The Transcriptional Activator Imp2p Maintains Ion Homeostasis in Saccharomyces cerevisiae

Corresponding author: Dindial Ramotar, Maisonneuve-Rosemont Hospital Research Center, 5415 de l’Assomption Boul., Montreal, Quebec, H1T 2M4, Canada, dramotar{at}hmr.qc.ca (E-mail).

Communicating editor: P. G. YOUNG

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast cells deficient in the transcriptional activator Imp2p are viable, but display marked hypersensitivity to a variety of oxidative agents. We now report that imp2 null mutants are also extremely sensitive to elevated levels of the monovalent ions, Na⁺ and Li⁺, as well as to the divalent ions Ca²⁺, Mn²⁺, Zn²⁺, and Cu²⁺, but not to Cd²⁺, Mg²⁺, Co²⁺, Ni²⁺, and Fe²⁺, as compared to the parent strain. We next searched for multicopy suppressor genes that would allow the imp2 mutant to grow under high salt conditions. Two genes that independently restored normal salt-resistance to the imp2 mutant, ENA1 and HAL3, were isolated. ENA1 encodes a P-type ion pump involved in monovalent ion efflux from the cell, while HAL3 encodes a protein required for activating the expression of Ena1p. Neither ENA1 nor HAL3 gene expression was positively regulated by Imp2p. Moreover, the imp2 ena1 double mutant was exquisitely sensitive to Na⁺/Li⁺ cations, as compared to either single mutant, implying that Imp2p mediates Na⁺/Li⁺ cation homeostasis independently of Ena1p.

ALL cells are equipped with uptake and efflux transport systems to effectively maintain both mono- and divalent ion homeostasis. Genetic defects that alter these systems can lead to imbalances in intracellular ion concentrations causing cells to rapidly lose their viability due to disruption of cell volume, intracellular pH, ionic strength, and/or enzyme-catalyzed reactions (FARCASANU et al. 1995 ; FERRANDO et al. 1995 ; VISICK and CLARKE 1995 ). When Saccharomyces cerevisiae is exposed to high NaCl concentrations, the Na⁺ cations can enter the cell through the K⁺ channels thereby interfering with cellular processes that require K⁺ (KO and GABER 1991 ; VARELA and MAGER 1996 ; HARO et al. 1991 ). However, yeast cells can prevent intracellular accumulation of toxic Na⁺ cations by (i) switching the K⁺ transport system from its low-affinity to a high-affinity state and (ii) expelling any excess of Na⁺ ions using a P-type ATPase encoded by the Na⁺/Li⁺ transporter gene ENA1/PMR2a (GARCIADEBLAS et al. 1993 ; FERRANDO et al. 1995 ; VARELA and MAGER 1996 ; PEDERSON and CARAFOLI 1987 ). Ena1p is the first open reading frame (ORF) in a tandem array of five ORFs and is one that is highly expressed and bears an inducible 5'-untranslated region (GARCIADEBLAS et al. 1993 ; FERRANDO et al. 1995 ). While deletion of the ENA1 gene produces mutants that are sensitive to Na⁺/Li⁺ cations, the phenotype of mutants lacking each of the other ORFs, PMR2b, PMR2c, PMR2d, and PMR2e, is unclear (WIELAND et al. 1995 ). Ena1p expression is stimulated by the calcium-activated protein phosphatase calcineurin, as well as by Sis2p/Hal3p (MENDOZA et al. 1994 ; FERRANDO et al. 1995 ). Mutants devoid of either calcineurin or Hal3p are moderately sensitive to Na⁺/Li⁺ cations, which is associated with decreased induction of Ena1p. Full salt tolerance is restored in the calcineurin- or the Hal3p-deficient mutants by overproduction of Ena1p, suggesting that Ena1p plays a central role in maintaining monovalent ion homeostasis. However, the mechanism of ENA1 gene activation by calcineurin or Hal3p remains unclear (FERRANDO et al. 1995 ). Recently, it has been shown that S. cerevisiae contains a Na⁺/H⁺ antiporter, encoded by the NHA1 gene, that can also eliminate excess Na⁺ ions (PRIOR et al. 1996 ). The Na⁺/H⁺ antiporter is operative mainly at acidic and neutral pH, and cells lacking the antiporter display increased sensitivity to Na⁺ and Li⁺ cations. Similar findings were first reported with the fission yeast Schizosaccharomyces pombe antiporter sod2p (JIA et al. 1992 ). Overproduction of either Nha1p or Sod2p antiporter functionally complements the Na⁺/Li⁺ sensitivity of the S. cerevisiae ena1 mutant (PRIOR et al. 1996 ; HAHNENBERGER et al. 1996 ). Thus, both the sodium pump and antiporter can be used in yeast cells to modulate internal Na⁺ levels.

It is less clear how yeast cells maintain divalent ion homeostasis. However, the broad substrate energy-dependent divalent ion transport system is responsible for the accumulation of biologically important trace metals, such as Mn²⁺, Mg²⁺, Co²⁺, Ca²⁺, and Zn²⁺ (FUHRMANN and ROTHSTEIN 1968 ). Divalent ions may also gain access via other uptake pathways, for example, the high affinity copper transporter, Ctr1p (DANCIS et al. 1994 ). Some of the transporters also function in the detoxification of divalent ions. The Cot1p and Zrc1p are believed to sequester Co²⁺ and Zn²⁺, respectively, in the mitochondrial matrix, thereby decreasing toxic accumulation of these ions in the cytoplasm (CONKLIN et al. 1994 ). cot1 and zrc1 mutants are hypersensitive, respectively, to Co²⁺ and Zn²⁺ (CONKLIN et al. 1994 ). The Golgi apparatus contains a P-type ion pump encoded by the PMR1 gene, which is capable of sequestering Ca²⁺ and Mn²⁺ and, to a lesser extent, Cu²⁺ and Zn²⁺ (PERKINS and GADD 1993 ; FARCASANU et al. 1995 ). A similar divalent ion pump, Pmc1p, is localized to the vacuoles, and mutants that lack both the Pmr1p and Pmc1p pumps are inviable at even low concentration of calcium (CUNNINGHAM and FINK 1996 ). Metal binding proteins also constitute an important process for maintaining divalent ion homeostasis. The yeast metallothionein, a small cysteine-rich polypeptide encoded by the CUP1 gene, can bind copper and zinc (HAMER et al. 1985 ; STRAIN and CULOTTA 1996 ). Indeed, cells lacking Cup1p are hypersensitive to copper.

Some of the genes involved in divalent ion homeostasis have been isolated as suppressors of yeast mutants that are sensitive to oxidative stress. The sod1 mutant, which lacks a functional Cu/Zn superoxide dismutase, is sensitive to atmospheric oxygen and exhibits a requirement for lysine and methionine during aerobic growth (GRALLA and VALENTINE 1991 ; LIU et al. 1992 ). sod1 mutant phenotypes are suppressed by mutation in the PMR1 gene (LAPINSKAS et al. 1995 ). Deficiency in the Pmr1p transporter causes yeast cells to accumulate higher levels of cytosolic Mn²⁺ in the cells. It is believed that the elevated level of Mn²⁺ stimulates a Mn²⁺-dependent antioxidant that scavenges the superoxide anion generated during aerobic growth of the sod1 mutant (LAPINSKAS et al. 1995 ).

IMP2 was initially isolated as a gene that restored to a yeast mutant the ability to grow on fermentable carbon sources, such as maltose, galactose, and raffinose (DONNINI et al. 1992 ). We recently re-isolated the yeast IMP2 gene and demonstrated that it is required to mediate cellular resistance to oxidative DNA damaging agents, including bleomycin and hydrogen peroxide (MASSON and RAMOTAR 1996 ). Furthermore, the hypersensitivity of the imp2 mutants to oxidants is due to the inability to repair oxidatively damaged DNA (MASSON and RAMOTAR 1996 ). Imp2p is believed to carry out its biological function as a transcriptional activator, particularly since it possesses an acidic domain characteristic of many transcriptional activator proteins. In addition, it is capable of activating the expression of ß-galactosidase in the classical test system, lexA-lacZ (MASSON and RAMOTAR 1996 ). A role in transcription is further supported by the finding that Imp2p is required to turn on the expression of at least three genes MALS, MALT, and GAL2, respectively, encoding maltase, maltose permease, and galactose permease (LODI et al. 1995 ). To date, however, there is no evidence indicating that Imp2p is regulating genes involved in oxidative stress response.

Because imp2 mutants are hypersensitive to drugs that generate free radicals, we decided to test if these mutants are also sensitive to free radicals released by macrophages in culture medium. Rather unexpectedly, we discovered that the imp2 mutants were rapidly killed by the macrophage culture medium, and not by the macrophages themselves. This preliminary study provided an indication that imp2 mutants might be hypersensitive to the higher salt content of the macrophage medium. We now extend this study and show that imp2 mutants are hypersensitive to both mono- and divalent ions and further demonstrate that Imp2p maintains Na⁺/Li⁺ ion homeostasis independently of Ena1p.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and media:
The S. cerevisiae strains used in this study are described in Table 1. Yeast cells were grown in either complete YPD or minimal synthetic (SD) medium (SHERMAN et al. 1983 ). When necessary, lithium chloride (125 mM), sodium chloride (800 mM), calcium chloride (300 mM), zinc chloride (6 mM), manganese chloride (6 mM), copper sulfate (10 mM), or bleomycin (3 µg/ml; generously provided by Bristol-Myers Squibb, Princeton, NJ) was added to YPD agar. The Escherichia coli strains used for plasmid maintainance were HB101 and DH5, and they were grown in either Terrific Broth or Luria Broth (SAMBROOK et al. 1989 ). Ampicillin (50 µg/ml) was added as required.

Plasmids:
The single copy plasmids, pHAL3-1 and pENA1-1, and the multicopy plasmids, pHAL3-2, pHAL1, pHAL2, pENA2-2, were kindly provided by RAMON SERRANO and KYLE CUNNINGHAM. The single copy and multicopy plasmids pIMP2-1 and pIMP2-2 were previously constructed in our laboratory and were derived from the single copy and multicopy vectors, YCp50 and YEp352 or YEp24, respectively. pIPR695 bears a 695-bp fragment consisting of the promoter region of IMP2 gene subcloned into the multicopy vector YCP356R (MASSON and RAMOTAR 1996 ). pKC201 bears the ENA1-LacZ fusion promoter probe vector (generously provided by KYLE CUNNINGHAM). The HAL3-LacZ fusion construct was provided by RAMON SERRANO.

Transformation and other recombinant DNA techniques:
Yeast cells were transformed by the lithium-acetate method (GIETZ and SCHIESTL 1991 ). Plasmid DNA purification, DNA cloning, and transformation of E. coli competent cells were done as previously described (SAMBROOK et al. 1989 ).

Crude extract preparation and ß-galactosidase assay:
Log phase cultures (typically 5 ml) grown in selective medium or YPD were centrifuged at 3000 rpm for 5 min and washed twice with 20 mM potassium phosphate buffer (pH 7.0). Crude protein extracts were prepared from the cell pellet by using a mini-bead beater (Xymotech, Montreal, Canada) and as previously described (MASSON and RAMOTAR 1996 ). The crude extracts were used to monitor ß-galactosidase activity (MILLER 1992 ).

Northern blot:
Total RNA was prepared by the rapid method (SCHMITT et al. 1990 ). Typically, 15 µg of RNA per lane was electrophoresed into a formaldehyde gel and transferred to a nylon N+ membrane (Amersham, Arlington Heights, IL). The blot was incubated overnight at 52° in prehybridization buffer (50% formamide, 0.1% SDS, 5x Denhardt's, 50 mM sodium phosphate buffer pH 6.5, 5x SSC, 0.1% sodium pyrophosphate, 100 µg/ml tRNA and 250 µg/ml denatured salmon sperm DNA). The membrane was probed (2 x 10⁶ cpm/ml) overnight at 52° with a random primed (T7 Quick Prime kit, Pharmacia, Piscataway, NJ) ³²P-labeled 2.5-kb EcoRI-HindIII fragment derived from the coding region of the HAL3 gene. The blot was washed two times in 1x SSC/0.1% SDS for 15 min at room temperature, followed by three washes in 0.2x SSC/0.1% SDS at 42°, 55°, and 65° for 30 min. The membrane was exposed for 72 hr at -80° and revealed by autoradiography.

Measurements of intracellular ion concentrations:
Both Na⁺ and Li⁺ cations were determined as described (FERRANDO et al. 1995 ). Briefly, cells were incubated in SD medium containing either 800 mM NaCl or 75 mM LiCl for the indicated time, harvested at 2000 g for 5 min at 4°, and resuspended in 20 mM MgCl₂ plus 1.5 or 0.2 M sorbitol, respectively. The cells were recovered as above, resuspended in the same solution, and collected on a glass filter (Whatman GF/C, Fisher Scientific, Ontario, Canada). The cells on the filter were washed twice and extracted by incubation with 1 ml of 20 mM MgCl₂ and heated at 95° for 15 min. Cells were pelleted and aliquots of the supernatant were analyzed for ionic content using an atomic absorption spectrometer. Na⁺ and Li⁺ cations were normalized to the dry weight of the sample. In the case of copper, manganese, and cobalt determinations, the cells were grown in 15 ml of SD medium to an OD600 of 1.0 and treated according to LAPINSKAS et al. 1995 . Cells were harvested and washed, and divalent ion contents were determined using atomic absorption spectrometer. The intracellular concentration of the divalent ions was expressed as nanomoles of ions per 10⁹ cells.

Gradient plate assay:
This assay was performed as previously described by RAMOTAR and MASSON 1996 . The gradient was prepared by using a 9 x 9-cm² petri plate (Falcon, Lincoln Park, NJ) placed at an inclined angle of 10°. The indicated concentration of NaCl or LiCl was added to 30 ml of YPD agar in a capped 50-ml sterile falcon tube, mixed by gentle inversion to avoid bubbles, and poured into the plate. After the medium solidified, the plate was placed horizontally followed by the addition of 35 ml of YPD agar without salt. Once the top layer solidified, the plate was dried at 37° for 40 min. Ten microliters of overnight culture was added to 1 ml of YPD agar (0.7% agar) kept at 55°, mixed, and poured along the entire length of a 2.5 x 8-cm microscope slide placed on top of a 55° heating block; the thin edge of another microscope slide was used to replicate the cells along the gradient so as to make a line. Cells that are resistant to the salt will grow all along the gradient and those cells that are sensitive to the salt will grow only a short distance along the gradient. A strain that grows all along the gradient (85 mm) is considered to show 100% growth. In general, this type of gradient provides a rapid way to assess salt tolerance of a given cell population.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

imp2 null mutants are hypersensitive to mono- and divalent ions:
Preliminary data suggested that Imp2p could protect yeast cells from stress conditions other than oxidative challenge. We therefore exposed the imp2 mutants to both mono- and divalent ions, as well as osmotic and heat shock. Both the parent and the imp2 strains were first streaked onto YPD solid agar plates containing increasing concentrations of NaCl. A concentration of 800 mM NaCl completely abolished growth of the imp2 mutant, but not the parent strain (Figure 1A). Since Li⁺ and Na⁺ cation homeostasis is known to be regulated by the same proteins in yeast, we tested whether the growth of the imp2 mutant was also affected by LiCl. Indeed a concentration of 125 mM LiCl totally retarded the growth of the imp2 mutant (Figure 1A). It is not surprising that sixfold less LiCl was required to achieve the same toxic effect as NaCl, since Li⁺ cations are more toxic than Na⁺. These concentrations of NaCl and LiCl also effectively prevented growth in three other wild-type strains (FY86, W303; MKp-o) that lacked the IMP2 gene (Table 1). No differences in cytotoxicity was observed between the imp2 mutant and the parent exposed to KCl, even at a concentration of 400 mM, where the growth of the parent began to diminish (data not shown). These data represent the first strong evidence that Imp2p is required to maintain monovalent ion homeostasis.

View larger version (111K): In this window In a new window Download PPT slide   Figure 1. —Growth inhibition of the parent and imp2 mutant strains challenged with mono- and divalent ions. The plate assay contained (A) either 0.8 M NaCl or 0.125 M LiCl and (B) 6 mM ZnCl2, 6 mM MnCl2, 10 mM CuSO4, or 300 mM CaCl2 in YPD medium. The photographs were taken 3 days after incubation at 30°.

We also examined whether Imp2p is essential for controlling divalent ion homeostasis. Interestingly, the imp2 mutant was unable to grow on YPD agar plates containing 6 mM of zinc chloride, 6 mM of manganese chloride, 300 mM of calcium chloride, or 10 mM of copper sulfate as compared to the parent (Figure 1B). The imp2 mutant was no more sensitive than the parent, when challenged with cadmium carbonate (2.5 mM), nickel chloride (10 mM), cobalt chloride (10 mM), magnesium chloride (100 mM), or iron sulfate (400 mM) (data not shown). This suggests that the mutant does not have a general defect in divalent ion metabolism. The observed hypersensitivity of the imp2 mutant to CaCl₂, ZnCl₂, MnCl₂, and CuSO₄ cannot be due to atomic size exclusion, since Ni²⁺ and Co²⁺, which are not toxic to the mutant, have nearly the same radii of approximately 0.075 nm as the toxic ions Mn²⁺ and Zn²⁺. Collectively, the above findings strongly suggest that Imp2p is essential for maintaining both mono- and divalent ion homeostasis, presumably by controlling the expression of independent proteins required to prevent toxicity caused by Na⁺/Li⁺ and Cu²⁺/Zn²⁺/Ca²⁺/Mn²⁺.

Northern blot analysis revealed that Imp2p levels were not altered in wild-type strains exposed to varying concentrations of mono- and divalents ions (data not shown). In addition, a plasmid construct bearing the presumptive IMP2 promoter, from nucleotides -695 to +1, fused to the lacZ reporter gene and introduced into either the parent or the imp2 mutant (MASSON and RAMOTAR 1996 ), showed a level of ß-galactosidase of ~120 units/mg protein in both strains, which was not altered when the cells were exposed to various concentrations of mono- and divalent ions. Imp2p was also not induced upon exposure to the oxidants, bleomycin and hydrogen peroxide (MASSON and RAMOTAR 1996 ). Thus, Imp2p appears to retain constitutive expression in yeast cells exposed to salt or oxidative stress.

As compared to the parent, the imp2 mutant showed normal growth on media containing 1 M sorbitol, excluding the possibility that Imp2p plays a role in osmotic shock. However, the imp2 mutant was sensitive to heat treatment for 2 hr at 50° (data not shown). Since imp2 mutants are known to be sensitive to agents generating oxygen-free radicals, its sensitivity to heat may be related to the burst of free radicals produced by the heat treatment (DAVISON et al. 1996 ).

imp2 mutant accumulate mono- and divalent ions:
The fact that imp2 mutants were hypersensitive to either mono- or divalent ions prompted us to test whether this could be due to intracellular accumulation of the ions. Both the parent and the mutant strains were grown in SD medium, supplemented with 0.8 M NaCl or 0.1 M LiCl. Samples were withdrawn at various times and subjected to atomic absorption spectroscopy to determine the amount of intracellular ions. Such experiments revealed that within 15 min the rates of Na⁺ and Li⁺ uptake were nearly the same in both strains, but differed beyond this time point (Figure 2A and B). While the parent strain achieved saturation of Na⁺ and Li⁺ uptake at around 45 min, the mutant continued to accumulate these ions. In general, the mutant accumulated nearly two- and threefold higher levels of, respectively, Na⁺ and Li⁺, as compared to the parent. Differences in intracellular divalent ions were also observed between the two strains grown in standard SD medium (Table 2). When compared to the parent, the imp2 mutant accumulated nearly four- and threefold higher intracellular level of, respectively, Mn²⁺ and Cu²⁺, but not Ni²⁺. No extensive studies were done to quantitate other mono- or divalent ions intracellular concentrations, as these experiments were too costly. Nonetheless, it appears that the toxicity of the mono- and divalent ions is a result of accumulation of these ions in the imp2 mutant.

View larger version (13K): In this window In a new window Download PPT slide   Figure 2. —Time-dependent accumulation of Na+ and Li+ cations in parent and imp2 mutant. Log phase cells grown in SD medium were supplemented with either 800 mM NaCl or 75 mM LiCl at time zero. At the indicated times, samples were taken and the intracellular accumulation of Na+, and Li+ cations was determined by atomic absorption spectroscopy. Open circle, the parent strain DBY747 (IMP2); and closed circle, strain DRY212 (imp2). The data are representative of three independent experiments. (A) Sodium, (B) lithium.

View this table: In this window In a new window   Table 2. Divalent ion accumulation in the parent and the imp2 mutant

Multicopy suppressor genes restore monovalent ions homeostasis in the imp2 null mutant:
Since Imp2p is a transcriptional activator, it may control one or more genes involved in salt tolerance. A number of salt tolerance genes are known, but none has been reported to be controlled by Imp2p. To identify such possible salt tolerance genes, we first sought multicopy suppressor genes that restore monovalent ion resistance to the imp2 mutant. A genomic DNA library constructed in the YEplac112 vector and bearing the auxothrophic selective marker gene TRP1 was introduced into the imp2 mutant. The resulting Trp⁺ colonies were replica plated and examined for growth on YPD solid agar plate containing 800 mM NaCl, which permitted wild-type growth, but not growth of the imp2 mutant. We identified two suppressor plasmids pSOS1 and pSOS2, from which subclones were derived and the DNA sequence determined. The suppressors were identical to previously reported genes, i.e., ENA1, which encodes the P-type ATPase pump involved in the efflux of sodium and lithium from yeast cell (WIELAND et al. 1995 ), and HAL3, which encodes a protein that allows wild-type cells to become more tolerant to sodium ions (FERRANDO et al. 1995 ).

A multicopy vector YEp352 carrying the entire promoter and coding regions of either the ENA1 or HAL3 gene restored full wild-type NaCl or LiCl resistance to the imp2 mutant, as determined by gradient plate assays that can measure the salt tolerance of a given strain (Figure 3A and B, bars 7 and 8 vs. 5, respectively). In contrast to the multicopy vector, the centromeric single copy vector YCp50 carrying either the ENA1 or HAL3 gene conferred no NaCl or LiCl resistance to the imp2 mutant (Table 3). These data clearly indicate that increased gene dosage of either ENA1 or HAL3 is essential for restoring Na⁺ and Li⁺ resistance to the imp2 mutant. Interestingly, the increased gene dosage provided no more than wild-type NaCl resistance to the imp2 mutant, contrasting a previous report indicating that overexpression of Hal3p confers additional salt resistance to a wild-type strain (FERRANDO et al. 1995 ). This disparity may be explained if in our case Hal3p is not expressed at a sufficiently high level.

View larger version (54K): In this window In a new window Download PPT slide   Figure 3. —Na+/Li+ resistance conferred to the wild-type, imp2, ena1, and imp2 ena1 strains carrying the indicated multicopy plasmids. Bars 1–4, wild-type strain K601 (IMP2, ENA1); bars 5–8, strain JYM1 (imp2::LEU2); bars 9–12, strain K633 (ena1::HIS3); and bars 13–16, strain JYM2 (imp2::LEU2; ena1::HIS3). All of the strains harbor either the 2-µm vector YEp352 or the multicopy plasmids pIMP2-2, or pENA1-2, and/or pHAL3-2 that were derived from YEp352. The results were obtained from gradient plate assays where the bottom layer contained either 1.2 M NaCl (A) or 0.2 M LiCl (B). Growth all along the gradient is considered to be 100%.

Imp2p does not regulate expression of either Ena1p or Hal3p:
Since Hal3p is known to stimulate the expression of Ena1p, we envisage that the gene dosage effect of ENA1 and HAL3 can be explained if Ena1p expression is reduced in the imp2 mutant. To directly test whether Imp2p exerts control on the expression of the ENA1 gene, total mRNA was isolated from both wild-type and the imp2 mutant and probed with the ENA1 gene. The result clearly indicated that the ENA1 message level was not altered in these strains (data not shown). As a corollary to this experiment, we introduced a plasmid construct, ena1::lacZ, bearing the promoter of ENA1 fused to the reporter gene lacZ, into the parent and the imp2 mutant strains and measured the level of ß-galactosidase expression. Extract prepared from either the wild-type or the imp2 mutant showed the same level of ß-galactosidase (60 units/mg protein). Furthermore, no dramatic difference in ß-galactosidase levels were observed in extracts derived from either the parent or imp2 mutant exposed to NaCl or LiCl. These data are consistent with the notion that Imp2p exerts no control on ENA1 gene expression. Similarly, Imp2p exerts no control on the HAL3 gene, as determined by Northern blot analysis (data not shown)

Imp2p confers salt resistance independently of Ena1p:
While the data clearly excluded the possibility that Imp2p regulates Ena1p, it remains possible that Imp2p could also control expression of a protein that stimulates Ena1p activity. To test this, we constructed a imp2 ena1 double mutant by deleting the IMP2 gene in the ena1 null mutant strain K633, which was derived from the parent K601 (Table 1). The isogenic single mutants ena1 and imp2, and the double mutant imp2 ena1 were examined for their sensitivity toward Na⁺ and Li⁺ using gradient plate assays. As shown in Figure 3A, both the imp2 and the ena1 single mutants showed nearly the same level of sensitivity to NaCl, as compared to the parent. However, the imp2 ena1 double mutant was exquisitely sensitive to NaCl (Figure 3A, bar 13). A similar result was also obtained when the strains were challenged with LiCl (Figure 3B). This finding eliminates the possibility that Imp2p controls the expression of a Ena1p-stimulatory protein, and instead indicates that Imp2p is acting independently of the Ena1p Na⁺/Li⁺ pump.

Because Hal3p restored Na⁺/Li⁺ resistance to the imp2 mutant and Hal3p is known to stimulate Ena1p expression, we decided to test whether Hal3p indeed acted via Ena1p. As a consequence, we introduced the multicopy plasmid pHAL3-2 into the imp2 ena1 double mutant and the ena1 single mutant and tested for salt resistance by gradient plate assays. Unexpectedly, pHAL-3-2 restored to the double mutant Na⁺ resistance to the level of that observed by the single imp2 mutant (Figure 3A, bar 16 vs. bar 5). The same plasmid also conferred a modest level of Na⁺ resistance to the ena1 single mutant (Figure 3A, bar 12). Interestingly, pHAL3-2 did not rescue the Li⁺ sensitivity of either ena1 single (Figure 3B, bar 12) or imp2 ena1 double mutant (Figure 3B, bar 16). These data suggest that Hal3p can promote Na⁺ resistance independently of Ena1p, but absolutely requires Ena1p to promote Li⁺ resistance. A simple interpretation of this finding is that Hal3p appears to stimulate another Na⁺ pump that has virtually little or no affinity for Li⁺. It should be noted that overexpression of Hal3p-related protein, the S. cerevisiae YKL088w protein or Ss22p, conferred no resistance to the imp2 mutant, despite the fact that both Hal3p and Ss22p have very acidic domains in their C-termini ends and share 38% identity over a stretch of 267 amino-acid residues (DI COMO et al. 1995 ).

Hal3p, but not Ena1p, confers copper and bleomycin resistance to the imp2 null mutant:
The fact that either Ena1p or Hal3p can replace Imp2p in protecting cells against monovalent ions prompted us to examine whether these two genes can also protect imp2 mutants from the toxic effects of divalent ions. imp2 mutants carrying the multicopy plasmid pENA1-2 remained hypersensitive to the divalent ions Cu²⁺, Zn²⁺, Mn²⁺, and Ca²⁺ (data not shown). Rather surprisingly, the multicopy plasmid pHAL3-2 conferred wild-type resistance to the imp2 mutant to Cu²⁺, but not to any of the other divalent ions, such as Zn²⁺, Mn²⁺, and Ca²⁺ to which the imp2 null mutants displayed hypersensitivity (Figure 1B; data not shown).

The fact that Cu²⁺ is a redox-active metal ion raised the possibility that the oxidant hypersensitivity of the imp2 mutant could be directly linked to copper toxicity. As a result, we tested whether overexpression of Hal3p would also confer oxidant resistance to the imp2 mutant. As shown in Figure 4, Hal3p conferred full bleomycin resistance to the imp2 mutant, but conferred no resistance to H₂O₂ (data not shown). To test if there is a relationship between copper and bleomycin toxicity, we examined whether the copper-sensitive mutant, cup1, that lacks the small cysteine protein metallothionien was also sensitive to bleomycin. We observed that the cup1 mutant was no more sensitive to either bleomycin or other oxidants than the parent strain (HAMER et al. 1985 ; data not shown). We conclude that the sensitivity of the imp2 mutant to bleomycin is unlikely to be linked to Cu²⁺ toxicity (see discussion).

View larger version (86K): In this window In a new window Download PPT slide   Figure 4. —Overexpression of Hal3p conferred bleomycin resistance to the imp2 mutant. The strains used were (clockwise from the top) DRY212 (imp2::LEU2), DBY747 (IMP2), DRY212/pHAL3-2, K633 (ena1::HIS3), W303-1A (ENA1), and DRY212/pENA1-2. The photographs were taken 3 days after incubation at 30°.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we demonstrate that mutants lacking the acidic transcriptional activator Imp2p are unable to grow when challenged with elevated levels of the monovalent ions Na⁺ and Li⁺, as well as the divalents ions Ca²⁺, Mn²⁺, Zn²⁺, and Cu²⁺. We previously showed that imp2 mutants are also hypersensitive to oxidative agents, including the antitumor drug bleomycin and the chemical oxidant hydrogen peroxide (MASSON and RAMOTAR 1996 ). From these combined studies, we conclude that Imp2p is required to control both salt and oxidative stresses.

The mechanism of gene activation by Imp2p does not involve its own induction (MASSON and RAMOTAR 1996 ), and perhaps pre-existing Imp2p is post-translationally modified in response to salt or oxidative stress. Imp2p has an arginine/serine (R/S)-rich domain encompassing amino acids 10 to 39 (MASSON and RAMOTAR 1996 ). Proteins with R/S domains are known to be phosphorylated in vivo thus influencing their biological function (ROSSI et al. 1996 ; COLLWILL et al. 1996 ). Imp2p also has a C-terminus leucine-rich repeat (LRR) domain that is 25 residues in length, but no obvious DNA binding domain. In a few cases, the LRR has been clearly documented to mediate protein-protein interactions, for example, that of the yeast transcriptional activator Ccr4 (DRAPER et al. 1994 ; KOBE and DEISENHOFER 1995 ). We therefore propose that in response to stress, Imp2p becomes activated by phosphorylation, which then turns on gene expression through protein-protein interaction. The activated form of Imp2p must be capable of discriminating between genes involved in salt and oxidative stress. Whether this distinction is made by different domains of Imp2p awaits structure/function analyses. Alternatively, Imp2p could simply serve to maintain basal level expression, as opposed to induced levels, of proteins involved in both salt and oxidative stress.

Several mutants (pos) isolated on the basis of their sensitivities toward H₂O₂ are also sensitive to NaCl (KREMS et al. 1995 ). While the molecular defect associated with this cross-sensitivity remains unclear, it is possible that endogenous oxidative stress is increased in some pos mutants, thus damaging the cell membrane and rendering the cell more permeable to NaCl. However, it is unlikely that salt stress results from oxidative stress in the imp2 mutant, since both stress responses were uncoupled when imp2 suppressor mutants that showed parental resistance to oxidants but retained full NaCl sensitivity were isolated (D. RAMOTAR, unpublished data). Furthermore, deficiency in the transcriptional activator Yap1p also results in hypersensitivities to oxidative stress, but causes no cross-sensitivity to NaCl (KUGE and JONES 1994 ).

We therefore propose that there is at least one other protein, besides Ena1p, that contributes significantly to the maintenance of Na⁺/Li⁺ tolerance in S. cerevisiae. Indeed, we show here that mutant cells bearing a deletion in both the ENA1 and IMP2 genes are exquisitely sensitive to Na⁺/Li⁺, whereas the single mutants ena1 or imp2, while equally sensitive, are less sensitive than the double mutant. The additive salt sensitivity of the imp2 ena1 double mutant implies that Imp2p does not mediate a stimulatory effect on Ena1p either at the gene or protein level and evokes the involvement of another Na⁺/Li⁺ pump, whose identity remains to be revealed. One such pump that could be controlled by Imp2p might be coded for by one of the four remaining members of the multigene cluster at the ENA1/PMR2 locus, i.e., PMR2b, PMR2c, PMR2d, and PMR2e. Deletion of the entire ENA1/PMR2 locus resulted in mutants that are extremely sensitive to salt (WIELAND et al. 1995 ). However, no detailed study has been done to examine the contribution of each of the Ena1p members to Na⁺/Li⁺ homeostasis, with the exception of Pmr2bp, which is nearly identical to Ena1p (WIELAND et al. 1995 ). Pmr2bp functionally replaces Ena1p when under the control of the ENA1 promoter that expresses the protein at a higher level (WIELAND et al. 1995 ). There is also the possibility that Imp2p could control a protein that functions in conjunction with ion channels to prevent entry of Na⁺/Li⁺ cations into the cell. Thus, a defective channel could allow influx of Na⁺/Li⁺ well beyond the operating capacity of Ena1p. In either case, loss of a second pump—or a channel protein—controlled by Imp2p would cause ena1 mutants to be even more sensitive to Na⁺/Li⁺. Recently, a Na⁺/H⁺ antiporter protein encoded by the NHA1 gene was isolated from yeast, and it was shown that nha1 mutants are sensitive to Na⁺ and Li⁺ (PRIOR et al. 1996 ). Increased NHA1 gene dosage via a multicopy vector also confers salt tolerance to the ena1 mutant. Moreover, the ena1 nha1 double mutant is extremely sensitive to NaCl (PRIOR et al. 1996 ). It is conceivable that Imp2p could also regulate NHA1 gene expression. In any case, loss of the Imp2p-controlled protein is not compensated for by the endogenous levels of Ena1p, unless Ena1p is overexpressed. Thus, the Imp2p-controlled protein cannot be an auxiliary protein.

How Imp2p controls divalent ion homeostasis is less clear. The toxicity of the imp2 mutant to a selective set of divalent ions cannot be explained by downregulation of a single transporter involved in divalent ion detoxification, such as Cot1p, Zrc1p, Pmc1p, and Pmr1p (CONKLIN et al. 1994 ; FARCASANU et al. 1995 ; LAPINSKAS et al. 1995 ). It is more likely that Imp2p regulates the expression of more than one of these proteins. We have not examined whether Imp2p could influence the expression of any of the above transporters involved in divalent ion homeostasis. However, it is reasonable to assume that Pmr1p and Pmc1p are potential targets, since pmr1 and pmc1 mutants are reported to be hypersensitive to Mn²⁺ and Ca²⁺, respectively (LAPINSKAS et al. 1995 ; CUNNINGHAM and FINK 1996 ). If so, one should find, for example, that the imp2 pmr1 double mutant will display the same sensitivity to Mn²⁺ as either the imp2 or pmr1 single mutant. Perhaps screening for multicopy genes that suppress the divalent ion toxicity of the imp2 mutant might prove a more valuable way to identify these genes.

Since Cu²⁺ and Mn²⁺ are redox-active metal ions, increased intracellular concentrations of these ions could potentiate the lethal effects of oxidants. From our data, it would appear that there is no link between the divalent ions' toxicities and the oxidant hypersensitivities of the imp2 mutant. However, the observation that overexpression of Hal3p confers Cu²⁺ as well as bleomycin resistance to the imp2 mutant may simply be coincidental. Both copper and bleomycin may either enter or be expelled from the cell by the same pathway regulated by Imp2p. Direct studies with the copper-sensitive mutant, cup1, clearly indicates that there is no link between copper toxicity and bleomycin.

How Hal3p replaces Imp2p:
The fact that hal3 null mutants do not share imp2 mutant phenotypes suggests that Hal3p has no direct role in either divalent ion homeostasis or defense against oxidative stress. We believe that the observed partial complementation of Imp2p phenotypes by overexpression of Hal3p could be explained if Hal3p is also acting as a transcriptional activator. This is supported by the fact that Hal3p possesses features resembling transcriptional activators, such as an acidic domain (FERRANDO et al. 1995 ) and the ability to stimulate expression of some genes, including ENA1 and the cyclin genes CLN1, CLN2, and CLB5 (DI COMO et al. 1995 ). It is not clear why Hal3p only suppresses some of the imp2 mutant phenotypes, Na⁺/Li⁺-, Cu²⁺-, and bleomycin sensitivities, and not Mn²⁺-, Ca²⁺-, Zn²⁺-, and H₂O₂-sensitivities. Whether this reflects more than one mechanism by which Imp2p activates expression of various different genes, and that Hal3p overexpression functions by only one of the mechanisms, remains to be investigated.

	ACKNOWLEDGMENTS

We thank Drs. ELLIOT DROBETSKY and HANS RUDOLPH for critically reviewing the manuscript. We also thank Drs. KIM ARNDT, KYLE CUNNINGHAM, HANS RUDOLPH, RAMON SERRANO, and HANA SRYCHROVA for providing strains and plasmids. This work was supported by a grant to D.R. from the National Cancer Institute of Canada with funds from the Canadian Cancer Society. J.-Y.M. received a graduate student fellowship from the Fonds pour la formation de Chercheurs et d'Aide à la Recherche du Québec and D.R. is a Career Scientist of the National Cancer Institute of Canada.

Manuscript received July 18, 1997; Accepted for publication March 12, 1998.

	LITERATURE CITED

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