pH-Responsive, posttranslational regulation of the Trk1 potassium transporter by the type 1-related Ppz1 phosphatase

Intracellular pH and K+ concentrations must be tightly controlled because they affect many cellular activities, including cell growth and death. The mechanisms of homeostasis of H+ and K+ are only partially understood. In the yeast Saccharomyces cerevisiae, proton efflux is mediated by the Pma1 H+-ATPase. As this pump is electrogenic, the activity of the Trk1 and -2 K+ uptake system is crucial for sustained Pma1p operation. The coordinated activities of these two systems determine cell volume, turgor, membrane potential, and pH. Genetic evidence indicates that Trk1p is activated by the Hal4 and -5 kinases and inhibited by the Ppz1 and -2 phosphatases, which, in turn, are inhibited by their regulatory subunit, Hal3p. We show that Trk1p, present in plasma membrane "rafts", physically interacts with Ppz1p, that Trk1p is phosphorylated in vivo, and that its level of phosphorylation increases in ppz1 and -2 mutants. Interestingly, both the interaction with and inhibition of Ppz1p by Hal3p are pH dependent. These results are consistent with a model in which the Ppz1-Hal3 interaction is a sensor of intracellular pH that modulates H+ and K+ homeostasis through the regulation of Trk1p activity.     

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.     

Histone acetyltransferase GCN5 contributes to cell wall integrity and salt stress tolerance by altering the expression of cellulose synthesis genes

Excess soluble salts in soil are harmful to the growth and development of most plants. Evidence is emerging that the plant cell wall is involved in sensing and responding to salt stress, but the underlying mechanisms are not well understood. We reveal that the histone acetyltransferase General control non‐repressed protein 5 (GCN5) is required for the maintenance of cell wall integrity and salt stress tolerance. The levels of GCN5 mRNA are increased in response to salt stress. The gcn5 mutants exhibited severe growth inhibition and defects in cell wall integrity under salt stress conditions. Combining RNA sequencing and chromatin immunoprecipitation assays, we identified the chitinase‐like gene CTL1, polygalacturonase involved in expansion‐3 (PGX3) and MYB domain protein‐54 (MYB54) as direct targets of GCN5. Acetylation of H3K9 and H3K14 mediated by GCN5 is associated with activation of CTL1, PGX3 and MYB54 under salt stress. Moreover, constitutive expression of CTL1 in the gcn5 mutant restores salt tolerance and cell wall integrity. In addition, the expression of the wheat TaGCN5 gene in Arabidopsis gcn5 mutant plants complemented the salt tolerance and cell wall integrity phenotypes, suggesting that GCN5‐mediated salt tolerance is conserved between Arabidopsis and wheat. Taken together, our data indicate that GCN5 plays a key role in the preservation of salt tolerance via versatile regulation in plants.     

Functional characterization of the Saccharomyces cerevisiae VHS3 gene: a regulatory subunit of the Ppz1 protein phosphatase with novel, phosphatase-unrelated functions

The yeast gene VHS3 (YOR054c) has been recently identified as a multicopy suppressor of the G(1)/S cell cycle blockade of a conditional sit4 and hal3 mutant. Vhs3 is structurally related to Hal3, a negative regulatory subunit of the Ser/Thr protein phosphatase Ppz1 important for cell integrity, salt tolerance, and cell cycle control. Phenotypic analyses using vhs3 mutants and overexpressing strains clearly show that Vhs3 has functions reminiscent to those of Hal3 and contrary to those of Ppz1. Mutation of Vhs3 His(459), equivalent to the supposedly functionally relevant His(90) in the plant homolog AtHal3a, did not affect Vhs3 functions mentioned above. Similarly to Hal3, Vhs3 binds in vivo to the C-terminal catalytic moiety of Ppz1 and inhibits in vitro its phosphatase activity. Therefore, our results indicate that Vhs3 plays a role as an inhibitory subunit of Ppz1. We have found that the vhs3 and hal3 mutations are synthetically lethal. Remarkably, lethality is not suppressed by deletion of PPZ1, PPZ2, or both phosphatase genes, indicating that it is not because of an excess of Ppz phosphatase activity. Furthermore, a Vhs3 version carrying the H459A mutation did not rescue the synthetically lethal phenotype. A conditional vhs3 tetO:HAL3 double mutant displays, in the presence of doxycycline, a flocculation phenotype that is dependent on the presence of Flo8 and Flo11. These results indicate that, besides its role as Ppz1 inhibitory subunit, Vhs3 (and probably Hal3) might have important Ppz-independent functions.     

The Schizosaccharomyces pombe Pzh1 protein phosphatase regulates Na+ ion influx in a Trk1-independent fashion.

We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis. pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ari?o, J. (1997) Eur. J. Biochem. 250, 476-483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells. K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions. trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.     

Accumulation of K+ in E. coli grown in aerobic conditions

The character of K+ accumulation in E. coli grown aerobilcally in the salt medium with succinate was studied. K+ uptake via the Trk system has Km 3.4 mM and Vmax 0.45 mM X g+1 X min-1. The initial rates of K+ uptake were not changes at different pH from 6.0 to 8.3 and temperature 17-37 degrees C. DCC did not block, protonophores and arsenate blocked the operation of Trk system. Valinomycin increased (or had no effect) K+ accumulation. K+ distribution is in good conformity with the measured membrane potential. The Trk system works at the utilization of lactic acid and glucose as well as of succinate. The Trk system is described. K+ ionophore by using the membrane potential and ATP regulates functioning of this system.     

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of beta1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect beta1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.     

Schizosaccharomyces pombe ehs1p is involved in maintaining cell wall integrity and in calcium uptake

The Schizosaccharomyces pombe mutant ehs1-1 mutant was isolated on the basis of its hypersensitivity to Echinocandin and Calcofluor White, which inhibit cell wall synthesis. The mutant shows a thermosensitive growth phenotype that is suppressed in the presence of an osmotic stabiliser. The mutant also showed other cell wall-associated phenotypes, such as enhanced sensitivity to enzymatic cell wall degradation and an imbalance in polysaccharide synthesis. The ehs1 + gene encodes a predicted integral membrane protein that is 30% identical to Saccharomyces cerevisiae Mid1p, a protein that has been proposed to form part of a calcium channel. As expected for such a function, we found that ehs1+ is involved in intracellular Ca2+ accumulation. High external Ca2+ concentrations suppressed all phenotypes associated with the ehs1 null mutation, suggesting that the cell integrity defects of ehs1 mutants result from inadequate levels of calcium in the cell. We observed a genetic relationship between ehs1+ and the protein kinase C homologue pck2+. pck2+ suppressed all phenotypes of ehs1-1 mutant cells. Overproduction of pck2p is deleterious to wild-type cells, increasing 1,3-beta-D-glucan synthase activity and promoting accumulation of extremely high levels of Ca2+. The lethality associated with pck2p, the increase in 1,3-beta-D-glucan synthase production and the strong Ca2+ accumulation are all dependent on the presence of ehs1p. Our results suggest that in fission yeast ehs1p forms part of a calcium channel that is involved in the cell wall integrity pathway that includes the kinase pck2p.     

Trk1 and Trk2 define the major K(+) transport system in fission yeast

The trk1(+) gene has been proposed as a component of the K(+) influx system in the fission yeast Schizosaccharomyces pombe. Previous work from our laboratories revealed that trk1 mutants do not show significantly altered content or influx of K(+), although they are more sensitive to Na(+). Genome database searches revealed that S. pombe encodes a putative gene (designated here trk2(+)) that shows significant identity to trk1(+). We have analyzed the characteristics of potassium influx in S. pombe by using trk1 trk2 mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics. trk2 mutants do not show altered K(+) transport and exhibit only a slightly reduced Na(+) tolerance. However, trk1 trk2 double mutants fail to grow at low K(+) concentrations and show a dramatic decrease in Rb(+) influx, as a result of loss of the high-affinity transport component. Furthermore, trk1 trk2 cells are very sensitive to Na(+), as would be expected for a strain showing defective potassium transport. When trk1 trk2 cells are maintained in K(+)-free medium, the potassium content remains higher than that of the wild type or trk single mutants. In addition, the trk1 trk2 strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K(+) transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.     

The presumed potassium carrier Trk2p in Saccharomyces cerevisiae determines an H+-dependent, K+-independent current

Ionic currents related to the major potassium uptake systems in Saccharomyces cerevisiae were examined by whole cell patch-clamping, under K+ replete conditions. Those currents have the following properties. They (1) are inward under all conditions investigated, (2) arise instantaneously with appropriate voltage steps, (3) depend solely upon the moderate affinity transporter Trk2p, not upon the high affinity transporter Trk1p. They (4) appear to be independent of the extracellular K+ concentration, (5) are also independent of extracellular Ca2+, Mg2+ and Cl- but (6) are strongly dependent on extracellular pH, being large at low pH (up to several hundred pA at -200 mV and pH 4) and near zero at high pH (above 7.5). They (7) increase in proportion to log[H+]o, rather than directly in proportion to the proton concentration and (8) behave kinetically as if each transporter cycle moved one proton plus one (high pH) or two (low pH) other ions, as yet unidentified. In view of background knowledge on K+ transport related to Trk2p, the new results suggest that the K+ status of yeast cells modulates both the kinetics of Trk2p-mediated transport and the identity of ions involved. That modulation could act either on the Trk2 protein itself or on interactions of Trk2 with other proteins in a hypothetical transporter complex. Structural considerations suggest a strong analogy to the KtrAB system in Vibrio alginolyticus and/or the TrkH system in Escherichia coli.     

N,N'-dicyclohexylcarbodiimide-sensitive K+-dependent ATPase activity in Escherichia coli

ATPase activity sensitive to N,N'-dicyclohexylcarbodiimide and dependent on K+ content in medium is observed only in anaerobically grown Escherichia coli and as the analysis of mutants with defects in different subunits of (F0F1) H+-ATPase and in potassium transport shows only under the structural integrity of both F0F1 and K+-ionophore (the Trk system). The obtained results confirm the data on the H+/K+-exchange and indicate that the F0F1 and Trk systems in anaerobically grown bacteria unite into the same membrane supercomplex inside which the direct energy transfer occurs without a mediation of delta-mu H+.     

Protein phosphatase Z modulates oxidative stress response in fungi

The genome of the filamentous fungus Aspergillus nidulans harbors the gene ppzA that codes for the catalytic subunit of protein phosphatase Z (PPZ), and the closely related opportunistic pathogen Aspergillus fumigatus encompasses a highly similar PPZ gene (phzA). When PpzA and PhzA were expressed in Saccharomyces cerevisiae or Schizosaccharomyces pombe they partially complemented the deleted phosphatases in the ppz1 or the pzh1 mutants, and they also mimicked the effect of Ppz1 overexpression in slt2 MAP kinase deficient S. cerevisiae cells. Although ppzA acted as the functional equivalent of the known PPZ enzymes its disruption in A. nidulans did not result in the expected phenotypes since it failed to affect salt tolerance or cell wall integrity. However, the inactivation of ppzA resulted in increased sensitivity to oxidizing agents like tert-butylhydroperoxide, menadione, and diamide. To demonstrate the general validity of our observations we showed that the deletion of the orthologous PPZ genes in other model organisms, such as S. cerevisiae (PPZ1) or Candida albicans (CaPPZ1) also caused oxidative stress sensitivity. Thus, our work reveals a novel function of the PPZ enzyme in A. nidulans that is conserved in very distantly related fungi.     

The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle

Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition.     

Interaction of H+ and K+ transport systems in E. coli growing under anaerobic and aerobic conditions

The interaction of H+-ATPase complex F1 X F0 with the Trk system of K+ accumulation in E. coli grown quasi-anaerobically in pepton media with glucose (anaerobia) and aerobically in the salt medium with succinate (aerobia) treated with cyanide was studied. The ratio of H+ fluxes via F1 X F0 and K+ fluxes via the Trk system is stable and equals 2 in anaerobia and is changed from 0.5 to 5.0 in aerobia treated with cyanide in response to pH variation, K+ activity and temperature variations. Q10 is about 2.8 both for F1 X F0 and the Trk system in anaerobia, but 2.4 and 1.0 respectively in aerobia. K+ distribution in anaerobia reaches high values, K+ equilibrium potential is much higher than the measured membrane potential. K+ distribution in aerobia is smaller, which is in conformity with the measured membrane potential. Structural association of F1 X F0 and the Trk system with the formation of H+--K+-pump is assumed to take place in anaerobia, and separate operation of these systems occurs in aerobia, transfer of K+ via Trk system being energized by the electric field on the membrane.     

Isolation and characterization of Zygosaccharomyces rouxii mutants defective in proton pumpout activity and salt tolerance

Two mutants defective in salt tolerance were identified among hygromycin B (HygB)-resistant mutants of Zygosaccharomyces rouxii. These mutants showed different phenotypes in terms of sensitivity towards high concentrations of glucose and KCl. Recovery of salt tolerance by the addition of KCl and CaCl₂ or by lowering pH (pH 4.0) was different for the two mutants. Moreover, both mutants showed lowered plasma membrane (PM-) ATPase activity and proton pumpout activity. They exhibited neither growth nor proton pumpout activity in a medium containing 5% NaCl. The proton pumpout activity was inhibited by vanadate, an inhibitor of PM-ATPase, only when cells were incubated in the presence of more than 1% NaCl. Damage of the proton pumpout activity seems to be the reason for the salt sensitivity of both mutants. We showed that it was essential for Z. rouxii cells to pump out protons under a high salt environment using mutants defective in this ability.     

An in Vivo Nuclear Magnetic Resonance Investigation of Ion Transport in Maize (Zea mays) and Spartina anglica Roots during Exposure to High Salt Concentrations

The response of maize (Zea mays L.) and Spartina anglica root tips to exposure to sodium chloride concentrations in the range 0 to 500 mM was investigated using 23Na and 31P nuclear magnetic resonance spectroscopy (NMR). Changes in the chemical shift of the pH-dependent 31P-NMR signals from the cytoplasmic and vacuolar orthophosphate pools were correlated with the uptake of sodium, and after allowing for a number of complicating factors we concluded that these chemical shift changes indicated the occurrence of a small cytoplasmic alkalinization (0.1-0.2 pH units) and a larger vacuolar alkalinization (0.6 pH units) in maize root tips exposed to salt concentrations greater than 200 mM. The data were interpreted in terms of the ion transport processes that may be important during salt stress, and we concluded that the vacuolar alkalinization provided evidence for the operation of a tonoplast Na+/H+-antiport with an activity that exceeded the activity of the tonoplast H+ pumps. The intracellular pH values stabilized during prolonged treatment with high salt concentrations, and this observation was linked to the recent demonstration (Y. Nakamura, K. Kasamo, N. Shimosato, M. Sakata, E. Ohta [1992] Plant Cell Physiol 33: 139-149) of the salt-induced activation of the tonoplast H+- ATPase. Sodium vanadate, an inhibitor of the plasmalemma H+- ATPase, stimulated the net uptake of sodium by maize root tips, and this was interpreted in terms of a reduction in active sodium efflux from the tissue. S. anglica root tips accumulated sodium more slowly than did maize, with no change in cytoplasmic pH and a relatively small change (0.3 pH units) in vacuolar pH, and it appears that salt tolerance in Spartina is based in part on its ability to prevent the net influx of sodium chloride.