Function and Regulation of the Saccharomyces cerevisiae ENA Sodium ATPase System

Despite the fact that sodium ions are necessary to assist in the function of specific macromolecules (65), for most eukaryotic cells an elevated intracellular concentration of sodium ions is deleterious, probably because they interfere with the correct functioning of certain cellular targets (83). Because of the abundance of sodium ions in natural environments, yeasts and many other eukaryotic cells have developed mechanisms to maintain a relatively low intracellular concentration of this cation and/or to sequester it into organelles. The yeast Saccharomyces cerevisiae is a model organism used very often for cation tolerance studies. In this organism, maintenance of intracellular low sodium when sodium is present in excess in the external medium relies heavily on the capacity to extrude the cation. This is achieved by two main extrusion mechanisms. The first one is an H⁺/Na⁺ antiporter encoded by the NHA1 gene, which is able to extrude Na⁺, Li⁺, and even K⁺ cations by exchange with protons, gaining biological significance at rather acidic external pHs (8, 73). The second is based on a P-type ATPase pump encoded by the ENA system, which is the subject of this review. Although both mechanisms are complementary, comparison of the phenotypes of strains lacking functional antiporter or ATPase genes suggests that under standard growth conditions, the ENA system is the major determinant for sodium detoxification. The importance of this efflux system is highlighted by the observation that it seems to be present in all fungi as well as in certain nonflowering plants, such as bryophytes. In addition, active research with S. cerevisiae has revealed a complex and sophisticated system of transcriptional regulation in response to specific environmental changes, which is the major issue of this review.

GENE STRUCTURE, FUNCTIONAL ROLES, AND SUBCELLULAR LOCALIZATION

P-type ATPases couple hydrolysis of ATP to transport of cations against electrochemical gradients by forming a typical phosphoenzyme intermediate (17). The ENA type (also known as IID) corresponds to a subfamily of P-type ATPases able to extrude Na⁺, Li⁺, and K⁺ with different capacities (10). Genes encoding putative or demonstrated functionally equivalent orthologs have been identified in other fungi, such as Schwanniomyces occidentalis (7) and Neurospora crassa (9), and these proteins are probably present in most fungi, although not in flowering plants (30). However, two Na⁺-ATPases have been characterized for the moss Physcomitrella patens (12), and at least one of them (PpENA1) appears to be functional as a sodium pump both in S. cerevisiae (12) and in planta (49). This led to the proposal that ENA-like sodium pump ATPases were present in early land plants (12).

A remarkable characteristic is that S. cerevisiae contains diverse copies of genes encoding identical or very similar ENA proteins (31, 36, 52, 94). ENA1 was initially identified as a putative calcium P-type ATPase gene on the basis of its sequence characteristics (78), and the locus was named PMR2 (for plasma membrane ATPase related). Two years later, its capacity to extrude Na⁺, Li⁺, and probably K⁺ was reported, thus defining its primary role in yeast biology (36). This experimental evidence is reflected in the name given by Rodriguez-Navarro and coworkers (36), i.e., exitus natru, for the latin words meaning “exit sodium.” ENA1 is currently the accepted gene name, whereas PMR2 and HOR6 are considered aliases. Notably, in S. cerevisiae the ENA genes are disposed on chromosome IV in tandem repeats with a variable number of copies. Thus, strains DBY746 and W303-1A contain four copies (31, 77), while strains S288C, A364A, and D273-10B contain five repeats (94). In contrast, other strains contain only two repeats, or even a single one. Strain FY1679, which was used for the systematic sequencing of the yeast genome, carries three ENA genes, namely, ENA1 (YDR040c), ENA2 (YDR039c), and ENA5 (YDR038c). All three encoded proteins contain 1,091 amino acids and are very similar in sequence (>97%), although ENA1 is slightly more distant from ENA2 and -5. Under standard growth conditions, Ena1, -2, and -5 are present in the cell at similar and rather low levels (around 600 molecules/cell) (33), although as discussed later in detail, ENA1 expression markedly increases in response to saline or alkaline pH stress. Other fungi, such as Candida albicans or Debaryomyces hansenii, also contain several ENA paralogs, although in most cases their sequences are less conserved, suggesting earlier gene duplication events (see below).

Lack of the entire ENA cluster in S. cerevisiae results in a dramatic phenotype of sensitivity to sodium and lithium cations (31, 36, 70, 77, 94) and a growth defect at alkaline pHs (36, 69) (Fig. 1). Tolerance to Li⁺ is largely restored by expression of ENA1 (31, 77), indicating that this gene is the most functionally relevant component of the cluster. Expression of S. cerevisiae ENA1 in other fungi, such as Schizosaccharomyces pombe, which lacks Na⁺-ATPase genes (6), or in plant cells (64) markedly increases tolerance to Na⁺ and Li⁺ and decreases the intracellular content of these cations, leaving little doubt about the importance of this ATPase in cation detoxification. It has been proposed, however, that Ena1/Pmr2A and Ena2/Pmr2B confer distinct sodium and lithium tolerances, with the former being more effective at extruding Na⁺ cations and the latter being more specific for Li⁺ (94). It was postulated that this behavior is caused by the slight divergence in the structures of the proteins, not by their different expression levels.

FIG. 1.

Lack of the ENA system dramatically decreases sodium, lithium, and alkaline pH tolerance in S. cerevisiae. The wild-type strain DBY746 and its isogenic derivative RH16.6, which lacks the entire ENA cluster (36), were grown for 3 days at 28°C under the indicated conditions. Three dilutions of the culture (1:10) are shown.

Although most of this review focuses on the role of the ENA system in detoxification of sodium cations, it is important to remark that in some fungi, these ATPases can play a major role in potassium homeostasis. This would be particularly relevant at higher-than-neutral external pHs, when the fungal K⁺/H⁺ antiporters would lose efflux capacity. The individual components of the ENA system in S. cerevisiae seem to be equally competent in extruding potassium and sodium, although in the presence of high external sodium, they preferentially extrude this cation (10). Both DhENA1 and DhENA2 from the halotolerant yeast Debaryomyces hansenii also appear able to protect cells from sodium or potassium stress (4). In contrast, SpCTA3, which has been proposed to be an ENA-like ATPase in Schizosaccharomyces pombe, acts essentially as a K⁺-ATPase (10). In some cases, such as in the soil yeast Schwanniomyces occidentalis, gene duplication seems to provide some degree of specialization. In this organism, which carries two Ena proteins, Ena1 appears to be an efficient sodium pump, whereas Ena2 acts as a K⁺-ATPase (7). A similar situation could exist in Neurospora crassa, which carries rather divergent Ena ATPases. In this case, NcEna1 would act as a Na⁺-ATPase, whereas the physiological function of the other two remains unknown (9, 10).

In comparison to our understanding of the transcriptional regulation of ENA1, little is known on the biochemistry of the Ena ATPases. The formation of the acyl phosphate intermediate characteristic of P-type ATPases was proved experimentally years ago with S. cerevisiae overexpressing ENA1 (11). Inspection of the amino acid sequences of the Ena proteins suggests the presence of 9 or 10 transmembrane domains. Localization at the cell membrane has been demonstrated for S. cerevisiae Pmr2a/Ena1 and Pmr2b/Ena2 (94) and for S. occidentalis (7), although it has been reported that overexpression of ENA1 also results in the presence of the protein in intracellular membrane structures (11). Interestingly, SRO7/SOP1, a homolog of the Drosophila Lgl tumor suppressor gene, encoding a protein involved in exocytic docking and fusion of Golgi apparatus-derived vesicles with the plasma membrane, was recently identified as being required for appropriate delivery of Ena1 to the plasma membrane (93). It is remarkable that the role of Sro7 could be relatively specific, since sensitivity to NaCl is the only evident phenotype of sro7 cells (45) and other membrane-directed proteins do not present altered membrane targeting (93).

TRANSCRIPTIONAL REGULATION OF ENA GENES

Early reports pointed out that the expression of ENA1, but not that of other members of the cluster, was potently increased by exposure to high Na⁺ or Li⁺ levels or alkaline pH (31, 60, 70, 94). The molecular basis of this response has been the subject of active investigation. We know now that exposure to salt or high pH causes the activation of diverse signaling pathways that are integrated in the ENA1 promoter, as demonstrated in studies pioneered by Marquez and Serrano more than 10 years ago (51). In addition, the gene is able to respond to defined nutritional signals, such as glucose or nitrogen availability. Altogether, the available information configures a picture clear enough to understand the major traits of the process (Fig. 2). Remarkably, the environmental challenges affecting ENA1 expression are rather specific. The ATPase gene does not respond transcriptionally to most stress conditions and does not belong to the genes involved in the so-called general environmental stress response. Similarly, despite initial suggestions on the regulation of ENA1 expression by stress-responsive element (STRE)-related sequences (38), further investigations ruled out this possibility (3, 75, 84).

FIG. 2.

Schematic depiction of the regulation of the ENA1 promoter in S. cerevisiae. Discontinuous lines denote regulatory interactions that remain to be documented. Regulatory sites in the promoter (which is not drawn to scale) are indicated in the inset. See the main text for details and references.

Regulation of ENA1 by the HOG osmoresponsive pathway.

Exposure of yeast cells to relatively low extracellular concentrations of NaCl (∼0.4 M) involves a situation of osmotic stress. Activation of the high-osmolarity glycerol (HOG) pathway in response to high osmolarity results in phosphorylation and activation of the Hog1 mitogen-activated protein (MAP) kinase (22). Under these circumstances, a lack of Hog1 largely attenuates the transcriptional response of ENA1 (51). Regulation of ENA1 by Hog1 is accomplished through the bZip transcription factor Sko1, one of the downstream targets of the kinase. Sko1 acts as a repressor by recruiting the general corepressor complex Ssn6-Tup1 to a cyclic AMP (cAMP) response element (CRE) site in the ENA1 promoter (positions −502 to −513) (75). In response to osmotic stress, activated Hog1 phosphorylates diverse residues at the Sko1 N-terminal region, and this phosphorylation disrupts the Sko1-Ssn6-Tup1 repressor complex (74). In contrast, phosphorylation of Sko1 sites near the bZIP domain by cAMP-dependent protein kinase (PKA) increases its repressor activity (74). Since exposure to NaCl decreases the level of cAMP in yeast cells (51), inhibition of PKA activity would further diminish the repressor capacity of Sko1. In addition to the Sko1-mediated mechanism, it has been shown that activated Hog1 recruits the specific Rpd3-Sin3 histone deacetylase complex to the promoter of ENA1 (among other osmotically responsive genes), leading to histone deacetylation, entry of RNA polymerase II, and induction of the expression of the gene upon saline stress (25).

Whereas the HOG pathway represents an important component of the transcriptional response of ENA1 to NaCl, exposure of yeast cells to relatively low concentrations of LiCl (50 to 100 mM) causes a growth defect that is not aggravated by deletion of HOG1 (79). Under these conditions, expression of ENA1 is already significantly increased (four- to eightfold), and the results are only modestly affected in hog1 mutants (A. Ruiz, A. González, and J. Ariño, unpublished results). Therefore, at sublethal Li⁺ concentrations, which pose a limited degree of osmotic stress to the cells, the HOG pathway may have minor relevance for ENA1 transcription.

The transcriptional response of ENA1 to alkaline pH is not decreased at all in a hog1 mutant (84). Moreover, a fragment of the ENA1 promoter containing the CRE region as the sole transcriptionally functional element is unable to elicit a high-pH response (69). These results, together with the evidence that a hog1 mutant is not sensitive to high pH, strongly suggest that the transcriptional response of ENA1 to alkaline pH does not involve activation of the Hog1 kinase.

Regulation of ENA1 by the calcineurin pathway.

Calcineurin, also known as type 2B Ser/Thr protein phosphatase, is a heterodimeric enzyme that in S. cerevisiae is composed of one of two redundant catalytic subunits (encoded by CNA1 and CNA2) and a single regulatory subunit (encoded by CNB1). Calcineurin is considered a stress-related enzyme, and its relevance to ionic homeostasis has been evidenced by the sensitivity to Na⁺, Li⁺, or high pH of Cnb1-deficient mutants or wild-type cells treated with the specific inhibitor FK506 (60, 63).

Under standard growth conditions, cytosolic calcium levels are low. Exposure of yeast cells to high sodium or pH provokes a rapid cytosolic calcium burst that results in activation of calcineurin (26, 55, 90). The origin of cytosolic calcium upon saline stress is somewhat controversial, as it has been proposed to be of vacuolar origin and mediated by the Yvc1 channel (26) or of extracellular origin and imported into the cells through the Mid1-Cch1 channel (55). Among other effects (some of them also relevant to saline tolerance), activation of calcineurin by external NaCl or calcium triggers the expression of more than 150 genes (99). This effect is caused mainly by dephosphorylation and entry into the nucleus of the transcription factor Crz1/Tcn1/Hal8 (54, 58, 86), which then binds to specific sequences, known as calcineurin-dependent response elements (CDREs), in calcineurin-responsible promoters. Phosphorylation of Crz1 by protein kinases such as Hrr25 or PKA promotes exit from the nucleus. Therefore, these kinases counteract the calcineurin-mediated activation of Crz1 (40, 41).

The link between calcineurin signaling and ENA1 regulation has been known for quite a long time. The accumulation of sodium or lithium cations in calcineurin-deficient cells could be attributed, at least in part, to a decrease in efflux capacity as a result of impaired induction of ENA1 expression (38, 51, 60). In contrast, hyperactivation of calcineurin increases ENA1 expression and improves salt tolerance (59, 66). Calcineurin activation of ENA1 is mediated by dephosphorylation of Crz1 (57, 86). Two Crz1 binding regions have been identified in the ENA1 promoter, at positions −813 to −821 and −727 to −719 (Fig. 2), with the downstream element being the most relevant for induction of the gene (57). As mentioned above, PKA can phosphorylate Crz1, thus opposing the action of calcineurin (40). This may explain the previously observed antagonistic regulation of ENA1 by the calcineurin and PKA pathways (38), which is supported by the observation that salt stress (0.25 to 1.0 M NaCl) decreases intracellular levels of cAMP (51).

Exposure of yeast cells to high pH triggers an almost immediate entry of external calcium into the cytosol, through the Cch1-Mid1 calcium channel (90), that results in activation of the calcineurin pathway and binding of Crz1 to the ENA1 promoter, most probably at the downstream CDRE (57, 84, 90). About 40% of the total response to high pH seems to be mediated by activation of calcineurin (69, 84). Very recently, it was shown that Mg²⁺ deprivation elicits a rapid uptake of calcium ions and that this results in induction of ENA1 caused by activation of the calcineurin/Crz1 pathway (95).

The regulatory role of calcium and calcineurin in ENA1 expression was also revealed by mutations affecting cytosolic calcium levels. For instance, cells lacking Pmc1, a vacuolar Ca²⁺-ATPase responsible for depleting cytosol of Ca²⁺ ions, display increased ENA1 expression that is dependent on calcineurin activity (21). Similarly, pmr1 mutants, which are deficient in a P-type ATPase required for Ca²⁺ and Mn²⁺ transport into the Golgi apparatus, show higher-than-normal ENA1 expression levels (66).

Regulation through the Rim101 pathway.

Rim101 is a C₂H₂ zinc finger transcription factor that was identified years ago as a positive regulator of the expression of genes involved in meiosis and sporulation (87). Rim101 also appears to be the S. cerevisiae homolog of PacC, an Aspergillus nidulans transcription factor that is a key element in the transcriptional activation of genes required for adaptation of this fungus to alkalinization of the environment (see reference 68 for a review). Yeast cells lacking Rim101 are sensitive to both high salt and high pH (44), and rim101 cells display decreased expression of ENA1 upon alkaline stress (44, 84). Remarkably, the mechanism of action of Rim101 on most of its target genes seems to be indirect, mediated by repression of other regulatory genes (43). Therefore, in S. cerevisiae this protein would essentially act as a transcriptional repressor, not an activator. In the case of ENA1, activation of Rim101 would repress the expression of Nrg1, a repressor protein that was shown to negatively control the expression of several glucose-repressed genes and invasive growth and that is regulated by the Snf1 protein kinase (92). Consistent with this model, the nrg1 mutation confers tolerance to Na⁺ and Li⁺ and suppresses the rim101 growth defect at high pH (43). Cells lacking Nrg1 express ENA1 at higher-than-normal levels, even in the absence of stress (43), and this effect is exacerbated by further deletion of NRG2, encoding a repressor protein partially redundant in function with Nrg1 (91). Initially, two putative binding sites for Nrg1 were suggested to be present in the ENA1 promoter (CCCTC and CCCCT), located at positions −725 to −729 and −651 to −647, respectively (43). It is remarkable that the upstream site partially overlaps with the most downstream and functionally relevant CDRE binding region (positions −727 to −719). The upstream Nrg1 binding site could be biologically important, as suggested by functional mapping of the ENA1 promoter in response to alkaline stress, whereas the downstream element appears to be less important or ineffective (84). Interestingly, the same report suggested the existence of an additional Rim101-regulated element that mapped in the so-called ARR2 region (−573 to −490). Recent work has proved that Nrg1 binds to this region in vivo and that alkaline stress promotes the loss of this specific protein-DNA interaction (69). Mapping of the ARR2 region suggests that binding occurs at positions −561 to −573, where an AGACCCT sequence can be found. This sequence closely matches the consensus binding sequence (GGACCCT) recently proposed for Nrg1 (50). All of this evidence suggests that Rim101 may control ENA1 expression in response to alkaline stress through the Nrg1 repressor protein. As mentioned above, a rim101 strain is sensitive to sodium and lithium, whereas a nrg1 mutant is tolerant (43, 91). The presence of functional Nrg1 binding sites in the ENA1 promoter would suggest that release of Nrg1-mediated repression would be an important component of the ENA1 transcriptional response to high salt and responsible, at least in part, for the observed phenotypes. However, it must be noted that deletion of Rim101 has little effect on the ENA1 response to 0.4 M NaCl (69), 0.9 M NaCl, or 0.2 M LiCl (M. Platara, A. Ruiz, and Ariño, unpublished observations), suggesting that this pathway might be more relevant under alkaline stress than under high-salt conditions.

Regulation of ENA1 by glucose and nitrogen sources.

The expression of ENA1 is substantially higher on derepressing carbon sources, such as galactose or raffinose, than on glucose (3, 76). Similarly, yeast cells lacking the Snf1 protein kinase, a key player in glucose repression, display enhanced sensitivity to sodium or lithium, and this can be attributed to the fact that induction of ENA1 by high salt is markedly impaired in a Snf1-deficient strain. The Snf1-mediated effect on ENA1 expression was determined to be independent of the HOG and calcineurin pathways (3). Proft and Serrano identified a URS_MIG-ENA1 element at promoter positions −533 to −544 to which the Mig1/2-Ssn6-Tup1 complex could bind, thus mediating ENA1 repression (75). It was proposed that glucose starvation would activate Snf1, which in turn would prevent Mig1/2-Ssn6-Tup1-mediated repression of ENA1. However, although it has been shown that Snf1 is phosphorylated and activated by saline stress (39, 56), this does not result in phosphorylation of Mig1 (56), and Snf1 remains excluded from the nucleus (39). The specific role of Snf1 in saline tolerance and ENA1 expression might be modulated by its functional link to Gis4, a recently uncovered component of the cation homeostatic mechanisms in yeast whose absence decreases saline tolerance, sodium and lithium efflux, and ENA1 expression (97). However, the precise form in which this happens is still obscure, and there are discrepancies on whether the function of Gis4 is specific for saline stress or also influences the role of Snf1 in glucose repression (46, 97).

Induction of ENA1 by high pH is markedly reduced in cells lacking SNF1 (3, 69). In contrast to what has been described for saline stress, Snf1 becomes enriched in the nucleus when cells are exposed to high pH, similar to what happens for carbon stress (39). Recent work (69) has demonstrated that activation of Snf1 affects the expression of ENA1 in two ways, by releasing repression mediated through the MIG element and by deactivating the Nrg1 repressor. In turn, the Nrg1 repressor would be under the control of two regulators, Snf1 and Rim101 (see above and Fig. 2). It is conceivable that exposure to high pH could actually interfere with normal glucose metabolism and hence mimic a glucose shortage situation, as deduced by the remarkable overlap in gene expression profile observed when both conditions are compared (90).

The TOR cascade plays an important role in regulating nutrient-responsive transcription (20). Less favorable nitrogen sources or rapamycin treatment inhibits Tor kinases, and this causes the subsequent dissociation of Ure2 from the GATA transcription factor Gln3 (upon dephosphorylation mediated by the Sit4 phosphatase) and the entry of Gln3 into the nucleus. Nuclear Gln3 would be able to bind to GATA-rich regions in the promoters of responsive genes and to induce their expression. It was reported some time ago that mutations in URE2 suppressed the sensitivity of calcineurin mutants to Na⁺ or Li⁺ cations and that this effect required the presence of the ENA1 gene (96). Crespo and coworkers demonstrated that treatment of yeast cells with rapamycin transiently increased ENA1 expression and that this effect virtually disappeared in strains lacking Gln3 and Gat1 (another Tor-regulated GATA factor) (19). Similarly, induction triggered by exposure to 0.4 M NaCl was markedly reduced in these mutants, while gln3 or gat1 cells were sensitive to sodium and the absence of the transcription factors eliminated the salt tolerance of ure2 cells (19). These observations were in agreement with the identification in the ENA1 promoter of six putative GATA motifs (GATAA and GATAAG), most of which are located far upstream of the known regulatory sites (positions −950 to −1,400 [approximately]), and sustained the notion that saline stress may induce activation of Gln3 and Gat1 and thus, upon entry into the nucleus, that these factors would cooperate in the induction of ENA1. However, the function of these GATA motifs has not been tested experimentally. In fact, a previous report proposed that ENA1 is still induced by monovalent cations in a sit4 background and that overexpression of SIT4 does not alter ENA1 mRNA levels (53). Nevertheless, it must be noted that the latter experiments were done with medium containing galactose as the carbon source, an experimental condition that has been reported to strongly affect the ENA1 basal expression level (3, 76). Furthermore, it was recently observed that treatment of cells grown under conditions in which Gln3 is normally nuclear (i.e., nitrogen starvation) with 1 M NaCl results in rapid relocalization of Gln3 to the cytoplasm (88). Therefore, the precise mechanism of regulation of ENA1 as a result of modulation of the TOR pathway is still unclear.

Regulation by the Hal3/Ppz system.

Ppz1 and Ppz2 represent examples of structurally atypical Ser/Thr protein phosphatases (47, 71, 72; reviewed in reference 5). Mutants lacking Ppz1 display a marked tolerance to Na⁺ and Li⁺ (70) that is further increased upon deletion of the PPZ2 gene (although single ppz2 mutants are not salt tolerant). ppz1 ppz2 mutants show increased ENA1 mRNA levels even in the absence of stress and an enhanced response in salt-stressed cells. The observation that the ppz mutation was unable to increase salt tolerance in an ena1 ena2 ena3 ena4 background was consistent with the notion that the main role of the encoded phosphatases in salt tolerance could be explained through their negative effect on ENA1 expression (70). However, later on it was shown (98) that Ppz1 and -2 also control potassium uptake by negatively regulating the Trk transporters and that this has a substantial affect on saline tolerance and intracellular pH (which may in turn affect ENA1 expression [see below]).

HAL3/SIS2 was identified almost simultaneously in two different laboratories as a gene conferring halotolerance when overexpressed (28) and rescuing the growth defect of a Sit4 phosphatase mutant (27). Overexpression of Hal3 increased potassium and decreased sodium contents, whereas hal3 mutants were halosensitive and accumulated more sodium than wild-type cells did (28). Hal3 was found to be necessary for full induction of ENA1 upon saline stress, suggesting that the beneficial effect of Hal3 could be mediated, at least in part, through its positive regulatory effect on the expression of this ATPase-encoding gene. The connection between Hal3 and the Ppz phosphatases was established by de Nadal and coworkers (24), who found that Hal3 was able to bind the C-terminal catalytic moiety of Ppz1 in vivo and in vitro, acting as a negative regulatory subunit of Ppz1 and thus controlling all known Ppz1 functions (18, 24). Hal3 is also able to bind Ppz2, and overexpression of Hal3 in a ppz1 single mutant increases ENA1 expression levels to values identical to those observed in ppz1 ppz2 cells. In contrast, no further increase is observed when Hal3 is expressed in a double phosphatase mutant (81). This suggests that Hal3 regulates both Ppz1 and Ppz2 function upon ENA1 expression. A screen for multicopy suppressors of the hal3 sit4 synthetically lethal phenotype allowed the characterization of Vhs3 as a second regulatory subunit of Ppz1 (80). Vhs3 also bind the C-terminal half of Ppz1 and inhibits its phosphatase activity in vitro, with a similar potency to that of Hal3. However, the phenotypes derived from mutation or overexpression of the VHS3 gene are much weaker than those for HAL3 mutation, probably because the former is expressed at lower levels (80).

The mechanisms by which the Ppz phosphatases influence ENA1 expression seem to be rather complex. It has been shown that the increase in the basal level of ENA1 expression in a ppz1 single mutant is fully mediated by constitutive activation of the calcineurin/Crz1 pathway (81). In contrast, in a ppz1 ppz2 mutant, additional regulatory mechanisms are activated, leading to higher expression of ENA1, even in the absence of stress. A likely explanation is that the lack of both phosphatases results in increased potassium uptake as a result of deregulated Trk transporter activity (98). This would cause intracellular alkalinization (not significant enough in a single ppz1 strain), which would further activate the ATPase promoter in a calcineurin-independent fashion. This scenario is consistent with the observation that a substantial part of the induction of the ENA1 gene in the ppz1 ppz2 background is insensitive to the calcineurin inhibitor FK506 and maps to a region of the promoter (ARR2) that does not contain CDREs (Fig. 2). Moreover, in a ppz1 ppz2 mutant (but not a ppz1 strain), increased expression of alkaline-inducible genes, such as PHO84 or PHO12, which do not contain CDREs and are not calcineurin regulatable, can be observed (81).

Other regulatory inputs affecting the ENA1 ATPase gene.

Different experimental approaches have revealed a number of genes whose deletion or overexpression affects saline tolerance, involving the Ena1 ATPase in one way or another. We discuss these observations below, trying to establish possible links with the main mechanism discussed above.

Betz and coworkers reported (14) that isc1 mutants, which lack inositol phosphosphingolipid phospholipase C activity, are sensitive to sodium or lithium stress but not to osmotic stress and retain larger-than-normal amounts of these cations. Expression of ENA1 under high sodium or lithium stress is decreased in such mutants, suggesting a role of Isc1 in regulation of the ATPase gene. It was proposed that a product derived from the Isc1 activity might increase intracellular calcium, similar to what has been suggested for sphingosine 1-phosphate-related compounds (16), and this would activate the calcineurin pathway. However, this possibility has not been explored further.

An additional link between saline tolerance and inositol metabolism was established by Lopez et al. after the identification of the inositol monophosphatases Imp1 and Imp2 as lithium- and sodium-sensitive enzymes (48). These authors observed that whereas the mutants had no obvious salt-sensitive phenotype, overexpression of the genes conferred tolerance to both cations in an ENA1-dependent fashion. However, overexpression of the IMP genes did not increase ENA1 expression. Since the authors observed that overexpression of Imp1 resulted in increased cytosolic calcium levels and seemed to be calcineurin independent, they invoked calcium/calmodulin posttranscriptional regulation, previously proposed by Wieland et al. (94), as a possible explanation for their findings.

CK2 is a conserved Ser/Thr protein kinase that in S. cerevisiae appears as an oligomer composed of two related catalytic subunits (α and α′, encoded by CKA1 and CKA2, respectively) and two regulatory subunits (β and β′, encoded by CKB1 and CKB2, respectively). The catalytic activity is essential, as illustrated by the fact that a cka1 cka2 mutant is unviable. The regulatory subunits are dispensable, but cells lacking CKB1 or CKB2 are sensitive to high sodium or lithium (15, 23, 89). A slight sensitivity of cka1 and cka2 mutants to high sodium has also been reported (13). In contrast, these mutants have not been found to be sensitive to high pH (89), although we observed a moderate sensitivity for ckb1 and ckb2 mutants in several genetic backgrounds (34; our unpublished observations). By using lacZ fusions to the ENA1 promoter, Tenney and Glover (89) proposed that a lack of Ckb1 or Ckb2 decreased ENA1 expression in cells stressed with 0.4 M NaCl. In contrast, using the same approach, de Nadal and coworkers (23) did not observe changes in ENA1 promoter activity in ckb1 cells challenged with larger amounts of NaCl (0.75 M) for 60 min. However, we recently detected decreased ENA1 expression in ckb1 cells exposed to 0.9 M NaCl for 4 h (M. Platara et al., unpublished data). This suggests that the apparent conflict in earlier reports may arise by comparison of different experimental conditions (intensity and timing of the stress). In any case, the mechanism by which CK2 may influence ENA1 expression remains unknown. A recent report proposed that Cka1 (but not Cka2) can phosphorylate Nrg1 in vivo in response to diverse stresses, including 1 M NaCl and alkaline pH (13). It remains to be tested whether or not this phosphorylation has any effect on the repressor capacity of Nrg1 on the ENA1 promoter.

Yeast cells lacking both Psr1 and Psr2, two similar membrane proteins with phosphatase activity, are sensitive to high sodium (but not to KCl or osmotic stress), and this phenotype is associated with the inability to achieve full induction of ENA1 (85). The experimental evidence suggests that the regulatory mechanism of ENA1 expression involving Psr1 and -2 is independent of calcineurin activation, at least in part. Remarkably, it has been demonstrated that Psr1 binds to Whi2 and that whi2 or psr1 psr2 cells contain a hyperphosphorylated form of the transcriptional activator Msn2 (42), which is required along with Msn4 for full activation of STRE-mediated gene expression in response to diverse forms of stress. Activation of STRE-mediated expression did require the phosphatase activity of Psr1, suggesting that it may dephosphorylate Msn2 (42). However, it must be kept in mind that activation of the ENA1 promoter by both high salt and alkaline pH seems to be fully independent of the Msn2/Msn4 transcription factors; that the ATPase gene is not induced by heat shock or oxidative stress (3, 75, 84), indicating that ENA1 is not induced through the mechanisms underlying the general stress response; and that the hypothetical STRE at position −651 in the ATPase promoter is not functional. Therefore, the mechanism for Psr1/Psr2 regulation of ENA1 expression is still an open question.

Type 2C protein phosphatases are conserved monomeric enzymes with diverse cellular roles. In yeast, there are seven possible isoforms (Ptc1-7), which have been shown to play a large variety of functions (see reference 35 and references therein). A common function for several type 2C protein phosphatases (Ptc1-4) is the negative regulation that they exert on the osmotically induced HOG pathway by dephosphorylating and inactivating the Hog1 MAP kinase (82). A recent report revealed that ptc1 cells were sensitive to Li⁺ (but not to Na⁺) and that this phenotype was not additive to that for ena1 ena2 ena3 ena4 mutation (79). Both basal and lithium-induced expression levels of ENA1 were decreased in the ptc1 strain, and this effect was not attributable to the role of Ptc1 in Hog1 regulation. A number of observations suggest that Ptc1 may regulate the Hal3/Ppz system. For instance, a lack of Hal3 provokes a similar decrease in ENA1 expression to that produced by mutation of PTC1, and both effects are not additive. Moreover, blocking the mechanism for Ppz-mediated activation of ENA1 (i.e., simultaneous deletion of the Trk potassium transporters and chemical inhibition of calcineurin) does not further increase the sensitivity of the ptc1 strain (79). How Ptc1 may regulate the Hal3/Ppz system is still unknown.

HAL9 was identified as a gene encoding a putative transcription factor containing a zinc finger that was able to confer, at a high copy number, increased tolerance to sodium or lithium ions (58). This phenotype was dependent on the presence of a functional ENA1-ENA2-ENA3-ENA4 cluster. Similarly, disruption of the gene decreased both salt tolerance and ENA1 expression (58). The mechanism for regulation of ENA1 expression by means of Hal9 is unknown. Hal9 may have another function(s) in the cell, as it has been reported that a hal9 mutant is hypersensitive to 4-nitroquinoline oxide and benomyl, is unable to grow on nonfermentable carbon sources (2), and has reduced expression of PDR16 and PDR5 (1), which encode proteins related to multidrug resistance. In any case, very little functional information is currently available for the Hal9 protein. HAL1 was identified in a similar way to HAL9. Cells lacking Hal1 are salt sensitive and display decreased ENA1 expression, whereas overexpression of HAL1 confers salt tolerance and increases expression of the ATPase gene (32, 76). Overexpression of HAL1 affects both the potassium and sodium contents of cells, although only in the latter case does the role of ENA1 seem important. Expression of HAL1 itself is increased in cells subjected to high concentrations of NaCl, KCl, or sorbitol, and this effect is mediated by two antagonistic transcription factors, namely, the Sko1 repressor and the Gcn4 activator, both of which compete for the CRE-like sequence present in the HAL1 promoter (67). The osmotic component of saline stress would activate the Hog1 pathway, triggering phosphorylation of Sko1 (74) and releasing HAL1 repression, while the Gcn4 activator would increase expression of this gene under salt stress. Activation of Gcn4 represents a link between salt stress and amino acid metabolism, which fits well with the impairment of amino acid uptake described for cells subjected to high Na⁺ or K⁺ concentrations (67). It is worth noting that a previous report described a dose-dependent effect of the STD1 and MTH1 genes on both HAL1 and ENA1 expression levels (29). STD1 and MTH1 encode proteins that act as negative regulators of the glucose-sensing signal transduction pathway and are required for repression of transcription by Rgt1. Yeast cells lacking Std1 or Mth1 were found to be sensitive to sodium, lithium, and high pH. Induction of HAL1 and ENA1 upon exposure of cells to 1 M NaCl was reduced in an std1 mth1 mutant, whereas high-copy-number expression of STD1 resulted in increased expression of both HAL1 and ENA1 (29). These dose-dependent effects on ENA1 expression were independent of those provoked by activation of the calcineurin pathway.

The nuclear phosphoprotein Lic4/Atc1 was related to cation homeostasis several years ago (37). Overexpression of Lic4 suppressed the Li⁺-sensitive phenotype of calcineurin mutants. Deletion of LIC4 resulted in lithium sensitivity and further decreased the expression of ENA1 in calcineurin mutants (but not in an otherwise wild-type background). ARL1 encodes a G protein that acts as a regulator of K⁺ influx (61), and it has been reported that the loss of ARL1 aggravates the Li⁺-sensitive phenotype of a lic4 mutant (62). This work also shows that constitutive expression of ENA1 suppresses the Li⁺-sensitive phenotype of the arl1 lic4 mutant and suggests the possibility that Arl1 (which has been related to regulation of membrane trafficking in yeast) may be involved in delivery of Ena1 (and other cation transporters) to the cell membrane. However, this possibility has not been substantiated further, and the function of LIC4 in ENA1 expression remains to be clarified.

CONCLUDING REMARKS

Ena1-related ATPases are conserved proteins in fungi that play a key role in monovalent cation homeostasis and that, in S. cerevisiae, are important determinants of salt and alkaline pH tolerance. Work in past years has revealed an intricate regulation of the expression of the ENA1 gene at the transcriptional level, based on a complex promoter which is able to integrate diverse signaling pathways. Whereas some regulatory inputs, such as those mediated by calcineurin or the HOG pathway, are relatively well defined, the nature of others is still obscure. In contrast with the relatively abundant knowledge on its transcriptional control, the biochemistry of the Ena1 ATPase remains to be developed. Despite initial reports raising the possibility of posttranscriptional regulation (48, 94), few advances have been made in this direction. The identification of posttranscriptional mechanisms for the regulation of Ena1 activity thus remains a major challenge in this field.