensitive diploid urements (Table I). CNBl transcriptlevels did not change unthat, after sporulation,produced 100% NaC1-sensitive haploid der these conditions. segregants of seven tetrads examined. Taken together, these Effect of cnblA on Rb' Influx-Disruption of the CNBl gene results provide strong evidence that strain SS2 has a mutated in strain DBY746 rendered cells more sensitive to Li' than predicted based on the extent of the defect in Li' efflux and a L. C. Mattheakis, F. Sor,and R. J. Collier, accession number ENAl expression and matched the sensitivity of the enal-deficient, isogenic strain RH16 (the 15,, were 0.15 and 0.17 M L01424.

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FIG.5. ENAl and CNBl mRNA levels in CNBl and cnbl cells. GRF167 (CNBl) and SS2 (cnbl)cells were incubated for 1 h in YPD buffered to pH7.0 or 8.5. Total RNA was subjected to Northern blot and hybridized successively to ENAl and CNBl probes. ENAl and CNBl mRNA sizes are 3.8 and0.85 kb, respectively.

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produce similar growth inhibition and Na' loading in both strains. DBY746 and DBY746cnblA cells grown in 150 mM Na' FIG.4. Net lithium efflux from cnblA cells. RH16.6 (circles) and and 50 mM Na', respectively, contained 250 Na+/300 K' nmol RH16.6cnblA (squares) cells, with (closed symbols) or without (open mg". Contents were 325 Na+/250 K+ nmol mg" when symbols) the plasmid pGB34, were loaded with Li+ in AP medium with DBY746 and DBY746cnbIA cells were grown in 300 mM Na' 0.2 mM KC1 and 50 mM LiCI. After 30 min, ion concentrations were brought to 50 mM KC1 and 10 mM LiCl. Samples were removed at the and 65 mM Na+, respectively. In these conditions where the indicated times, and the Li+content of cells was measured. Values were intracellular Na+/K+ratio wasclose to 1,wild-type cells exhibdry weight of the sample. ited the high affinity mode of K+ uptake whereas cnblA failed to do it. At the two Na' levels, the K , for Rb' in DBY746 cells TARIXI was 80 10 PM (and Ki K' of was10 -c 2 VM) and in Expression of a ENAl-Lac2 fusion in cnblA cells DBY746cnbIA cells, 1.6 2 0.2 mM (Ki of K' was 0.2 f 0.02 mM). Strains GRF167, GRF167cnbl A, RH16.6, and RH16.6cnbA carrying DBY746cnblA cells growing in standard AP (3mMK)' medium, a n integrated copy of pFR70i (a ENAl-Lac2 fusion) were used, and in theabsence of Na+, showed wild-type low affinity Rb' influx their relevant genotypes are illustrated (first column). Cells were incubated in the indicated medium (second column)for 30 min before de- kinetics (IC,,* of 6 mM and Ki of K' of 3 mM). Therefore, calfor the transition of the K' termination of 6-galactosidase activity (third column). P-Galactosidase cineurinactivitywasrequired units were obtained from three or four experiments. The data are pre- transport system from the low affinity to the high affinity state sented as the mean f S.E., and n indicates the number of samples in response to Na' stress. Thehigh affinitymode for K' uptake measured. 6-Galactosidase units are normalized per cell number (27). in yeast resultsfrom exposing cells to Na+ (29),or K+-depleted Genotype Medium 6-Galactosidase medium (24) andcalcineurin might be a common entity in this transition. However, experiments measuringRb' influx in K+ENAl CNBl YPD, pH 7.0 0.9 * 0.7 ( n = 7 ) 11.0 f 4.2 ( n = 7 ) YPD + 0.4 M NaCl starved DBY746 and DBY746cnblA cells indicated that cal15.5 f 2.7 ( n = 7 ) YPD, pH 8.5 cineurin was not involved in the transition to the high affinity ENAl cnbl YPD, pH 7.0 0.5 f 0.2 ( n = 7 ) mode of K' transport that ismediated by K+ starvation. 4.3 f 1.0 ( n = 7 ) YPD + 0.4 M NaCl The effect of a cnbl disruption in thestrain TE12 9.1 f 0.7 (n= 7) YPD, pH 8.5 enal CNBl 20.6 f 4.4 ( n = 9) AP (3 mM K') (enalAtrhlA) was investigated. TE12 and TEcnblA cells were AP + 50 mM Li* 21.2 f 5.2 ( n = l l ) indistinguishable in their sensitivity to Na' and Li' in either enal cnbl 5.6 f 2.2 (n= 9) AP (3 mM K*) YPD or AP media. Therefore, no additional mechanisms by 8.4 f 1.2 ( n = l l ) AP + 50 mM Li' which cnbl contributes toNa' sensitivity could be observed.

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NaCI, respectively). Furthermore, disruptionof CNBl in strain RH16.6 decreased significantly the Na' tolerance in low K' medium. In AP (0.5 mM K') RH16 cells showed an Iso of 54 mM NaCl whereas that of RH16cnblA was 21 mM. Because both strains lack any mechanism for Na+ emux, this result suggested an increased Na+ uptake in cnbl cells. In addition to Na' e m u , discrimination between K' and Na' influx is an important determinant of Na' tolerance (2). In yeast cells uptake of K', Na', and Li' is through the same transport system (1).The K , values for Na', Li', and K' influx vary depending on the growth conditions. Under Na', but not Li' stress, the transport system changes to a high affinity K' state that has little difference in theaffinity for Na' or for Li', thereby allowing a better discrimination between K' and Na' (2). To test theeffect of the CNBl disruptionon K+/Na+selectivity, DBY746 and DBY746cnblA were grown in AJ? medium supplemented with 0.5 mMK' and different concentrations of Na'. Several concentrations of Na' were selected, in pairs, to

DISCUSSION

Complementation of the highly NaC1-sensitive mutant SS2 led to the isolation of the CNBl gene, encoding the regulatory B subunit of calcineurin. We also demonstrated that SS2 was indeed a cnbl mutant.Because CNBl is theonly gene encoding the regulatory subunit of calcineurin and the catalytic CNA subunit requires the regulatory CNB subunit for function, a cnbl mutantshould not have calcineurin activity. In support of this assumption, Cyert and Thorner (13) have shown that a cnblA mutant is functionally equivalent to null mutations in both CNAl and CNA2, the genes encoding the two isoforms of the catalytic subunit in S. cerevisiae. However, we observed that the cnbl mutation carriedby the strain SS2 produced a more severe phenotypethan thecomplete absence of the CNBl protein in the otherwise isogenic cnblA strain. This finding could be explained by a basal, CNB-independent activity of the catalytic subunits as hasbeen described for the CNA subunits of Neurospora crassa (30). This basal activity wouldbe im-

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paired by the CNB subunit present in the strainSS2. rylation-dependent inactivation of the Ca2+channel in neurons Cells lacking a functional calcineurin had a 2-fold reduced (10). rate of net Li' efflux, a process mediated by the ENAl gene, Other genes involved in protein phosphorylation are also because its disruption abolished Li+ efflux (Fig. 4). The tran- required for NaCl toleranceof S. cereuisiae. Increased dosage of scription of the ENAl gene is induced by Na+, Li', and alkaline YCKl or YCK2, genes encoding casein kinase I homologues, pH in wild-type S. cerevisiae cells (28). AcnblA strain contain- improved tolerance to extreme salinity (5). The disruption of ing anENAl-lacZ fusion expressed only half of the P-galacto- the YCRlOl gene, encoding a putative protein kinase, resulted sidase activity detected in control CNBl cells after induction in Na+sensitivity (32).However, further characterizationof the with NaCl or alkaline pH. These data were consistent with physiological determinants of these phenotypes was not dethose obtained by quantitation of ENAl autoradiographic sig- scribed. The identification of protein kinases andcalcineurin as nals from Northern blots (Fig.5). Furthermore, theinduction of important determinants in NaCl tolerance implicates protein the ENAl promoter that takes place after culture in AP me- phosphorylation among the cellularmechanisms controlling dium (28) was not observed in cnblA cells. Therefore, cal- adaptation to high salinity.The importantrole that calcineurin cineurin seemed to be required for adequate transcription of plays on NaCl tolerance and its strict dependence on Ca2' for ENAl. No additional effect of calcineurin on translation or activity point to changes in cytoplasmic Ca2+ as a triggering enzymatic activitycan be inferred because the expression of the factor for the adaptation process. NaCl stress might increase ENAl-lacZ fusion in the same conditions used for Li+ efflux the concentration of cytosolic Ca2+ that, in turn, would activate measurement correlated with the decrease of the rate ofLi' the Ca2+-dependent protein phosphatase activity of calefflux. Our results indicate that some induction of the ENAl cineurin. Our results suggest that calcineurin plays a key role promoter takes place in theabsence of CNB, but its activity is in a common pathway leading to theefficient expression of the important to the steady-state mRNA level that occurs in the Na+ efflux system and theconversion of the K+ uptake system cells. It is worth notingthat a 2-fold reduction in theexpression to thehigh affinity state, thuscoordinating both cellularadapof a ENAl gene with a truncated promoter resulted in signifitation responses t o NaCl stress. cant sensitivity toNa' and Li' (4). Thus, a reduced expression of ENAl in cnbl cells largely contributes to their Na+ andLi' Acknowledgment-We thank Professor P. M. Hasegawa for critical sensitivity. In lymphocytes, calcineurin modulates lymphokine comments. gene expression by dephosphorylation of specific transcription REFERENCES factors (9). This protein phosphatase may have a similar function in the regulation of ENAl gene transcription in S. cereui1. Borst-Pauwels, G. (1981)Biochirn. Biophys. Acta 660, 88-127 2. Haro, R., Baiiuelos, M. A,, Quintero,F. J., Rubio, F.,and Rodriguez-Navarro, A. siae. (1993) Physiol. Plant. 89, 868-874 In addition to its role in the Na+ and Li' efflux system, 3. Gaber, R. F., Styles, C. A,, and Fink, G. R. (1988)Mol. Cell. Biol.8, 2848-2859 calcineurin seems to be essential for the transition of the K' 4. Garciadeblas, B., Rubio, F., Quintero, F, J., Baiiuelos, M. A,, Haro, R., and Rodriguez-Navarro. A. (1993) Mol. & Gen. Genet. 236,363-368 uptake systemfrom the low affinity to thehigh affinity state in 5. Robinson, L. C., Hubbard, E. J. A,, Graves, P. R., DePaoli-Roach,A. A,, Roach, response to Na+ stress. In an environment that produced an P. J., Kung, C., Haas, D. W., Hagedorn, C. H., Goebl, M., Culbertson, M. R., and Carlson, M. (1992)Proc. Nutl. Acad. Sei. U. S. A. 89,28-32 intracellular Na+/K+ratio close to 1, CNBl cells had a K , for 6. Brewster,J. L., de Valoir, T., Dwyer, N. D.,Winter, E., and Gustin, M. C. (1993) Rb+ of 80 VM and cnblA cells remained at 1.6 mM. Presumably, Science 269, 1760-1763 K+/Na+discrimination during ion uptake depends on the rela- 7. Klee, C. B., Draetta, G. F., and Hubbard, M. J. (1988) Adu. Enzyrnol. Relat. Areas Mol. B i d . 61, 149-200 tive affinity for these ions. In thehigh affinity state, the transS. J., Tamura, J., Kincaid, R. L., Tocci, M.J., and ONeill, E.A. (1992) port system has substantiallyincreased affinity for K+, but the 8. 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(1991) Methods Enzyrnol. 194,273-281 brane protein implicated to be a K' transporter (3). In trkl 17. Becker, D. M., and Guarente, L. (1991) Methods Enzyrnol. 194, 182-187 mutants the K' uptake system is permanently in the low af- 18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring finity state (31). Because the transition to the high affinity Harbor, NY state does not requireprotein synthesis (261, it isprobable that 19. Carlson, M., and Botstein, D. (1982) Cell 28, 145-154 the TRKl protein is controlled posttranslationally. Our results 20. Delcasso-Tremousaygue,D., Grellet, F., Panabieres, F.,Ananiev, E. D., and Delseny, M. (1988) Eur J . Biochern. 172,767-776 indicatethatprotein dephosphorylationmediated by cal21. Myers,A. M., Tzagoloff, A,, Kinney, D. M., and Lusty, C.J. 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