A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.     

The Arabidopsis HAL2-like gene family includes a novel sodium-sensitive phosphatase

The yeast HAL2 gene encodes a lithium- and sodium-sensitive phosphatase that hydrolyses 3'-phosphoadenosine-5'-phosphate (PAP). Salt toxicity in yeast results from Hal2 inhibition and accumulation of PAP, which inhibits sulphate assimilation and RNA processing. We have investigated whether the model plant Arabidopsis thaliana contains sodium-sensitive PAP phosphatases. The Arabidopsis HAL2-like gene family is composed of three members: AtAHL and AtSAL2, characterized in the present work, and the previously identified AtSAL1. The AtAHL and AtSAL2 cDNAs complement the auxotrophy for methionine of the yeast hal2 mutant and the recombinant proteins catalyse the conversion of PAP to AMP in a Mg(2+)-dependent reaction sensitive to inhibition by Ca2+ and Li+. The PAP phosphatase activity of AtAHL is sensitive to physiological concentrations of Na+, whereas the activities of AtSAL1 and AtSAL2 are not. Another important difference is that AtAHL is very specific for PAP while AtSAL1 and AtSAL2 also act as inositol polyphosphate 1-phosphatases. AtAHL constitutes a novel type of sodium-sensitive PAP phosphatase which could act co-ordinately with plant sulphotransferases and serve as target of salt toxicity in plants.     

Crystal structure of an enzyme displaying both inositol-polyphosphate-1-phosphatase and 3'-phosphoadenosine-5'-phosphate phosphatase activities: a novel target of lithium therapy.

Lithium cations exert profound and selective psychopharmacological effects on ameliorate manic-depressive psychosis. Although lithium is an effective drug for both treatment and prophylaxis of bipolar disorder, the precise mechanism of action is not well understood. Lithium acts as both an uncompetitive and non-competitive inhibitor of several lithium- sensitive phosphatases with regard to substrate and magnesium cofactor, respectively. In this work, we report the crystal structure and reaction mechanism of Rattus norvegicus 3'-phosphoadenosine 5'-phosphate and inositol 1,4-bisphosphate phosphatase (RnPIP), a recently identified target of lithium therapy. This Li(+)-sensitive enzyme plays a crucial role in several cellular processes, such as RNA processing, sulphation reactions and probably inositol recycling. RnPIP specifically removes the 3'-phosphate group of 3'-phosphoadenosine 5'-phosphate (PAP) and the 1'-phosphate group of inositol 1,4-bisphosphate (I(1),(4)P(2)) producing AMP and inositol 4'-phosphate, respectively. The crystal structure of RnPIP complexed with AMP, Pi and magnesium ions at 1.69 A resolution provides insight into the reaction mechanism of the hydrolysis of PAP. The core fold of the enzyme is equivalent to that found in other Li(+)-sensitive phosphatases, such as inositol monophosphatase, but molecular modelling of I(1),(4)P(2) in the RnPIP active site reveals important structural determinants that accommodate this additional substrate. RnPIP is potently inhibited by lithium and, as the accumulation of PAP inhibits a variety of proteins, including sulphotransferases and RNA processing enzymes, this dual specificity enzyme represents a potential target of lithium action, in addition to inositol monophosphatases.     

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.     

Novel 3'-phosphoadenosine-5'-phosphatases from extremely halotolerant Hortaea werneckii reveal insight into molecular determinants of salt tolerance of black yeasts

The 3'-phosphoadenosine-5'-phosphatase encoded by HAL2 gene, is a ubiquitous enzyme required for the removal of the cytotoxic 3'-phosphoadenosine-5'-phosphate produced during sulfur assimilation in eukaryotes. Salt toxicity in yeast and plants results from Hal2 inhibition by sodium or lithium ions. Two novel HAL2-like genes, HwHAL2A and HwHAL2B, have been cloned from saltern-inhabited extremely halotolerant black yeast Hortaea werneckii. Expression of both HwHAL2 isoforms was differentially inducible upon salt. When the HwHAL2 genes were transferred from such a halotolerant species into the salt sensitive Saccharomyces cerevisiae, the resulting organism can tolerate 1.8M NaCl or 0.8M LiCl, the highest reported salt concentrations at which S. cerevisiae can grow. With genetic and biochemical validation we demonstrated the critical HwHal2B sequence motif--the META sequence--common only to Dothideales fungi, with evident effect on the HwHal2B-dependent salt tolerance. These results may have significance for biosaline agriculture in coastal environments.     

Purification and biochemical characterization of Mycobacterium tuberculosis SuhB, an inositol monophosphatase involved in inositol biosynthesis

Phosphatidylinositol is an essential component of mycobacteria, and phosphatidylinositol-based lipids such as phosphatidylinositolmannosides, lipomannan, and lipoarabinomannan are major immunomodulatory components of the Mycobacterium tuberculosis cell wall. Inositol monophosphatase (EC 3.1.3.25) is a crucial enzyme in the biosynthesis of free myo-inositol from inositol-1-phosphate, a key substrate for the phosphatidylinositol synthase in mycobacteria. Analysis of the M. tuberculosis genome suggested the presence of four M. tuberculosis gene products that exhibit an inositol monophosphatase signature. In the present report, we have focused on SuhB, which possesses the highest degree of homology with human inositol monophosphatase. SuhB gene was cloned into an E. coli expression vector to over-produce a His-tagged protein, which was purified and characterized. SuhB required divalent metal ions for functional inositol monophosphatase activity, with Mg(2+) being the strongest activator. Inositol monophosphatase activity catalyzed by SuhB was inhibited by the monovalent cation lithium (IC(50) = 0.9 mM). As anticipated, inositol-1-phosphate was the preferred substrate (K(m) = 0.177 +/- 0.025 mM; k(cat) = 3.6 +/- 0.2 s(-)(1)); however, SuhB was also able to hydrolyze a variety of polyol phosphates such as glucitol-6-phosphate, glycerol-2-phosphate, and 2'-AMP. To provide further insight into the structure-function relationship of SuhB, different mutant proteins were generated (E83D, D104N, D107N, W234L, and D235N). These mutations almost completely abrogated inositol monophosphatase activity, thus underlining the importance of these residues in inositol-1-phosphate dephosphorylation. We also identified L81 as a key residue involved in sensitivity to lithium. The L81A mutation rendered SuhB inositol monophosphatase activity 10-fold more resistant to inhibition by lithium (IC(50) = 10 mM). These studies provide the first steps in the delineation of the biosynthesis of the key metabolite inositol in M. tuberculosis.     

Salt tolerance and methionine biosynthesis in Saccharomyces cerevisiae involve a putative phosphatase gene.

The progressive salinization of irrigated land poses a threat to the future of agriculture in arid regions. The identification of crucial metabolic steps in salt tolerance is important for the understanding of stress physiology and may provide the tools for its genetic engineering. In the yeast Saccharomyces cerevisiae we have isolated a gene, HAL2, which upon increase in gene dosage improves growth under NaCl and LiCl stresses. The HAL2 protein is homologous to inositol phosphatases, enzymes known to be inhibited by lithium salts. Complementation analysis demonstrated that HAL2 is identical to MET22, a gene involved in methionine biosynthesis. Accordingly, methionine supplementation improves the tolerance of yeast to NaCl and LiCl. These results demonstrate an unsuspected interplay between methionine biosynthesis and salt tolerance.     

Expression of yeast INM1 encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate

Inositol monophosphatase plays a vital role in the de novo biosynthesis of inositol and in the phosphoinositide second messenger signalling pathway. We cloned the Saccharomyces cerevisiae open reading frame (ORF) YHR046c (termed INM1), which encodes inositol monophosphatase, characterized the protein Inm1p and analysed expression of the INM1 gene. INM1 was expressed in bacteria under the control of the lacZ promoter. The purified protein has inositol monophosphatase activity that is inhibited by the antibipolar drug lithium, but not valproate. In the inm1Delta:URA3 null mutant, inositol monophosphatase activity was reduced but not eliminated. The disruption had little effect on growth in the presence of lithium or valproate and no effect on growth in the absence of inositol. To characterize the regulation of INM1, we examined the effects of inositol, carbon source, growth phase, and the antibipolar drugs lithium and valproate on INM1 expression using an INM1-lacZ reporter gene. Unlike all other phospholipid biosynthetic enzyme-encoding genes studied, which contain the UASINO regulatory element, INM1 expression is increased in the presence of inositol. In addition, INM1 expression was repressed during growth in glycerol and derepressed as glucose-grown cells entered stationary. Both lithium and valproate, which cause a decrease in intracellular inositol, effect a decrease in INM1 expression. A model is presented to account for regulation of INM1 expression.     

Regulation of inositol monophosphatase in Saccharomyces cerevisiae

Inositol monophosphatase is a key enzyme in the de novo biosynthesis of inositol and in the phosphoinositide second-messenger signalling pathway. Inhibition of this enzyme is a proposed mechanism for lithium's pharmacological action in bipolar illness (manic depression). Very little is known about how expression of this enzyme is regulated. Because the yeast Saccharomyces cerevisiae has been shown to be an excellent model system in which to understand the regulation of inositol metabolism, we characterized inositol monophosphatase in this yeast. Lithium inhibited monophosphatase activity in vitro . Growth in the presence of inositol resulted in increased expression of the enzyme in vivo , although inositol had no effect on enzyme activity in vitro . The inositol effect was apparent when cells were grown in glucose but not in glycerol/ethanol. Monophosphatase activity was derepressed as cells entered stationary phase. This effect was apparent only during growth in glucose plus inositol. The results demonstrate that S. cerevisiae monophosphatase is inhibited by lithium and regulated by factors affecting phospholipid biosynthesis.     

The yeast inositol monophosphatase is a lithium- and sodium-sensitive enzyme encoded by a non-essential gene pair

Inositol monophosphatases (IMPases) are lithium-sensitive enzymes that participate in the inositol cycle of calcium signalling and in inositol biosynthesis. Two open reading frames (YHR046c and YDR287w) with homology to animal and plant IMPases are present in the yeast genome. The two recombinant purified proteins were shown to catalyse inositol-1-phosphate hydrolysis sensitive to lithium and sodium. A double gene disruption had no apparent growth defect and was not auxotroph for inositol. Therefore, lithium effects in yeast cannot be explained by inhibition of IMPases and inositol depletion, as suggested for animal systems. Overexpression of yeast IMPases increased lithium and sodium tolerance and reduced the intracellular accumulation of lithium. This phenotype was blocked by a null mutation in the cation-extrusion ATPase encoded by the ENA1/PMR2A gene, but it was not affected by inositol supplementation. As overexpression of IMPases increased intracellular free Ca2+, it is suggested that yeast IMPases are limiting for the optimal operation of the inositol cycle of calcium signalling, which modulates the Ena1 cation-extrusion ATPase.     

A novel and conserved salt-induced protein is an important determinant of salt tolerance in yeast.

We have isolated a novel yeast gene, HAL1, which upon overexpression improves growth under salt stress. In addition, disruption of this gene decreases salt tolerance. Therefore HAL1 constitutes a rate-limiting determinant for halotolerance. It encodes a polar protein of 32 kDa located in the yeast cytoplasm and unrelated to sequences in data banks. The expression of this gene is increased by high concentrations of either NaCl, KCl or sorbitol. On the other hand, the growth advantage obtained by overexpression of HAL1 is specific for NaCl stress. In cells overexpressing HAL1, sodium toxicity seems to be counteracted by an increased accumulation of potassium. The HAL1 protein could interact with the transport systems which determine intracellular K+ homeostasis. The HAL1 gene and encoded protein are conserved in plants, being induced in these organisms by salt stress and abscisic acid. These results suggest that yeast serves as a convenient model system for the molecular biology of plant salt tolerance.     

Role of protein phosphatases 2C on tolerance to lithium toxicity in the yeast Saccharomyces cerevisiae

Protein phosphatases 2C are a family of conserved enzymes involved in many aspects of the cell biology. We reported that, in the yeast Saccharomyces cerevisiae, overexpression of the Ptc3p isoform resulted in increased lithium tolerance in the hypersensitive hal3 background. We have found that the tolerance induced by PTC3 overexpression is also observed in wild-type cells and that this is most probably the result of increased expression of the ENA1 Na(+)-ATPase mediated by the Hog1 MAP kinase pathway. This effect does not require a catalytically active protein. Surprisingly, deletion of PTC3 (similarly to that of PTC2, PTC4 or PTC5) does not confer a lithium-sensitive phenotype, but mutation of PTC1 does. Lack of PTC1 in an ena1-4 background did not result in additive lithium sensitivity and the ptc1 mutant showed a decreased expression of the ENA1 gene in cells stressed with LiCl. In agreement, under these conditions, the ptc1 mutant was less effective in extruding Li(+) and accumulated higher concentrations of this cation. Deletion of PTC1 in a hal3 background did not exacerbate the halosensitive phenotype of the hal3 strain. In addition, induction from the ENA1 promoter under LiCl stress decreased similarly (50%) in hal3, ptc1 and ptc1 hal3 mutants. Finally, mutation of PTC1 virtually abolishes the increased tolerance to toxic cations provided by overexpression of Hal3p. These results indicate that Ptc1p modulates the function of Ena1p by regulating the Hal3/Ppz1,2 pathway. In conclusion, overexpression of PTC3 and lack of PTC1 affect lithium tolerance in yeast, although through different mechanisms.     

Overexpression of NtHAL3 genes confers increased levels of proline biosynthesis and the enhancement of salt tolerance in cultured tobacco cells

The Hal3 protein of Saccharomyces cerevisiae inhibits the activity of PPZ1 type-1 protein phosphatases and functions as a regulator of salt tolerance and cell cycle control. In plants, two HAL3 homologue genes in Arabidopsis thaliana, AtHAL3a and AtHAl3b, have been isolated and the function of AtHAL3a has been investigated through the use of transgenic plants. Expressions of both AtHAL3 genes are induced by salt stress. AtHAL3a overexpressing transgenic plants exhibit improved salt and sorbitol tolerance. In vitro studies have demonstrated that AtHAL3 protein possessed 4'-phosphopantothenoylcysteine decarboxylase activity. This result suggests that the molecular function of plant HAL3 genes is different from that of yeast HAL3. To understand the function of plant HAL3 genes in salt tolerance more clearly, three tobacco HAL3 genes, NtHAL3a, NtHAL3b, and NtHAL3c, from Nicotiana tabacum were identified. NtHAL3 genes were constitutively expressed in all organs and under all conditions of stress examined. Overexpression of NtHAL3a improved salt, osmotic, and lithium tolerance in cultured tobacco cells. NtHAL3 genes could complement the temperature-sensitive mutation in the E. coli dfp gene encoding 4'-phosphopantothenoyl-cysteine decarboxylase in the coenzyme A biosynthetic pathway. Cells overexpressing NtHAL3a had an increased intracellular ratio of proline. Taken together, these results suggest that NtHAL3 proteins are involved in the coenzyme A biosynthetic pathway in tobacco cells.     

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na⁺/Li⁺-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li⁺-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li⁺ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li⁺-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to?cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast.     

A lithium-sensitive and sodium-tolerant 3'-phosphoadenosine-5'-phosphatase encoded by halA from the cyanobacterium Arthrospira platensis is closely related to its counterparts from yeasts and plants

3'-Phosphoadenosine-5'-phosphatase (PAPase) is required for the removal of toxic 3'-phosphoadenosine-5'-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg2+ for its activity and could use PAP or 3'-phosphoadenosine-5'-phosphosulfate as a substrate. HalA is sensitive to Li+ (50% inhibitory concentration [IC50] = 3.6 mM) but only slightly sensitive to Na+ (IC50 = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li+ and Na+, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li+ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.     

Cloning and expression of bovine brain inositol monophosphatase.

Inositol monophosphatase is a key enzyme of the inositol phosphate second messenger signaling pathway. It is responsible for the provision of inositol required for synthesis of phosphatidylinositol and polyphosphoinositides and has been implicated as the pharmacological target for lithium action in brain. Using oligonucleotide probes based on partial amino acid sequence data for the bovine brain enzyme, several overlapping cDNA clones of 2-3 kilobases in length have been isolated. All contain an open reading frame encoding a 277-amino acid protein. No significant sequence homology was found with any known protein. The open reading frame was inserted into a bacterial expression vector in order to confirm the presumed identity of the protein. The expressed protein reacted with an anti-inositol monophosphatase monoclonal antibody. In addition, the protein was enzymically active and indistinguishable from the bovine brain enzyme with respect to Km values for substrate and Li+ sensitivity of inositol 1-phosphate hydrolysis.     

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na⁺/Li⁺-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li⁺-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li⁺ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li⁺-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to␣cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast. Received: 17 August 1998 / Accepted: 7 December 1998