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• W2059699772 abstract "Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, which is required for modulation of Na+ uptake and efflux mechanisms. Five Na+-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H+-ATPase. One mutant, pma1-α4, which has the single amino acid change Glu367 → Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na+ tolerance. After exposure to Na+ in the culture medium,22Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity. Efflux of 22Na from intact cells was relatively unchanged in the pma1 mutant. However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na+ within a slowly exchanging pool. We show that NHX1, a novel gene homologous to the mammalian NHE family of Na+/H+exchangers, is required for Na+ sequestration in yeast and contributes to the Na+-tolerant phenotype ofpma1-α4. Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, which is required for modulation of Na+ uptake and efflux mechanisms. Five Na+-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H+-ATPase. One mutant, pma1-α4, which has the single amino acid change Glu367 → Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na+ tolerance. After exposure to Na+ in the culture medium,22Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity. Efflux of 22Na from intact cells was relatively unchanged in the pma1 mutant. However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na+ within a slowly exchanging pool. We show that NHX1, a novel gene homologous to the mammalian NHE family of Na+/H+exchangers, is required for Na+ sequestration in yeast and contributes to the Na+-tolerant phenotype ofpma1-α4. Living cells actively maintain low cytoplasmic sodium ion concentrations against large, inwardly directed Na+gradients. In animal cells, the intracellular Na+/K+ ratio is largely dependent on the plasma membrane Na+/K+-ATPase, a P-type ion pump, that drives Na+ ions out of the cell in exchange for K+ ions (1Skou J.C. Esmann M. J. Bioenerg. Biomembr. 1992; 24: 249-261PubMed Google Scholar). The resultant Na+ gradient serves as the primary energy source for the transport of other ions and metabolites via an array of secondary, Na+-coupled carriers. In lieu of sodium, plants and fungi utilize H+-coupled circuits, driven by the plasma membrane H+-ATPase, PMA1, also a member of the P-ATPase family (2Serrano R. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989; 40: 61-94Crossref Google Scholar,3Rao R. Slayman C.W. Brambl R. Marzluf G.A. The Mycota III. Springer-Verlag, Berlin1996: 29-56Google Scholar). Mechanisms for dealing with toxic concentrations of Na+have only recently begun to be elucidated at a molecular level, but are of increasing urgency as soil salinity rises and poses a significant threat to agricultural production worldwide (4Serrano R. Gaxiola R. Crit. Rev. Plant Sci. 1994; 13: 121-138Crossref Scopus (196) Google Scholar, 5Serrano R. Int. Rev. Cytol. 1996; 165: 1-52Crossref PubMed Google Scholar). Because of a basic similarity in ion transport processes, the use of yeast as a model system to identify genes involved in halotolerance is of particular applicability to higher plants. The recent availability of the complete genome sequence from Saccharomyces cerevisiae has brought to light unexpected homologs of both prokaryotic and eukaryotic Na+ transporters, providing a convenient starting point for dissecting the individual contributions of these transporters toward sodium tolerance. Multiple transport pathways, all of which are under complex regulation, appear to mediate cellular Na+ homeostasis in S. cerevisiae. One route of Na+ entry is thought to be the K+ transporters, TRK1 and TRK2 (6Gaber R.F. Styles C.A. Fink G.R. Mol. Cell. Biol. 1988; 8: 2848-2859Crossref PubMed Scopus (231) Google Scholar, 7Ko C.H. Buckley A.M. Gaber R.F. Genetics. 1990; 125: 305-312Crossref PubMed Google Scholar, 8Ko C.H. Gaber R.F. Mol. Cell. Biol. 1991; 11: 4266-4273Crossref PubMed Scopus (235) Google Scholar, 9Haro R. Banuelos M.A. Quintero F.J. Rubio F. Rodriguez-Navarro A. Physiol. Plant. 1993; 89: 868-874Crossref Scopus (1) Google Scholar, 10Ramos J. Alijo R. Haro R. Rodriguez-Navarro A. J. Bacteriol. 1994; 176: 249-252Crossref PubMed Google Scholar). Under conditions of Na+ stress or K+ starvation, the principal cation carrier is TRK1, which has been proposed to limit Na+ entry by increasing K+/Na+discrimination (9Haro R. Banuelos M.A. Quintero F.J. Rubio F. Rodriguez-Navarro A. Physiol. Plant. 1993; 89: 868-874Crossref Scopus (1) Google Scholar, 11Rodriguez-Navarro A. Ramos J. J. Bacteriol. 1984; 159: 940-945Crossref PubMed Google Scholar). Activation of calcineurin, the Ca2+- and calmodulin-dependent protein phosphatase, is required for the transition in response to Na+ stress (12Mendoza I. Quintero F.J. Bressan R.A. Hasegawa P.M. Pardo J.M. J. Biol. Chem. 1996; 271: 23061-23067Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The primary pathway for Na+extrusion from the cell is through the P-type ion pumps encoded by thePMR2/ENA locus, consisting of an unusual tandem array of nearly identical genes (PMR2A-E; Refs. 13Rudolph H.K. Antebi A. Fink G.R. Buckley C.M. Dorman T.E. LeVitre J. Davidow L.S. Mao J.I. Moir D.T. Cell. 1989; 58: 133-145Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 14Haro R. Garciadeblas B. Rodriguez-Navarro A FEBS Lett. 1991; 291: 189-191Crossref PubMed Scopus (309) Google Scholar, 15Wieland J. Nitsche A.M. Strayle J. Steiner H. Rudolf H.K. EMBO J. 1995; 14: 3870-3882Crossref PubMed Scopus (162) Google Scholar). Expression of PMR2A is induced by high pH and Na+ stress (16Garciadeblas B. Rubio F. Quintero F.J. Banuelos M.A. Haro R. Rodriguez-Navarro A. Mol. Gen. Genet. 1993; 236: 363-368Crossref PubMed Scopus (176) Google Scholar) and is modulated by a host of factors, including calcineurin (17Mendoza I. Rubio F. Rodriguez-Navarro A. Pardo J.M. J. Biol. Chem. 1994; 269: 8792-8796Abstract Full Text PDF PubMed Google Scholar, 18Nakamura T. Liu Y. Harata D. Namba H. Harada S. Hirokawa T. Miyakawa T. EMBO J. 1993; 12: 4063-4071Crossref PubMed Scopus (229) Google Scholar). Three distinct genes encoding putative Na+/H+antiporters have been identified by the genome sequencing project. One of these, NHA1, was recently cloned by selection for increased NaCl tolerance from a multicopy genomic library (19Prior C. Potier S. Souciet J.-L. Sychrova H. FEBS Lett. 1996; 387: 89-93Crossref PubMed Scopus (149) Google Scholar). Although activity and localization of the NHA1 protein has not yet been established, it is homologous (40% identity) to a plasma membrane Na+/H+ exchanger, encoded by thesod2 gene in the fission yeast Schizosaccharomyces pombe, which mediates Na+ extrusion at acidic to neutral pH (20Jia Z.-P. McCullough N. Martel R. Hemmingsen S. Young P.G. EMBO J. 1992; 11: 1631-1640Crossref PubMed Scopus (158) Google Scholar). Disruption of NHA1 confers significant Na+ sensitivity in a strain lacking the PMR2locus, but only a weak phenotype in a wild-type background. A protein with homology to putative Na+/H+ exchangers from Enterococcus hirae and Lactococcus lactis, as well as to a putative K+/H+ exchanger (KefC) from Escherichia coli (22Waser M. Hess-Bienz D. Davies K. Solioz M. J. Biol. Chem. 1992; 267: 5396-5400Abstract Full Text PDF PubMed Google Scholar, 23Martinussen J. Hammer K. J. Bacteriol. 1994; 176: 6457-6463Crossref PubMed Google Scholar, 24Munro A.W. Ritchie G.Y. Lamb A.J. Douglas R.M. Booth I.R. Mol. Microbiol. 1991; 5: 607-616Crossref PubMed Scopus (79) Google Scholar), is encoded by the yeast geneYJL094c (21Misoga T. Witzel A. Zimmermann F.K. Yeast. 1994; 10: 965-973Crossref PubMed Scopus (26) Google Scholar). A third yeast gene, YDR456w, encodes a protein sharing significant homology (∼30% identity) with the amiloride-sensitive Na+/H+ exchangers (NHE1–4) in animal cells (25Andre B. Yeast. 1995; 11: 1575-1611Crossref PubMed Scopus (207) Google Scholar). The latter play physiologically vital roles in the regulation of intracellular pH, in Na+concentration, and in cell volume control. Activation by a variety of growth factors, hormones, and cytoplasmic acidosis leads to H+ extrusion from the plasma membrane, in exchange for an influx of Na+ ions (reviewed in Refs. 26Wakabayashi S. Sardet C. Fafournoux P. Counillon L. Meloche S. Pages G. Pouyssegur J. Rev. Physiol. Biochem. Pharmacol. 1992; 119: 157-186Crossref PubMed Scopus (76) Google Scholar and 27Tse C.-M. Levine S.A. Yun C.H.C. Brant S.R. Nath S. Pouyssegur J. Donowitz M. Cell. Physiol. Biochem. 1994; 4: 282-300Crossref Scopus (37) Google Scholar). In this work, we report that mutations in the plasma membrane H+-ATPase, PMA1, confer Na+ tolerance in yeast. We show that the mutant cells sequester Na+ in a slowly exchanging pool, and that this sequestration is mediated via the yeast homolog of the mammalian amiloride-sensitive Na+/H+ exchanger, which we term NHX1. All strains used in this study are isogenic to W303 (ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura3-1) (28Wallis J.W. Chrebet G. Brodsky G. Rolfe M. Rothstein R. Cell. 1989; 58: 409-419Abstract Full Text PDF PubMed Scopus (453) Google Scholar). Spontaneous Na+-tolerant suppressors of calcineurin deficiency were isolated independently in two strains described previously (29Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (364) Google Scholar), K473-2 (MATa pmc1::LEU2 cnb1-2) and K482-3 (MATα pmc1::TRP1 cnb1-3), by selection on YPD agar medium supplemented with 1.0 m NaCl. After 3 days at 30 °C, a single Na+-tolerant colony was picked from each patch, purified, and subjected to complementation testing. Of 41 independent suppressors, 35 were recessive and defined two complementation groups of 33 and 2 alleles, respectively. The remaining six suppressors were dominant or semidominant; after crossing with the parent of the opposite mating type, four alleles were tightly linked topmc1::TRP1 and one allele was tightly linked topmc1::LEU2 (zero recombinants in 26 complete tetrads). The PMC1 gene encodes a vacuolar Ca2+pump, which plays no detectable role in Na+ tolerance and is adjacent to the PMA1 gene. Complementation tests with well characterized alleles of pma1 (30Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar) showed all five suppressors linked to PMC1 are alleles of PMA1. Strains K638 (PMA1) and K804 (pma1-α4) areMATα derivatives of W303 carryingpmc1::TRP1 and pmr2::HIS3(Δpmr2A-E) null alleles. YR89 (Δpmr2A-E) was a gift from Hans Rudolph (University of Stuttgart, Germany). YPD medium contained 2% glucose, 1% yeast extract, and 2% peptone (all from Difco), and was adjusted to pH 7 with sodium phosphate, where indicated. APG is a synthetic minimal medium containing 10 mm arginine, 8 mm phosphoric acid, 2% glucose, 2 mm MgSO4, 1 mm KCl, 0.2 mm CaCl2, and trace minerals and vitamins, at pH 6.7, as described (11Rodriguez-Navarro A. Ramos J. J. Bacteriol. 1984; 159: 940-945Crossref PubMed Google Scholar). Where indicated, NaCl was added, or the pH was adjusted to 5.0 by addition of acetic acid. Growth assays were performed by inoculating 1 ml of APG medium in a multiwell plate with 2–5 μl of a saturated seed culture. Growth was monitored by measuring absorbance at 600 nm after culturing for 48 h at 30 °C. The chromosomal pma1alleles were rescued by digestion of genomic DNA with PstI, to release a ∼23-kilobase pair fragment containing the disruptedpmc1 gene (29Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (364) Google Scholar) and the adjacent pma1 gene. After treatment with DNA ligase and transformation into E. coli, AmpR plasmids were recovered, and a 4.8-kilobase pairHindIII fragment containing intact pma1 was cloned into the yeast centromeric vector YCplac111 (31Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2522) Google Scholar). Plasmids were transformed into strain YR89 (Δpmr2A-E) using the lithium acetate procedure (32Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). A series of YCplac111-based plasmids containing various fragments of the pma1-α4 allele, subcloned into wild-type PMA1, were generated and also introduced into strain YR89. By comparison of the Na+-tolerant phenotype conferred by the plasmids, the mutation in the pma1-α4allele was localized to a 615-base pair fragment betweenBstEII and EcoRI, and identified by DNA sequencing. A single base change, G → A, resulting in the substitution Glu367 → Lys, was found. The NHX1 gene was cloned by amplification of genomic DNA from K638 using the polymerase chain reaction and the following primers: sense primer 5′-CGCCATTGTGTATCCATTTATGC-3′ at −598 from the initiating codon ATG, and the antisense primer 5′-CTCACCAATTATACGAGTAG-3′ at +350 from the terminating codon TAG. The amplified product was digested with HindIII andNheI, and the resultant 2662-base pair fragment cloned into the HindIII and XbaI sites of pBluescript II KS (Stratagene). A null allele (nhx1::URA3) was constructed by replacement of the 1286-base pairEcoRV-SpeI fragment of NHX1 with a 1-kilobase pair SmaI-HindIII fragment of URA3, after treatment of DNA fragments with Klenow enzyme to create blunt ends. Yeast strains were grown in APG medium, supplemented where indicated with 20 mmNaCl, to a density of 0.9–1.4 OD600 units/ml. Cells were harvested by centrifugation, washed once in APG medium ± 20 mm NaCl (depending on growth conditions), and resuspended in the same medium at a density of 4–8 × 108cells/ml. Uptake was initiated by diluting the cells with an equal volume of APG medium containing NaCl at a final concentration of 20 mm and 22NaCl (NEN Life Science Products) at 13–30 μCi/ml. Samples were incubated at 23 °C, and at the indicated times aliquots were withdrawn and the reaction quenched by the addition of ice-cold AP medium containing 75 mmpotassium gluconate. The reaction mixture was rapidly filtered through 0.45-μm HAWP membranes (Millipore) and washed twice with 6 ml of quench buffer. Radioactivity retained on the filters was measured by liquid scintillation counting. In assays of 22Na efflux, cells were loaded with 22NaCl exactly as above. After incubation for 45 min, cells were collected by centrifugation, washed twice in ice-cold Buffer A (10 mm Tris-HCl, pH 6.0, 2 mm MgCl2, 1 mm KCl, 1% glucose, 0.6 m sorbitol), and resuspended in the same buffer at room temperature. At the indicated times, aliquots were withdrawn, filtered, and processed as described above. Steady-state labeling of cells with22Na was performed in 1 ml of APG medium containing 1–4 μCi of 22NaCl and varying concentrations of nonradioactive NaCl. Cultures were incubated at 23 °C for 96 h, after which the cells were collected by rapid filtration. Filters were washed twice with 6 ml of APG medium, and radioactivity assessed by liquid scintillation counting. The optical density of a duplicate, nonradioactive culture was measured to quantitate growth. After labeling with 22Na as described above, cells were collected by centrifugation, washed twice with ice-cold Buffer A (see above), and resuspended at 1 × 108cells/ml in Buffer A, essentially as described by Anraku and co-workers (33Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar). One half of the suspension was permeabilized by addition of CuSO4 to a final concentration of 500 μm, while the other half received an equal volume of water (22.5 μl). After incubation at 23 °C for the indicated times, aliquots were withdrawn, filtered, and processed as described above. To assay22Na influx in permeabilized cells, cultures (2 × 108 cells/ml) were preincubated with 500 μmCuSO4 in Buffer A for 1 h as above. Uptake was initiated by diluting cells with an equal volume of Buffer A supplemented to give a final concentration of 20 mm NaCl, 9 μCi/ml 22Na, 2 mm Tris-ATP, and 500 μm CuSO4. At the indicated times, aliquots were withdrawn for filtration as above. Calcineurin promotes Na+ tolerance by increasing expression of thePMR2A/ENA1 gene (17Mendoza I. Rubio F. Rodriguez-Navarro A. Pardo J.M. J. Biol. Chem. 1994; 269: 8792-8796Abstract Full Text PDF PubMed Google Scholar, 18Nakamura T. Liu Y. Harata D. Namba H. Harada S. Hirokawa T. Miyakawa T. EMBO J. 1993; 12: 4063-4071Crossref PubMed Scopus (229) Google Scholar) and by converting the K+ transport system to a high affinity, Na+discriminatory state (12Mendoza I. Quintero F.J. Bressan R.A. Hasegawa P.M. Pardo J.M. J. Biol. Chem. 1996; 271: 23061-23067Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Loss of calcineurin function, as incnb1 mutants, therefore causes sensitivity to high salt conditions. To learn more about salt tolerance in yeast, we isolated spontaneous Na+-resistant suppressors of thecnb1 mutants by selection on YPD medium supplemented with 1.0 m NaCl. Five dominant mutations were mapped to thePMA1 locus as described under “Experimental Procedures.” All five mutations in PMA1 conferred different degrees of Na+ tolerance (Fig.1 A), and Li+tolerance (data not shown), relative to the parent strains. All five Na+-tolerant mutations in PMA1 also conferred sensitivity to H+ (low pH) and were recessive to wild type for this phenotype. Several pma1 alleles isolated in genetic screens not involving high salt (30Chang A. Fink G.R. J. Cell Biol. 1995; 128: 39-49Crossref PubMed Scopus (94) Google Scholar) were also found to be recessive for H+ sensitivity and dominant or semidominant for Na+ tolerance when calcineurin is inactivated (data not shown). These results suggest the Na+-tolerant alleles ofPMA1 are loss-of-function mutations with regard to H+ pumping and thus are referred to as recessivepma1 alleles. Because of its strong Na+-tolerant phenotype,pma1-α4 was chosen for further analysis. The mutant allele was rescued from the chromosome, subcloned, and partially sequenced (see “Experimental Procedures”) to reveal the nature of the mutation: a single amino acid substitution, Glu367 → Lys, within the highly conserved phosphorylation domain of the ATPase (Fig. 1 B). ATPase activity measured in total membrane preparations, was reduced by 45%, relative to the PMA1 control (data not shown). Mutations in the immediate vicinity of this residue (Ser368) have been reported to cause a similar reduction in ATPase activity, H+ pumping, and defects in membrane potential (34Perlin D.S. Brown C.L. Haber J.E. J. Biol. Chem. 1988; 263: 18118-18122Abstract Full Text PDF PubMed Google Scholar, 35Perlin D.S. Harris S.L. Seto-Young D. Haber J.E. J. Biol. Chem. 1989; 264: 21857-21864Abstract Full Text PDF PubMed Google Scholar). Thus, it was unlikely that the pma1-α4mutant had gained a novel ability to pump Na+ ions, despite its dominance over PMA1 in Na+ tolerance. The Na+-tolerant phenotype of the pma1-α4 mutation was also found to be independent of the entire PMR2 locus, in the absence or presence of calcineurin function (Fig.1 C). This ruled out the possibility that PMR2 expression or function was improved by the pma1 mutant. Finally, it should be noted that the effects of pma1-α4 on Na+tolerance were found to be completely independent of the vacuolar Ca2+ transporters encoded by PMC1 andVCX1 (data not shown), mutations in which were included in some experiments for technical reasons. These results suggest thatpma1-α4 promotes Na+ tolerance through processes independent of calcineurin and the PMR2-encoded Na+ efflux pumps. Because the plasma membrane H+-ATPase generates the driving force for active transport, we tested the hypothesis that reduced influx of Na+ in the pma1-α4 mutant contributes to Na+ tolerance. Fig.2 A shows the time course of22Na uptake by the pma1-α4 mutant (K804) and the isogenic PMA1 strain (K638), after culture in APG medium lacking NaCl (see “Experimental Procedures”). Previous work has shown that under these conditions, uptake of monovalent cations (K+, Rb+) is in the low affinity mode and is unaffected by dissipation of the H+ gradient in response to protonophore addition or ATP depletion (11Rodriguez-Navarro A. Ramos J. J. Bacteriol. 1984; 159: 940-945Crossref PubMed Google Scholar). Consistent with these observations, initial rates of 22Na uptake were similar in both mutant and control, although at longer time points, uptake in the mutant consistently exceeded that of the PMA1 control cells (Fig. 2 A). A shift in the cation uptake system to a high affinity, K+-selective mode has been reported to occur within 4 h of K+ depletion from the growth medium; under these conditions, transport is sensitive to protonophore addition and ATP depletion and is dependent on the TRK1 transporter (11Rodriguez-Navarro A. Ramos J. J. Bacteriol. 1984; 159: 940-945Crossref PubMed Google Scholar). Induction of the K+-selective mode has also been reported to occur after exposure to Na+ or Li+ in the growth medium (9Haro R. Banuelos M.A. Quintero F.J. Rubio F. Rodriguez-Navarro A. Physiol. Plant. 1993; 89: 868-874Crossref Scopus (1) Google Scholar). Fig. 2 B shows that, after growth in APG medium containing 20 mm NaCl, 22Na uptake in the control strain K638 was drastically reduced, consistent with increased discrimination against Na+ ions. Furthermore, uptake was reduced by an additional 2-fold in the pma1-α4mutant, relative to control. The results demonstrate that, after salt adaptation, the pma1-α4 mutation increases Na+tolerance by limiting Na+ influx, possibly through modulation of the TRK1 transporter. External pH had a striking effect on Na+ tolerance; at neutral pH, the pma1-α4mutant (K804) grew to higher cell densities (10-fold relative to control, K638) in response to increasing concentrations of NaCl (Fig.3 A). However, Na+tolerance increased with decreasing pH and was similar in bothpma1-α4 mutant and control strains at pH 5 (Fig.3 B). To assess intracellular Na+ levels, cells were grown to saturation in APG medium containing 22NaCl (0.1 μm) and varying concentrations of unlabeled NaCl (see “Experimental Procedures”). Surprisingly, accumulation of Na+ in the sodium-tolerant mutant exceeded that of control cells, by 2–5-fold, in media containing tracer 22Na alone (Fig. 3, C and D, inset). As external NaCl concentrations increased, intracellular Na+ in the mutant reached stable levels at both pH values tested (Fig. 3,C and D) and correlated with a similar stability in cell density. Overall, a 7-fold increase in external NaCl concentrations elicited only a 2-fold increase in intracellular Na+ in the mutant. In PMA1 control cells, a similar response was observed at pH 5; in contrast, at neutral pH, intracellular Na+ rose by 12-fold over the same range and was accompanied by a drastic decline in cell density. Thus, the ability to limit intracellular Na+ levels correlates with sodium-tolerant growth. To determine the intracellular distribution of sodium, plasma membranes were selectively permeabilized by exposure to Cu2+ ions, as described by Anraku and co-workers (33Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar). At least 50% of intracellular Na+ in the pma1-α4 mutant appeared to be retained in a Cu2+-resistant pool under all conditions tested (Fig. 3, E and F). By comparison, in the PMA1 control, approximately 90% of intracellular Na+ was released after Cu2+ treatment of cells grown in media containing low NaCl. Based on a previous demonstration (33Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar) that the Cu2+ permeablization technique allows the specific extraction of cytosolic pools of ions, amino acids, and other metabolites, our results strongly suggest a cytoplasmic localization of sodium in the PMA1 control cells. After exposure to higher concentrations of NaCl, particularly at acidic pH, Na+sequestration in the PMA1 control increased to levels comparable to the pma1 mutant. A range of control experiments, including assays of 45Ca2+ uptake, release of nucleotides, and turbidity changes in the cell suspension (33Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar), were used to confirm that both mutant and control cells were equally permeabilized by the treatment with Cu2+ ions (data not shown). Therefore, these results demonstrate that thepma1-α4 mutation enhances the ability to sequester Na+ in an intracellular pool, and that this sequestration may contribute significantly to halotolerance. Based on previous observations that reduced H+ pumping inpma1 mutants leads to reductions in cytoplasmic pH (36Vallejo C.G. Serrano R. Yeast. 1989; 5: 307-319Crossref PubMed Scopus (96) Google Scholar), we hypothesized that cytosolic pH may be a controlling factor in sodium tolerance. Because cytoplasmic acidosis has been shown to activate the amiloride-sensitive Na+/H+ exchanger in mammalian cells (37Aronson P.S. Nee J. Suhm M.A. Nature. 1982; 299: 161-163Crossref PubMed Scopus (466) Google Scholar), it seemed likely that activation of a similar exchanger might mediate Na+ tolerance in yeast. Systematic sequencing of the yeast genome has uncovered a gene present on chromosome IV (YDR456w), encoding a protein of 633 amino acids that we have named NHX1 on the basis of homology with the family of Na+/H+ exchangers (NHE1–4) found in mammals and other vertebrates, as well as in the nematode Caenorhabditis elegans, and the euryhaline crab Carcinus maenas (Fig.4). We analyzed the effect of targeted disruptions of this gene in the following isogenic set of yeast strains: K601 (W303- derivative; see “Experimental Procedures”), K638 (Δpmc1 Δpmr2 PMA1), and K804 (Δpmc1 Δpmr2 pma1-α4). In each of these genetic backgrounds, disruption ofNHX1 led to a significant decrease in Na+tolerance at pH 4 or 5 (Fig. 5,B, D, and F), substantiating its prominent role in mediating salt tolerance at acid pH. At neutral pH, however, the disruption had little or no effect on sensitivity to Na+ in the PMA1 strains (Fig. 5, Aand C). In striking contrast, disruption of NHX1effectively nullified the Na+-tolerant phenotype of thepma1-α4 mutant at neutral pH (Fig. 5 E). The results demonstrate that Na+ tolerance in thepma1-α4 mutant is largely mediated by the putative Na+/H+ exchanger, NHX1.Figure 5Effect of NHX1 gene disruption on Na+ tolerance. Growth was monitored as a function of NaCl concentrations in APG medium at the indicated pH. Open symbols denote the Δnhx1 strain; the isogenic parent strain is shown in closed symbols. Parent strains and relevant genotypes are as follows. A and B, K601 (wild type); C and D, K638 (Δpmc1 Δpmr2 PMA1); E and F, K804 (Δpmc1 Δpmr2 pma1-α4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine if sodium sequestration in thepma1-α4 mutant was mediated by NHX1, we followed the time course of 22Na efflux from Cu2+-treated cells, relative to untreated cells (Fig.6 A). In the PMA1control strain, addition of Cu2+ elicited an immediate efflux of 22Na, with less than 10% remaining in 20 min. By contrast, efflux of 22Na was slow and biphasic in thepma1-α4 mutant, with approximately 50% remaining in the cells after treatment with Cu2+ for 2 h. Disruption ofNHX1 in the pma1-α4 mutant effectively nullified the ability to sequester sodium, resulting in almost complete release of intracellular 22Na upon permeabilization of the plasma membrane. The data also indicated that, in the absence of Cu2+ treatment, 22Na efflux was low or absent in all three strains (Fig. 6 A). Thus, there was no evidence that NHX1 was involved in a Na+ efflux mechanism in thepma1 mutant cells; instead, the data suggest that thePMR2/ENA pumps are the major route for Na+efflux. Uptake of 22Na was also monitored in Cu2+-permeabilized cells (Fig. 6 B). The results demonstrate a substantial enhancement of Na+ uptake into Cu2+-resistant compartment(s) in thepma1-α4 mutant, relative to the PMA1control, that is completely abolished by the disruption inNHX1. Thus, a putative Na+/H+exchanger is required for sequestration of Na+ within an intracellular, Cu2+-resistant pool, possibly the vacuole. One possibility is that mislocalization of the mutant pma1protein to an intracellular compartment may stimulate Na+/H+ exchange by contribution of a proton motive force, although a plasma membrane localization for pma1-α4 has been confirmed by immunofluorescence (data not shown). We suggest that cytoplasmic acidosis, caused by the pma1-α4 mutation or by growth in low pH conditions, increases expression or function of NHX1 to promote Na+ tolerance. Mutations in the plasma membrane H+-ATPase confer sensitivity to weak acids and low extracellular pH, reflecting a reduced ability to pump protons from cells (39McCusker J.H. Perlin D.S. Haber J.E. Mol. Cell. Biol. 1987; 7: 4082-4088Crossref PubMed Scopus (138) Google Scholar). The resultant defect in the electrochemical H+ gradient is also believed to be responsible for the observed resistance of pma1 mutants to cytotoxi" @default.
• W2059699772 created "2016-06-24" @default.
• W2059699772 creator A5017278189 @default.
• W2059699772 creator A5017285382 @default.
• W2059699772 creator A5055002773 @default.
• W2059699772 date "1997-10-01" @default.
• W2059699772 modified "2023-10-17" @default.
• W2059699772 title "Intracellular Sequestration of Sodium by a Novel Na+/H+ Exchanger in Yeast Is Enhanced by Mutations in the Plasma Membrane H+-ATPase" @default.
• W2059699772 cites W1488890226 @default.
• W2059699772 cites W1503948998 @default.
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