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Potassium and Na + effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. In fungal cells, these effluxes are mediated by cation/H + antiporters and ENA ATPases. We have cloned and studied the functions of the two ENA ATPases of Ustilago maydis , U. maydis Ena1 (UmEna1) and UmEna2. UmEna1 is a typical K + or Na + efflux ATPase whose function is indispensable for growth at pH 9.0 and for even modest Na + or K + tolerances above pH 8.0. UmEna1 locates to the plasma membrane and has the characteristics of the low-Na + /K + -discrimination ENA ATPases. However, it still protects U. maydis cells in high-Na + media because Na + showed a low cytoplasmic toxicity. The UmEna2 ATPase is phylogenetically distant from UmEna1 and is located mainly at the endoplasmic reticulum. The function of UmEna2 is not clear, but we found that it shares several similarities with Neurospora crassa ENA2, which suggests that endomembrane ENA ATPases may exist in many fungi. The expression of ena1 and ena2 transcripts in U. maydis was enhanced at high pH and at high K + and Na + concentrations. We discuss that there are two modes of Na + tolerance in fungi: the high-Na + -content mode, involving ENA ATPases with low Na + /K + discrimination, as described here for U. maydis , and the low-Na + -content mode, involving Na + -specific ENA ATPases, as in Neurospora crassa .

HvHKT1 mediates K + or Na + uniport in yeast cells if the expression promoter is joined directly to the HvHKT1 cDNA, and Na + -K + symport if a 59 nucleotide polylinker is inserted. Our results show that three ATG triplets in the polylinker decreased the synthesis of the transporter and that the lower amount of transporter caused the functional change. With the rice HKT1 cDNA, the 59 nt polylinker changed the mode of Na + uptake from K + -insensitive to K + -inhibitable. These two modes of Na + uptake also occurred in rice plants.

Twenty-day-old sunflower plants (Helianthus annuus L. cv Sun-Gro 380) grown hydroponically under controlled conditions were used to study the effect of transpiration on Na(+) compartmentalization in roots. The plants were exposed to low Na(+) concentrations (25 mM NaCl) and different environmental humidity conditions over a short time period (8.5 h). Under these conditions, Na(+) was accumulated primarily in the root, but only the Na(+) accumulated in the root symplast was dependent on transpiration, while the Na(+) accumulated in both the shoot and the root apoplast exhibited a low transpiration dependence. Moreover, Na(+) content in the root apoplast was reached quickly (0.25 h) and increased little with time. These results suggest that, in sunflower plants under moderate salinity conditions, Na(+) uptake in the root symplast is mediated by a transport system whose activity is enhanced by transpiration.

A cDNA that encodes a transporter from the NHAD family was identified in Physcomitrella patens. Computer-based searches using the amino acid sequence of PpNHAD1 revealed that, in addition to being expressed in flowering plants, highly conserved transporters of this family are expressed in red algae, green algae, mosses, liverworts, and photosynthetic stramenopiles, but not in heterotrophic stramenopiles. A chloroplast transit peptide was detected in PpNHAD1 and in most of the related sequences, indicating that PpNHAD1 is a chloroplast transporter. A PpNHAD1-GFP fusion localized to the chloroplast in Physcomitrella protoplasts, and truncation of the N-terminus of the protein dispersed the fluorescence signal outside the chloroplast. PpNHAD1 did not show functional expression in either yeast or bacterial mutants, but truncated proteins with shorter N-termini, PpNHAD1-1 and PpNHAD1-2, could be functionally expressed in bacteria. PpNHAD1-1 alleviated the Li + intolerance of a Na + -efflux Escherichia coli mutant at acidic pH values. Both PpNHAD1-1 and PpNHAD1-2 reduced the K + requirements of a K + -influx E. coli mutant more actively at high pH values. PpNHAD1 seems to be an important transporter that mediates ionic homeostasis in chloroplasts from red algae to flowering plants.

Salinity tolerance in many plants is inversely related to the extent of Na+ accumulation in the shoot, notably in the major cereals such as wheat and rice [1]. In Arabidopsis and rice, there is evidence indicating a central role for members of the HKT gene family of Na+ and Na+/K+ transporters in controlling Na+ accumulation [2–6] and, thus, in determining salinity tolerance. However, in heterologous systems, whereas the wheat TaHKT1 protein transports both Na+ and K+, AtHKT1 is more Na+-specific [7,8] and their sequences are not particularly closely related.

All living cells have an absolute requirement for K+, which must be taken up from the external medium. In contrast to marine organisms, which live in a medium with an inexhaustible supply of K+, terrestrial life evolved in oligotrophic environments where the low supply of K+ limited the growth of colonizing plants. In these limiting conditions Na+ could substitute for K+ in some cellular functions, but in others it is toxic. In the vacuole, Na+ is not toxic and can undertake osmotic functions, reducing the total K+ requirements and improving growth when the lack of K+ is a limiting factor. Because of these physiological requirements, the terrestrial life of plants depends on high-affinity K+ uptake systems and benefits from high-affinity Na+ uptake systems. In plants, both systems have received extensive attention during recent years and a clear insight of their functions is emerging. Some plant HAK transporters mediate high-affinity K+ uptake in yeast, mimicking K+ uptake in roots, while other members of the same family may be K+ transporters in the tonoplast. In parallel with the HAK transporters, some HKT transporters mediate high-affinity Na+ uptake without cotransporting K+. HKT transporters have two functions: (i) to take up Na+ from the soil solution to reduce K+ requirements when K+ is a limiting factor, and (ii) to reduce Na+ accumulation in leaves by both removing Na+ from the xylem sap and loading Na+ into the phloem sap.

The function of HKT1 in roots is controversial. We tackled this controversy by studying Na+ uptake in barley (Hordeum vulgare) roots, cloning the HvHKT1 gene, and expressing the HvHKT1 cDNA in yeast (Saccharomyces cerevisiae) cells. High-affinity Na+ uptake was not detected in plants growing at high K+ but appeared soon after exposing the plants to a K+-free medium. It was a uniport, insensitive to external K+ at the beginning of K+ starvation and inhibitable by K+ several hours later. The expression of HvHKT1 in yeast was Na+ (or K+) uniport, Na+-K+ symport, or a mix of both, depending on the construct from which the transporter was expressed. The Na+ uniport function was insensitive to external K+ and mimicked the Na+ uptake carried out by the roots at the beginning of K+ starvation. The K+ uniport function only took place in yeast cells that were completely K+ starved and disappeared when internal K+ increased, which makes it unlikely that HvHKT1 mediates K+ uptake in roots. Mutation of the first in-frame AUG codon of HvHKT1 to CUC changed the uniport function into symport. The expression of the symport from either mutants or constructs keeping the first in-frame AUG took place only in K+-starved cells, while the uniport was expressed in all conditions. We discuss here that the symport occurs only in heterologous expression. It is most likely related to the K+ inhibitable Na+ uptake process of roots that heterologous systems fail to reproduce.

Plants take up large amounts of K from the soil solution and distribute it to the cells of all organs, where it fulfills important physiological functions. Transport of K from the soil solution to its final destination is mediated by channels and transporters. To better understand K movements in plants, we intended to characterize the function of the large KT-HAK-KUP family of transporters in rice (Oryza sativa cv Nipponbare). By searching in databases and cDNA cloning, we have identified 17 genes (OsHAK1–17) encoding transporters of this family and obtained evidence of the existence of other two genes. Phylogenetic analysis of the encoded transporters reveals a great diversity among them, and three distant transporters, OsHAK1, OsHAK7, and OsHAK10, were expressed in yeast (Saccharomyces cerevisiae) and bacterial mutants to determine their functions. The three transporters mediate K influxes or effluxes, depending on the conditions of the experiment. A comparative kinetic analysis of HAK-mediated K influx in yeast and in roots of K -starved rice seedlings demonstrated the involvement of HAK transporters in root K uptake. We discuss that all HAK transporters may mediate K transport, but probably not only in the plasma membrane. Transient expression of the OsHAK10-green fluorescent protein fusion protein in living onion epidermal cells targeted this protein to the tonoplast., This work was supported by the Consejería de Educación y Cultura de la Comunidad de Madrid (Programa de Grupos Estratégicos) and by the Ministerio de Ciencia y Tecnología (grant no. BIO2000– 0938).

The cDNAs CnHAK1 and CnHAK2, encoding K+ transporters, were amplified from the leaves of the seagrass Cymodocea nodosa. None of these transporters suppressed the K+ deficiency of a Saccharomyces cerevisiae mutant, but both suppressed the equivalent defect of an Escherichia coli mutant. Overexpression of the transporter CnHAKI, but not CnHAK2, mediated very rapid K+ or Rb+ influxes in the E. coli mutant. The concentration dependence of these influxes demonstrated that CnHAK1 is a low-affinity K+ transporter, which does not discriminate between K+ and Rb+. CnHAK1 expressed in E. coli worked in reverse when the external K+ concentrations were low, and we established the condition of a simple functional test of K+ loss for transporters of the Kup-HAK family. In comparison with its homologue barley transporter HvHAK2, CnHAKI was insensitive to Na+.

Plant K(+) transporters of the HAK family belong to four rather divergent phylogenetic clusters, although most of the transporters belong to clusters I or II. A simple phylogenetic analysis of fungal and plant HAK transporters suggests that an original HAK gene duplicated even before fungi and plants diverged, generating transporters that at present fulfill different functions in the plant. The HvHAK1 transporter belongs to cluster I and mediates high-affinity K(+) uptake in barley roots, but no function is known for the cluster II transporter, HvHAK2, which is not functional in yeast. The function of HvHAK2 was investigated by constructing HvHAK1-HAK2 chimeric transporters, which were not functional even when they included only short fragments of HvHAK2. Then, amino acids characteristic of cluster II in the N terminus and in the first transmembrane domain were introduced into HvHAK1. All of these changes increased the Rb(+) K(m), introducing minimal changes in the Na(+) K(m), which suggested that HvHAK2 is a low-affinity, Na(+)-sensitive K(+) transporter. Using a K(+)-defective Escherichia coli mutant, we functionally expressed HvHAK2 and found that the predicted characteristics were correct, as well as discovering that the bacterial expression of HvHAK2 is functional at pH 5.5 but not at 7.5. We discuss whether HvHAK2 may be a tonoplast transporter effective for vacuolar K(+) depletion in K(+) starved plants.

Recent progress has been made in the characterization of cation transporters that maintain ion homeostasis during salt stress in plants. Sodium-proton antiporters at the vacuolar (NHX1) and plasma membrane (SOS1) have been identified in Arabidopsis. SOS1 is regulated by the calcium-activated protein kinase complex SOS2-SOS3. In yeast, a transcription repressor, Sko1, mediates regulation of the sodium-pump ENA1 gene by the Hog1 MAP kinase. The recent visualization at the atomic level of the inhibitory site of sodium in the known target Hal2 has helped identify the interactions determining Na(+) toxicity.

Using PCR, reverse transcription-PCR (RT-PCR) and colony hybridization in a genomic library, we isolated six genes which encode type II P-type ATPases in Neurospora crassa. The six full-length cDNAs were cloned in a yeast expression vector and transformed into Saccharomyces cerevisiae null Ca2+- or Na+-ATPase mutants. Three cDNAs suppressed the defect of the Ca2+ mutant and two of these protected from Mn2+ toxicity. One cDNA suppressed the defect of the Na+ mutant and two cDNAs were not functional in S. cerevisiae. The expression of the transcripts of the six genes in the presence of Ca2+, Na+, high pH or supporting an osmotic shock indicated that, with the exception of one of the Ca2+-ATPases, the main function of the cloned ATPases is the adaptation to stress conditions. The relationship between the cloned fungal Ca2+- and Na+-ATPases and plant type II P-ATPases is discussed.

The high-affinity K+ uptake system of plants plays a crucial role in nutrition and has been the subject of extensive kinetic studies. However, major components of this system remain to be identified. We isolated a cDNA from barley roots, HvHAK1, whose translated sequence shows homology to the Escherichia coli Kup and Schwanniomyces occidentalis HAK1 K+ transporters. HvHAK1 conferred high-affinity K+ uptake to a K(+)-uptake-deficient yeast mutant exhibiting the hallmark characteristics of the high-affinity K+ uptake described for barley roots. HvHAK1 also mediated low-affinity Na+ uptake. Another cDNA (HvHAK2) encoding a polypeptide 42% identical to HvHAK1 was also isolated. Analysis of several genomes of Triticeae indicates that HvHAK1 belongs to a multigene family. Translated sequences from bacterial DNAs and Arabidopsis, rice, and possibly human cDNAs show homology to the Kup-HAK1-HvHAK1 family of K+ transporters.

The ENA1 gene of Saccharomyces cerevisiae encodes a putative ATPase necessary for Na+ efflux. Plasma membranes and intracellular membranes of a yeast strain overexpressing the ENA1 gene contain significant amounts of ENA1 protein. Consequences of the overexpression with reference to the wild-type strain are: (1) a 5-fold higher content of the ENA1-protein in plasma membranes; (2) lower Na+ and Li+ effluxes; (3) slightly higher Na+ tolerance; and (4) much higher Li+ tolerance. The ENA1-specific ATPase activity in plasma membrane preparations of the overexpressing strain was low, but an ENA1 phosphoprotein was clearly detected when the plasma membranes were exposed to ATP in the presence of Na+ or to Pi in the absence of Na+. The characteristics of this phosphoprotein, which correspond to the acyl phosphate intermediaries of P-type ATPases, the absolute requirement of Na+ or other alkali cations for phosphorylation, and the Na+ and pH dependence of phosphorylation from ATP and Pi suggest that the product of the ENA1 gene may be a Na,H-ATPase, which can also pump other alkali cations. The role of the intracellular membranes structures produced with the overexpression of ENA1 in Na+ and Li+ tolerances and the existence of a β-subunit of the ENA1 ATPase are discussed.

A cDNA library in a yeast expression vector was prepared from roots of Arabidopsis exposed to salt and was used to select LI+-tolerant yeast transformants. The cDNA SAL1 isolated from one of these transformants encodes a polypeptide of 353 amino acid residues. This protein is homologous to the HAL2 and CysQ phosphatases of yeast and Escherichia coli, respectively. Partial cDNA sequences in the data bases indicate that rice produces a phosphatase highly homologous to SAL1 and that a second gene homologous to SAL1 exists in Arabidopsis. The SAL1 protein expressed in E. coli showed 3[prime](2[prime]),5[prime]-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. In yeast, SAL1 restored the ability of a hal2/met22 mutant to grow on sulfate as a sole sulfur source, increased the intracellular Li+ tolerance, and modified NA+ and LI+ effluxes. We propose that the product of SAL1 participates in the sulfur assimilation pathway as well as in the phosphoinositide signaling pathway and that changes in the latter may affect Na+ and Li+ fluxes., This work was funded in part by the EuropeanCommunities’ BIOTECH Programme, as part of the Projectof TechnologicalPriority 1993-1996, and by project PB92-O907 from Dirección General de Investigación Científica y Técnica, Spain.

Fermentable sugars activated the K+ uptake system, increasing the Vmaxs of Rb+, Na+, and Li+ influxes, but sugars did not affect the effluxes of these cations. This activation seems to be a direct effect of fermentation and not the consequence of the H+ pump ATPase activation or internal pH decrease produced by fermentation.

The gene ENA1 was cloned by its ability to complement the Li+ sensitivity of a low Li+-efflux strain. The nucleotide sequence of the cloned DNA fragment showed that there are two almost identical genes in tandem, and predicts that they encode P-ATPases. Disruption of both genes originated a strain defective in Na+ and Li+ effluxes, and sensitive to Na+, to Li+ and to alkaline pH. By transformation with ENA1 the defective effluxes and tolerances were repaired.

To investigate the regulation of K+ fluxes in Saccharomyces cerevisiae the dependence of K+ efflux and Rb+ influx on [K+]i, pHi, [Na+]i, membrane potential, cell volume, and turgor pressure were studied in cells with different K+ contents. By decreasing the cell volume with osmotic shocks and the cellular pH with butyric acid the following was found. (1) The K+ efflux induced by uncouplers decreases simultaneously with the decrease of the K+ content of the cell, but the process was insensitive to [K+]i, pHi, cell volume and turgor pressure. The internal presence of Na+ inhibited this K+ efflux. (2) The increase of the Vmax of Rb+ influx observed in low-K+ cells is due to the decrease of the pHi and probably mediated by the increase of the activity of the plasma membrane ATPase. The Vmax is independent of [K+]i, [Na+]i, cell volume and turgor pressure. (3) The decrease in the Km of Bt+ influx observed in low-K+ cells does not depend directly on [K+]i, pHi, cell volume or turgor pressure. If Na+ is present, [Na+]i might be directly involved in the regulation of the Km.

In a newly formulated growth medium lacking Na+ and NH4+, Saccharomyces cerevisiae grew maximally at 5 microM K+. Cells grown under these conditions transported K+ with an apparent Km of 24 microM, whereas cells grown in customary high-K+ medium had a significantly higher Km (2 mM K+). The two types of transport also differed in carbonyl cyanide-m-chlorophenyl hydrazone sensitivity, response to ATP depletion, and temperature dependence. The results can be accounted for either by two transport systems or by one system operating in two different ways.

Rb + transport in low-K + cells of Neurospora crassa is biphasic, transport at millimolar Rb + being added to a transport process which saturates in the micromolar range. Both processes exhibit Michaelis-Menten kinetics, but in the micromolar phase the kinetic parameters depend on the K + content of the cell (the lower the K + content the lower the K m and the higher the V max ). Normal-K + cells, suspended in a buffer with millimolar K + , do not present Rb + transport in the micromolar range. Millimolar transport in these cells presents kinetics which depend on the K + in buffer (the higher the K + the higher the K m ), although the K + content of the cells is constant. Na + inhibits competitively Rb + transport in low-K + and normal-K + cells, but, even when the differences between the Rb + K m values are more than three orders of magnitude, the apparent dissociation constant for Na + is the same, and millimolar, in both cases.

In growing cells, K+ and Rb+ effluxes followed first order kinetics, took place with slight differences between K+ and Rb+ and were stimulated at acidic pH values. In uncoupled cells and ATP-depleted cells, K+ efflux was higher than Rb+ efflux. In ATP-depleted cells, the effluxes were not stimulated at acidic pH values. Two different K+ effluxes are proposed: (i) the normal efflux, which may be a K+/H+ antiport and does not function in ATP-depleted cells, and (ii) the backward reaction of the uptake system, which can be high in depolarized cells. The role of the normal efflux system in K+ content and pH regulation is discussed.

A mutant in Saccharomyces cerevisiae required one hundred times more K+ than wild type for the same half maximal growth rate. Mutant cells and wild type cells grown at millimolar K+ did not show significant differences in Rb+ transport. In the mutant, a rapid K+ loss induced by azide or incubation (4 h) in K+-free medium decreased the Rb+ transport K m by one half; in the wild type, those treatments decreased the Rb+ K m twenty and one hundred times, respectively. Mutant and wild type did not show significant differences in Na+ transport and in the Na+ inhibition of Rb+ transport, either in normal-K+ cells or in K+-starved cells. The results suggest that either two systems or one system with two interacting sites mediate K+ transport in S. cerevisiae.

The growth rate ofSaccharomyces cerevisiae was dependent on K+ content in culture medium in a certain range of K+ concentrations. Above the upper limit of the range, growth did not respond to K+ increase, and below the lower limit, yeast died. Rb+ and Na+ enhanced growth in the range of K+ dependence and decreased the K+ concentration below which cells died. Both Rb+ and Na+ became toxic above a certain Rb+/K+ and Na+/K+ cellular ratio.

Low-K+, high-Na+ cells of strain RL21a of Neurospora crassa, in steady state with 25 mM Na+, were used to study K+/Na+ exchanges in the presence or absence of Ca2+ and Mg2+. In the presence of Ca2+ and Mg2+, a low concentration of K+ (0.3 mM) triggered a rapid exchange, but in the absence of the divalents, a high K+ concentration (30 mM) was required to initiate the exchange at a rapid rate. In the absence of Ca2+ and Mg2+, K+ uptake did not occur at low K+ concentration, internal K+ did not regulate Na+ influx in the presence of external K+, and the efflux of Na+ proceeded at maximum activity at very low-K+ contents.

Combined ion flux and electrophysiological measurements have been used to characterized active transport of potassium by cells of Neurospora crassa that have been moderately starved of K+ and then maintained in the presence of millimolar free calcium ions. These conditions elicit a high-affinity (K1/2 = 1-10 microM) potassium uptake system that is strongly depolarizing. Current-voltage measurements have demonstrated a K+-associated inward current exceeding (at saturation) half the total current normally driven outward through the plasma membrane proton pump. Potassium activity ratios and fluxes have been compared quantitatively with electrophysiological parameters, by using small (approximately 15 micron diam) spherical cells of Neurospora grown in ethylene glycol. All data are consistent with a transport mechanism that carries K ions inward by cotransport with H ions, which move down the electrochemical gradient created by the primary proton pump. The stoichiometry of entry is 1 K ion with 1 H ion; overall charge balance is maintained by pumped extrusion of two protons, to yield a net flux stoichiometry of 1 K+ exchanging for 1 H+. The mechanism is competent to sustain the largest stable K+ gradients that have been measured in Neurospora, with no direct contribution from phosphate hydrolysis or redox processes. Such a potassium-proton symport mechanism could account for many observations reported on K+ movement in other fungi, in algae, and in higher plants.

Na+ influx and efflux in Neurospora crassa RL21a can be studied separately to calculate net Na+ movements. In the absence of external K+, Na+ influx was independent of the K+ content of the cells, but when K+ was present, the inhibition of Na+ influx by external K+ was higher the higher the K+ content. Efflux depended on the K+ and Na+ content, and on the history of the cells. Efflux was higher the higher the Na+ and K+ contents, and, in low-K+ cells, the efflux was also higher in cells grown in the presence of Na+ than when Na+ was given to cells grown in the absence of Na+. Addition of K+ to cells in steady state with external Na+ resulted in a net Na+-loss. In cells grown without Na+ this loss was a consequence of the inhibition of Na+ influx. In Na+-grown cells, addition of K+ inhibited Na+ influx and increased Na+ efflux.

The Rb+-uptake kinetics in K+-starved sunflower (Helianthus annuus) plants can be explained by the addition of two Michaelis-Menten equations. In contrast, Rb+ uptake can be described by a single Michaelis-Menten equation in normal-K+ plants. Differences in the Kms and in the Arrhenius plots of Rb+ uptake in the two types of plants suggest two uptake modes.

(1) The temperature- and pH-dependence of Rb + uptake in low-K + and normal-K + cells were studied to accumulate information on the systems involved. Arrhenius plots of V max values in the two phases of low K + cells were identical, and different from the Arrhenius plot of normal-K + cells' V max . The pH-dependences of Rb + uptake in the two phases of low-K + cells and in normal-K + cells were all different. (2) The Rb + uptake in low-K + cells was not affected by a rapid exchange of external medium, ruling out the idea of the dual-uptake isotherm being an artifact due to unstirred layers. (3) Metabolic blockage induced net K + loss in normal-K + cells but not in low-K + cells. That loss had the characteristics of a K + diffusion. It is concluded that in low-K + cells, an Rb + -H + cotransport occurs at low Rb + and an Rb + -Rb + cotransport at high Rb + . In normal-K + cells, Rb + uptake may be mediated by the system which mediates the K + loss induced by metabolic blockage.

Eukaryotic KcsA-related K + transporters mediate physiologically relevant K + and Na + fluxes in fungi and plants. ScTRK1 is a characteristic member of the group, and here we report a mutational analysis of the unique M2 D helix of this transporter. Our results support the theoretical models placing this helix in a relevant position in the pore and interacting with P segments. Most single mutations eliminating positively charged or introducing negatively charged residues reduced the V max of Rb + influx to a half, several together showed an additive effect, and four practically suppressed transport. In contrast, the introduction of only one positively charged residue practically abolished the function of the transporter. Almost all mutations in the M2 D helix affected the two Rb + binding sites of the transporter, mimicking mutations in the selectivity filter.

5 páginas, 5 figuras, 1 tabla, 32 referencias., NaC1-sensitive yeast mutants were isolateod i dentify genes essential for NaCl tolerance. Complementation of a mutant highly sensitive to Na+ and Li+ led to the isolation of the CNBl gene. This gene encodes the regulatory subunit (CNB) of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Cells deficienitn CNB accumulated Li+ due to reduced expression of ENAl, a gene encoding a P-type ATPase involved in Na+ and Li+ efflux. In addition, the K+ transport system of cnblA cells was not converted to the high affinity state that facilitates better discriminationo f K+ over Na+. Thust he cnblA strain resembled a trk1 mutant. These results indicate that adaptation to NaCl stress in Saccharomyces cerevisiae requires a signal transduction pathway involving Ca2+ and protein phosphorylation-dephosphorylation. In this pathway, calcineurin would coordinate gene expression and activity of ion transporters to facilitate ion homeostasis., This work was supported by grants PB89-0180 from Dirección General de Investigación Científica y Técnica (to A. R.-N.)and AGR91-0858 from Plan Nacional de Investigación Agraria (to J. M. P.).