©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The PPZ Protein Phosphatases Are Important Determinants of Salt Tolerance in Yeast Cells (*)

Francesc Posas (§) , Manel Camps (¶) , Joaqun Ario (**)

From the (1) Departament de Bioqumica i Biologia Molecular, Facultat de Veterinria, Universitat Autnoma de Barcelona, Bellaterra 08193, Barcelona, Spain

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein phosphatases PPZ1 and PPZ2 represent a novel form of Ser/Thr phosphatases structurally related to type 1 phosphatases and characterized by an unusual amino-terminal region. We have found that the deletion of PPZ1 gene results in increased tolerance to Na and Li cations. Simultaneous deletion of PPZ2 gene results in an additional increase in salt tolerance. After exposure to high concentration of Li, the intracellular content of the cation was markedly decreased in ppz1 ppz2 mutants when compared to wild type cells. No significant differences were observed between both strains when the Li influx was measured, but ppz1 ppz2 mutants eliminated Li more efficiently than wild type cells. This can be explained by the fact that expression of the ENA1 gene, which encodes the major component of the efflux system for these cations, is strongly increased in ppz1 ppz2 cells. As expected, the disruption of the PPZ genes did not complement the characteristic hypersensitivity for Na and Li of a ena1 strain. The lack of protein phosphatase 2B (calcineurin) has been found to decrease salt resistance by reducing the expression of the ENA1 gene. We have observed that the disruption of the PPZ genes substantially enhances the resistance of the hypersensitive calcineurin-deficient mutants. Since PPZ phosphatases have been found to be functionally related to the protein kinase C/mitogen-activated kinase pathway, we have tested bck1 or mpk1/slt2 deletion mutants and found that they do not display altered salt sensitivity. However, disruption of PPZ1 fails to increase salt resistance in a mpk1/slt2 background. In conclusion, we postulate the existence in yeast of a novel PPZ-mediated pathway involved in salt homeostasis that is opposite to and independent of the recently described calcineurin-mediated pathway.


INTRODUCTION

The yeast Saccharomyces cerevisiae is able to maintain a suitable intracellular concentration of Na, even in the present of relatively high concentrations of this cation in the medium, through the coordinate regulation of uptake and efflux systems. Influx of Na (and Li) occurs through the K uptake system. Exposure to sodium stress causes the conversion of the K uptake system to a high affinity mode, in which the entrance of Na is reduced (Haro et al., 1993). Two components of this system, TRK1 and TRK2, have been identified and cloned (Gaber et al., 1988, Ko and Gaber, 1991), and it has been postulated that TRK1 is required for the expression of the high K affinity mode. The efflux of Na is mainly mediated by a putative P-type ATPase encoded by the gene ENA1 (for a recent review, see Rodrguez-Navarro et al.(1994)). This gene is the first unit of a tandem array of four closely related genes, namely ENA1-ENA4 (Rudolph et al., 1989; Haro et al., 1991; Martnez et al., 1991). ENA2, ENA3, and ENA4 present a very weak constitutive expression, while ENA1, although poorly expressed on basal conditions, is induced upon exposure to high concentrations of Li or Na, as well as to alkaline pH. Therefore, deletion of ENA1 results in cells hypersensitive to Na or Li (Haro et al., 1991; Garciadeblás et al., 1993).

Reversible protein phosphorylation is a major regulatory mechanism for the modulation of protein function, and protein phosphorylation mechanisms are likely to be involved in the control of Na homeostasis. For instance, increased dosage of YCK1 or YCK2, the genes encoding casein kinase I homologues, has been reported to increase tolerance to salt (Robinson et al., 1992). Similarly, disruption of the YCR101 gene, encoding a predicted protein kinase, confers sensitivity to salt (Skala et al., 1991).

Protein phosphatases reverse the action of kinases. Traditionally, Ser/Thr phosphatases have been classified into four groups, namely types 1, 2A, 2B, and 2C (see Cohen(1989) for a review), and this classification is useful for a large variety of organisms, including yeast. A role for the Ca-dependent Ser/Thr protein phosphatase 2B (calcineurin) in the regulation of salt homeostasis has been suggested very recently (Nakamura et al., 1993; Mendoza et al., 1994), since disruption of the genes encoding either the catalytic (CMP1/CNA1 and CMP2/CNA2) or the regulatory (CNB1) subunits of this phosphatase results in increased sensitivity to salt. It has been proposed that, at least in part, this increase in sensitivity is due to the reduced expression of the ENA1 gene in a calcineurin-deficient background (Mendoza et al., 1994).

In addition to the mentioned Ser/Thr phosphatases, the genome of S. cerevisiae encodes a number of proteins structurally related to (but clearly different from) type 1 and 2A phosphatases. Almost without exception, the regulation and the biological functions of these novel phosphatases are largely unknown. A few years ago our laboratory identified and cloned a gene named PPZ1, encoding a 692-residue putative Ser/Thr phosphatase characterized by a carboxyl-terminal half related to the protein phosphatase-1 family and a large amino-terminal extension unrelated to protein phosphatases (Posas et al., 1992). We found that the simultaneous disruption of PPZ1 and a second, related gene designated PPZ2 results in increased sensitivity to caffeine that lead to cell lysis, pointing to a role for these phosphatases in the maintenance of cellular integrity (Posas et al., 1993). PPZ2 also encodes a large protein (710 residues) sharing most of the structural characteristics of PPZ1 (Lee et al., 1993a; Hughes et al., 1993). It has been recently suggested that the PPZ1/PPZ2 phosphatases might be related to the SLT2/MPK1 pathway, since overexpression of gene PPZ2 suppresses the lytic defect of mpk1 strains and deletion of PPZ1/PPZ2 genes is additive with the mpk1 defect (Lee et al., 1993b).

In this paper we present evidence that the PPZ phosphatases are important determinants in salt tolerance by regulating the efflux of cations and that these phosphatases and calcineurin play opposite functional roles.


MATERIALS AND METHODS

Strains, Media, and Growth Conditions

S. cerevisiae strains used in this work are listed in . Strains were grown in S.D. or in complex YPD medium (1% yeast extract, 2% peptone, 2% glucose) at 28 °C for routine work and storage (Sherman et al., 1986). YPD was supplemented with 20 mM TAPS() when used at pH 8.5. For Northern blot experiments YPD was supplemented with 50 mM HEPES and adjusted to pH 7.0.

Escherichia coli NM522 strain was used as bacterial host for plasmids and was grown in LB medium supplemented with the appropriate antibiotic when needed.

Gene Disruption Methods

The one-step gene disruption method (Rothstein, 1983) was used in all cases. ppz1 and ppz2 deletion mutants were obtained as described previously (Posas et al., 1992, 1993). cnb1 disruption mutants were obtained in our laboratory exactly as described previously (Mendoza et al., 1994). Yeast cells were transformed by using a modification of the lithium acetate method (Hill et al., 1991). All mutations were confirmed by Southern blot analysis using digoxigenin-labeled DNA probes.

Salt Sensitivity Assays

The sensitivity of the cells to NaCl, LiCl, or other salts was evaluated on freshly prepared YPD medium containing various concentrations of the salts. For drop tests cells were grown for 2 days in liquid culture without salt and then 3 µl of a 1/100 dilution were plated on YPD-agar medium with salt. Growth was recorded after 3 days at 28 °C. Relative growth was measured in liquid cultures as growth, recorded by measuring the A, of overnight cultures in different salt concentrations in comparison with growth monitored in the same medium without added salts.

Measurement of Intracellular LiConcentrations

For measurement of ion fluxes, cells were grown in YPD until an A of 1 was reached. Then LiCl was added from a 5 M stock solution to achieve a final concentration of 200 mM (only of 50 mM in the case of strain RH16.6, because of its very high sensitivity to this cation). At the indicated intervals, samples were harvested by filtration and washed with three volumes of ice-cold water containing 1.5 M sorbitol and 20 mM MgCl, rinsed with cold deionized water, and extracted by incubation at 95 °C for 30 min as described (Gaxiola et al., 1992). Li concentrations of the clarified extract were determined by flame spectrophotometry using a Corning 810 apparatus.

Northern Blot Analysis

For Northern blot analysis, cells from a stationary culture were grown as described until the culture reached an optical density of 1. Total RNA was prepared as described (Treco, 1989), electrophoresed on 0.7% agarose-formaldehyde gels (20-30 µg/lane) and transferred to nylon membranes (Hybond N, Amersham Corp.) under vacuum. Membranes were hybridized at 42 °C in the presence of 50% (v/v) formamide and 10 cpm/ml of the appropriate P-labeled DNA fragment. The probes used were the 3.7-kilobase pair XhoI-PstI fragment of the ENA1 gene, kindly provided by Prof. A. Rodrguez-Navarro (Haro et al., 1991) and a 1.75-kilobase pair fragment of the ACT1 gene (Gallwitz and Sures, 1980). Filters were washed in 0.1 SSC (1 SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 0.1% SDS at 55 °C.


RESULTS

Disruption of PPZ Genes Results in Increased Tolerance to Salt

During our studies on the role of the PPZ phosphatases in the maintenance of cellular integrity, we observed that the ppz1 ppz2 double mutant JA20-1C displayed a surprisingly vigorous growth compared to the wild type JA20-1D when exposed to moderate saline stress. This initial observation was further confirmed when the double disruption was carried out in other genetic backgrounds. The effect of increasing concentrations of salt on cell growth was studied by growing wild type as well as ppz1 and ppz1 ppz2 cells in plates containing different concentrations of LiCl. Lithium was used in these and other experiments instead of NaCl because these cations share the same uptake and efflux systems. Because Li is more toxic than Na, the former cation could be used at lower concentrations, thus avoiding the osmotic effects derived of the use of very high concentrations of NaCl. As can be observed in Fig. 1 (panelA), wild type DBY746 cells grows poorly at 100 mM LiCl and cannot grow at all at 300 mM, whereas strain JA30 (which lacks a functional copy of gene PPZ1) can still grow at 300 mM LiCl. Simultaneous disruption of PPZ1 and PPZ2 genes, as in the case of strain JA31, allows cell growth even in the presence of 600 mM LiCl. This double disruptant was able to grow in YPD plates containing up to 800 mM LiCl (not shown). The effect of lack of PPZ phosphatases has also been observed in other genetic backgrounds, as in the case of the haploid cells derived from the diploid strain DL790 (ppz1/PPZ1 ppz2/PPZ2). Interestingly, in all cases the disruption of PPZ2 alone resulted in an almost negligible increase in salt tolerance when compared with wild type cells (not shown).


Figure 1: Growth of wild type and ppz mutant strains in the presence of salt. PanelA, wild type DBY746 cells as well as ppz1 PPZ2 (JA30) and ppz1 ppz2 (JA31) strains were inoculated in YPD plates, containing the indicated concentrations of LiCl, as described under ``Materials and Methods.'' PanelB, wild type DBY746 (), JA30 (), and JA31 () cells were grown in YPD medium to saturation and then aliquots transferred to YPD supplemented with the indicated concentrations of LiCl or NaCl. Growth was monitored spectrophotometrically at 660 nm and is expressed as percentage of the growth measured in the absence of added salts. Data are presented as the mean ± S.E. of three to five independent experiments.



The increased tolerance to lithium conferred to the cells by the disruption of the PPZ1 and PPZ2 genes can be also observed in liquid cultures (Fig. 1, panelB). Growth was also improved in the mutants in the presence of increasing concentrations of NaCl (rightpanel). When the pH of the medium was raised to 8.5, the resistance to Li and Na of the ppz mutants was similar to than that observed when the medium was not buffered at alkaline pH (not shown). The described effects of the ppz1 ppz2 mutations were specific for Li or Na, since growth was not improved in the presence of equivalent concentrations of KCl.

The Lack of PPZ Phosphatases Leads to Decreased Internal Li Content as a Result of Increased Cation Efflux

To learn whether the increased salt tolerance of ppz mutants was due to the fact that these cells contain a reduced amount of cations, we grew wild type and ppz1 ppz2 cells for 90 min in YPD medium containing 200 mM LiCl. When the intracellular content of Li was measured, it became evident that mutant cells contained a lower amount of the cation (30.6 ± 1.9 nmol/mg dry cells) than wild type cells (48.1 ± 2.5 nmol/mg dry cells). The intracellular concentration of Na was similarly reduced in the mutant strain when cells were preloaded for 90 min with 0.96 M NaCl.

The possibility that an altered cation uptake might be responsible for the observed differences was tested by determining the initial rate of Li uptake in both wild type and ppz1 ppz2 cells. As presented in Fig. 2 , both strains take up Li at approximately the same rate (about 2 nmolmgmin), suggesting that alterations in the uptake mechanisms are not responsible for the observed differences in the intracellular content of Li. Changes in accumulation might also be caused by an altered efflux rate. To test this possibility, cells were incubated for 90 min in YPD medium in the presence of 200 mM LiCl and then centrifuged and resuspended in Li-free medium. The internal Li content was then determined in both wild type and ppz1 ppz2 cells at different times after removal of the cation. As presented in Fig. 3, the rate of elimination of Li was clearly higher in mutant cells, suggesting that the decreased intracellular levels observed in these cells are essentially the result of an increased Li efflux.


Figure 2: Time dependence of Li accumulation in DBY746 and JA31 strains. Wild type DBY746 () and JA31 () cells were grown in YPD until the optical density reached about 1-2. The medium was then made 200 mM LiCl, and samples were removed at the indicated times for determination of intracellular Li concentration. Data are presented as the mean ± S.E. of four independent experiments.




Figure 3: Determination of lithium efflux in wild type and ppz cells. DBY746 () and JA31 () cells were grown as described in Fig. 2. Then, the medium was made 200 mM LiCl and growth resumed for 90 min. The cells were centrifuged and resuspended in fresh YPD medium, samples were taken at the indicated times, and the intracellular lithium content determined as described. Data are presented as the mean ± S.E. of four independent experiments.



Disruption of the PPZ Genes Results in Increased Expression of the ENA1 Gene

The putative P-type ATPase encoded by the ENA1 gene is believed to constitute the major system for Na (and Li) efflux in yeast cells. We compared the sensitivity to Na and Li of strains RH16.6 (ena1-ena4 PPZ1 PPZ2) and JA35 (ena1-ena4 ppz ppz2) in both YPD liquid cultures and YPD-agar plates. We found that in a ena1 background, the ppz1 ppz2 mutation was unable to increase the tolerance to the cations (). Furthermore, when RH16.6 and JA35 cells were loaded for 45 min in YPD medium containing 50 mM LiCl and the amount of internal Li was determined, no significant differences were found between both strains (32.4 ± 0.5 nmol/mg dry cells versus 30.2 ± 0.7, respectively). Both results were compatible with the notion of an involvement of the ENA1 gene product in the mechanism of action of the PPZ phosphatases. Since it is known that exposure to salt stress results in induction of gene ENA1, which otherwise is strongly repressed, we tested by Northern blot experiments the mRNA levels of the ENA1 gene in wild type and ppz1 ppz2 mutants. As shown in Fig. 4, exposure of wild type cells to Li cations results in a transient increase of ENA1 mRNA levels, which is clearly higher in ppz1 ppz2 cells. Remarkably, ppz-deficient cells contain more ENA1 message even in the absence of salt stress, suggesting an effect of the ppz1 ppz2 mutation in basal conditions.


Figure 4: Effect of lack of PPZ phosphatases in ENA1 mRNA levels. Wild type DBY746 cells (openbars) as well as JA31 mutants (ppz1 ppz2) (stripedbars) were grown in YPD medium supplemented with 50 mM HEPES (pH 7.0) as described and then made 200 mM LiCl. Growth was resumed, and cells collected at the indicated times for total RNA extraction. Northern blots were performed, membranes hybridized with a P-labeled ENA1 probe, and films exposed for 48 h. The membranes were then stripped and reprobed with the ACT1 gene as control for the amount of RNA transferred. The levels of ENA1 mRNA were determined by densitometric scanning of the films. Data are referred to the signal obtained from wild type cells collected immediately prior to the addition of LiCl and is expressed as the mean ± S.E. from four independent experiments. Inset shows an autoradiogram obtained from a typical experiment.



The Lack of PPZ Phosphatases Counteracts the Defect Associated to the Absence of Calcineurin

Since it has been shown recently that the lack of the type 2B phosphatase (calcineurin) leads to a salt-sensitive phenotype, we constructed strains JA40 (PPZ1 PPZ2 cnb1) and JA41 (ppz1 ppz2 cnb1) and tested their sensitivity to LiCl in comparison with wild type and JA31 (ppz1 ppz2 CNB1) cells (Fig. 5, upperpanel). As reported previously, the lack of CNB1 (encoding the regulatory subunit of calcineurin) in an otherwise wild type background leads to hypersensitivity to salt. Simultaneous disruption of PPZ1 and PPZ2 in a cnb1 background results in higher tolerance than wild type cells, but this mutant is less resistant than a ppz1 ppz2 CNB1 strain, indicating that the effects of these mutations are independent and additive. Furthermore, exposure of strain YPH499 carrying the double disruption cna1 cna2 (the genes encoding the catalytic subunits of calcineurin) to 0.8 M NaCl prevents growth in rich medium plates. However, when these cells carry the ppz1 disruption, growth was restored (Fig. 5, middlepanel). Growth under higher concentrations of NaCl indicated that cna1 cna2 ppz1 cells were slightly less tolerant than wild type cells (data not shown). When the levels of ENA1 mRNA were determined, it was observed that, as expected, they were lower in cnb1 than in wild type cells. However, the levels of ENA1 message were dramatically increased when the cnb1 cells also contains the ppz1 ppz2 mutation (Fig. 5, lowerpanel). These observations indicate that the lack of PPZ phosphatases is able to increase salt tolerance in calcineurin-depleted cells and suggest that the mechanism of action of the PPZ phosphatases is not mediated by calcineurin.


Figure 5: Complementation of the salt sensitivity phenotype of calcineurin-deficient cells by the ppz mutations. Upperpanel, wild type DBY746 (PPZ1 PPZ2 CNB1), JA40 (PPZ1 PPZ2 cnb1), JA31 (ppz1 ppz2 CNB1), and JA41 (ppz1 ppz2 cnb1) were inoculated on YPD plates or YPD plates containing the indicated concentrations of LiCl. Growth was scored after incubation at 28 °C for 3 days. Strain JA40 was constructed in our laboratory but should be identical to strain DBY746cnb1 described previously (Mendoza et al., 1994). Middle panel, wild type YPH499 (CNA1 CNA2 PPZ1) and its isogenic derivatives cna1 cna2 PPZ1 (Cyert et al., 1991) and cna1 cna2 ppz1 (strain JA45) were streaked onto YPD plates or YPD plates supplemented with 0.8 M NaCl. Growth was scored after incubation at 28 °C for 4 days. Lower panel, wild type DBY746 (lanesA) as well as JA40 (lanesB), JA31 (lanesC), and JA41 (lanesD) mutants were grown and incubated with 200 mM LiCl as described in Fig. 4. Samples were taken at the indicated times, total RNA prepared, and Northern blot experiments performed using P-labeled ENA1 and ACT1 probes.



Saline Stress and the Relationship between PPZ Phosphatases and the PKC/MAP Kinase Pathway

PPZ phosphatases appear to be related to some of the effects derived of blockage of the PKC/MAP kinase pathway (i.e. cellular lysis under temperature or caffeine stress). However, when strain DL456, which carries a disruption of the MAP kinase gene (SLT2/MPK1), is grown on YPD plates containing different concentrations of LiCl, it does not present a significantly altered salt tolerance when compared with the wild type strain (Fig. 6). Disruption of the bck1 gene, encoding a kinase acting upstream the MAP kinase gene product, equally results in absence of phenotypic change (not shown). Interestingly, the disruption of PPZ1 in a mpk1 background (strain DL823) does not result in improved growth in the presence of different concentrations of LiCl. Since DL823 cells must be grown in plates supplemented with 10% sorbitol, it is important to note that the failure of these cells to grow was due to Li toxicity and not to the rather high osmolarity of the medium, since this strain did survive when identical concentrations of the less toxic salt NaCl were used instead of LiCl (Fig. 6).


Figure 6: Comparison of the effect of the ppz mutation on salt resistance in wild type and mpk1 backgrounds. The wild type strain 1788 (PPZ1 PPZ2 MPK1), as well as strains DL823 (PPZ1 PPZ2 mpk1) and DL456 (ppz1 ppz2 mpk1), were inoculated in YPD plates or YPD plates containing different concentrations of LiCl or NaCl. In all cases 10% sorbitol was included in the plates. A haploid ppz1 MPK1 strain derived from strain 1788 is included for comparison. Plates were incubated at 28 °C for 3 days.




DISCUSSION

In the last few years, a number of genes involved in the modulation of cellular sensitivity to Na and Li toxicity have been identified in yeast. Some of these genes encode components of the uptake and efflux systems located at the plasma membrane, as it is the case of genes TRK1/TRK2 and ENA1, respectively. Other genes would include regulatory components of the above mentioned systems, as it has been suggested in the case of calcineurin (Mendoza et al., 1994). Finally, a number of genes have been described to produce a salt-sensitive phenotype when mutated or to confer increased salt tolerance when overexpressed, as it is the case of HAL1 and HAL2 (Gaxiola et al., 1992; Gläser et al., 1993; Murgua et al., 1995), LIS1/ERG6 (Welihinda et al., 1994), and the protein kinases encoded by genes YCK1/YCK2 (Robinson et al., 1992) and YCR101 (Skala et al., 1991).

It is remarkable that, in all cases described so far, the lack of function of the identified genes results in increased sensitivity to salt. Here, we present the opposite situation and, as far as we know, this is the first case of genes that, when disrupted, yields a phenotype of strongly increased salt tolerance. Both PPZ1 and PPZ2 genes encode very similar proteins, characterized by a carboxyl-terminal half related to type 1 phosphatases and by a large amino-terminal half rich in Ser/Thr residues. Despite this similarity, our results suggest that both proteins do not contribute equally to the phenotype studied, since deletion of PPZ1, but not of PPZ2, already results in a noticeable increase in salt tolerance. However, since the double disruption gives a more dramatic phenotype, it has to be concluded that the lack of PPZ2 also provokes alterations in salt homeostasis. This situation is very much alike to the one previously observed in our laboratory regarding cell integrity of the ppz mutants under caffeine stress (Posas et al., 1993) and might be due to a number of factors, including lower levels or activity of the PPZ2 gene product.

We postulate that the increased cation efflux observed in ppz1 ppz2 cells is due to the fact that the PPZ phosphatases negatively control the function of the ENA1 gene. This notion is based on several lines of evidence. First, we have observed that growth is improved in ppz mutants even when the extracellular pH is high enough to block the function of the proposed Na/H antiporter system (Rodrguez-Navarro et al., 1981, 1994). Therefore, involvement of this hypothetical antiporter in the mechanism of action of PPZ phosphatases can be discarded. Second, the fact that the lack of PPZ cannot improve growth in the presence of salt of a hypersensitive ena1-ena4 strain suggests that the mechanism of action of PPZ might be mediated by the ENA1 gene product. Finally, the observation that ENA1 mRNA levels are increased in ppz mutants suggests that the increase in cation efflux is due, at least in part, to the existence of higher amounts of the ENA1 protein. A remarkable fact is that an increase in the expression of ENA1 can be detected in a PPZ-deficient background even when the cells have not been exposed to saline stress. This could be interpreted as if the PPZ phosphatases would negatively affect the expression of ENA1 under basal conditions, thus avoiding an unnecessarily excessive expression of ENA1, which is, by itself, detrimental for cell growth.() Exposure of ppz cells to salt would trigger a signaling pathway that would cause an additional increase in ENA1 mRNA levels. Of course, our present data cannot rule out the possibility of a negative control of the activity of the ENA1 protein by the PPZ phosphatases, conceivably through phospho-dephosphorylation reactions.

It has been reported recently that calcineurin plays a role in salt homeostasis and that, at least in part, this occurs through the control of cation efflux. Loss of calcineurin function can be provoked by deletion of the single CNB1 gene (encoding the regulatory subunit of the phosphatase) or by deletion of the two genes encoding the catalytic polypeptides (CNA1/CMP1 and CNA2/CMP2). Both types of mutation result in decreased cation efflux (Nakamura et al., 1993; Mendoza et al., 1994), most probably as a result of a decrease in the expression of ENA1 (Mendoza et al., 1994). Our observations that the lack of PPZ phosphatases can counteract the lack of calcineurin by increasing ENA1 mRNA levels indicates that the effect of the PPZ phosphatases on sodium and lithium efflux is not mediated by calcineurin and provides a very interesting example of two different phosphatases playing opposites roles in a given biological system. This situation is reminiscent of the growth restoration observed in cAMP-dependent protein kinase-deficient strains upon disruption of the gene encoding the YAK1 kinase (Garret and Broach, 1989; Ward and Garret, 1994) and is compatible with the notion of both phosphatases acting in parallel pathways, with overlapping but antagonistic effects. While the involvement of calcineurin in adaptation to high salt stress conditions clearly suggests a role for a calcium-mediated signaling pathway, very little is known at present about the activity of PPZ1/PPZ2 and their regulation.

We recently described that disruption of both PPZ genes resulted in cell lysis upon caffeine stress (Posas et al., 1993). Cell lysis is also a characteristic phenotype of mutants in the PKC/MAP kinase pathway (Levin and Errede, 1993). The role of the PPZ phosphatases in the maintenance of cell integrity was reinforced after the isolation of PPZ2 as a multicopy suppressor of the lytic effect of the mpk1 mutation (Lee et al., 1993b), thus suggesting that these phosphatases may function within this PKC1-mediated pathway. However, our finding that neither the lack of the BCK1 gene (Lee and Levin, 1992), encoding a putative MAP kinase kinase kinase that is believed to act directly downstream from PKC, nor the disruption of the SLT2/MPK1 gene, encoding the MAP kinase homologue of the pathway (Torres et al., 1991; Lee et al., 1993a), results in significant changes in salt sensitivity suggests that this pathway is not involved in the regulation of salt homeostasis. However, it is remarkable that the disruption of PPZ1 fails to increase the tolerance for Li or Na of a mpk1 strain. This observation suggests that a functional MAP kinase would be a requisite for the phenotypic expression of the lack of PPZ function and provides additional evidence for a functional link between both proteins.

Two main conclusions can be drawn from the results presented here. First, the PPZ phosphatases negatively regulate the efflux of cations in yeast cells, most probably through the repression of the ENA1 gene. Second, this event is independent of the existence of calcineurin, thus suggesting that PPZ and calcineurin are two Ser/Thr phosphatases acting on parallel pathways. A tentative model for PPZ function is depicted in Fig. 7. In this model we favor the idea that the PPZ phosphatases do not directly regulate the expression of the ENA1 gene, since these phosphatases do not show the structural features of known transcriptional regulatory proteins and, in addition, we have found that PPZ1 is not present in nuclear fractions.() Instead, a regulatory protein (denoted as X in Fig. 7) might act as a transcriptional repressor when dephosphorylated by PPZ1/PPZ2. Therefore, the lack of PPZ phosphatases would result in inactivation of the repressor and in increase of the expression of the ENA1 gene even in the absence of salt stress. Under saline stress, an independent signaling pathway would be activated and induce ENA1 expression, thus overriding the effect of the repressor. Finally, the combination of saline stress and lack of PPZ would result in stimulation of a positive effect and blocking of a negative one and would account for the strong expression of ENA1. It is worth noting that our results indicate that ENA1 is still induced in significant extent in the absence of the CNB1 gene, suggesting that calcineurin must not be solely responsible for salt-induced increased in ENA1 transcription. Alternatively, the PPZ phosphatases might inactivate a transcriptional activator. In this situation, the absence of PPZ would also result in an increased transcription of ENA1 in basal conditions that would be further enhanced by activation of the salt-induced pathway upon exposure to saline stress. In any case, the dramatic increase in salt resistance featured by ppz strains reveals that these novel phosphatases, whose biological functions were essentially unknown, are important determinants in salt homeostasis in yeast cells.


Figure 7: A tentative model for PPZ function in salt homeostasis. See details in the main text.



  
Table: Saccharomyces cerevisiae strains used in this work


  
Table: Effect of ppz and ena1 mutations on Li tolerance

Tolerance is expressed as the concentration of Li that reduces 50% growth of liquid cultures. Data are expressed as mean ± S.E. from 3 to 5 independent experiments.



FOOTNOTES

*
This work was supported in part by Grant PB92-0585 from the Direccion General de Investigacion Cientifica y Tecnica (Spain) (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a fellowship from the Plan de Formacion del Personal Investigador (Ministerio de Educacion y Ciencia, Spain).

Recipient of a fellowship from the Formació de Personal Investigador (Generalitat de Catalunya).

**
To whom all correspondence should be addressed: Departament de Bioqumica i Biologia Molecular, Facultat de Veterinria, Ed. V, Universitat Autnoma de Barcelona, Bellaterra 08193, Barcelona, Spain. Tel.: 34-3-5812182; Fax: 34-3-5812006; E-mail: IVBQ0@cc.uab.es.

The abbreviations used are: TAPS, N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid; PKC, protein kinase C; MAP, mitogen-activated protein.

A. Rodrguez-Navarro, personal communication.

F. Posas and J. Ario, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. M. S. Cyert and D. E. Levin for strains, Dr. T. Miyakawa and Dr. J. M. Pardo for plasmids, and Dr. R. Serrano for helpful discussion. We acknowledge the skillful technical support of A. Vilalta and R. Martnez. We express our gratitude to Dr. A. Rodrguez-Navarro for the generous gift of strains, plasmids, and continuous scientific advice.


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