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. 1999 May;19(5):3328-37.
doi: 10.1128/MCB.19.5.3328.

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

J M Mulet  1 , M P Leube, S J Kron, G Rios, G R Fink, R Serrano
Affiliations

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

J M Mulet et al. Mol Cell Biol. 1999 May.

Abstract

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.

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Figures

FIG. 1
FIG. 1
Isolation and halotolerance phenotypes of the HAL4 and HAL5 genes. (A) Genomic structure of the inserts of genomic clones PM47 and PM42 containing the HAL4 and HAL5 genes, respectively. ORFs are shown as boxes. Primers (not to scale) and restriction sites used for subcloning are indicated. (B) Overexpression of HAL4 and HAL5 confers Li+ and Na+ tolerance. The SacI-HpaI genomic fragment containing HAL4 and the PCR fragment amplified with the P42-2 and P42-1 primers containing HAL5 (see panel A) were subcloned into multicopy plasmid YEp351 (26) to give rise to overexpression plasmids YEp-HAL4 and YEp-HAL5, respectively. After transformation into strain W303-1A (wt) and its ena1-4::HIS3 derivative (ena1-4), salt tolerance was determined by drop tests in solid YPD containing the indicated concentrations of LiCl or NaCl. Cells transformed with empty YEp351 plasmid served as controls.
FIG. 2
FIG. 2
Yeast hal4 and hal5 mutants are sensitive to lithium and sodium. Strain W3031-A (lane 1) and its derivatives SKY697 (ena1-4::HIS3) (lane 2), SKY655 (hal4::LEU2) (lane 3), SKY656 (hal5::HIS3) (lane 4), and SKY637 (hal4::LEU2 hal5::HIS3) (lane 5) were tested for salt tolerance in YPD plates containing KCl, LiCl, or NaCl as indicated.
FIG. 3
FIG. 3
Effect of ENA1-4 and HAL4-HAL5 on Na+ uptake and efflux. (A) Sodium uptake. Cells from wild-type yeast (W303-1A) the ena1-4 mutant (SKY697), and the hal4 hal5 mutant (SKY637) were grown overnight in YPD, and 0.8 M NaCl was added at time zero. Samples were taken at the indicated times for determination of the intracellular sodium concentration. (B) Sodium efflux. Cells from the yeast strains described for panel A were loaded with either 0.8 M (wild type) or 0.4 M (ena1-4 and hal4 hal5 mutants) NaCl for 2.5 h, washed, and resuspended in NaCl-free medium at time zero. Samples were taken at the indicated times for determination of the intracellular sodium concentration. Results are the averages of three determinations, and the standard deviations were less of 15%. The experiment was repeated twice with similar results.
FIG. 4
FIG. 4
Overexpression of HAL4 and HAL5 is unable to confer salt tolerance in a trk1 trk2 mutant. Strain WΔ3 (trk1 trk2 double mutant) was transformed with control plasmid YEp352 (2μm origin, URA3 marker) (26), with YEp24 (2μm origin, URA3 marker) carrying HAL4 (YEpHAL4) or HAL5 (YEpHAL5), and with YCp50 carrying ENA1 (YCpENA1) (17). Salt tolerance was measured by drop test in YPD containing NaCl or LiCl as indicated. As a control for wild-type tolerance, we used strain W303-1A (TRK1 TRK2) transformed with empty plasmid YEp352.
FIG. 5
FIG. 5
Growth of hal4 hal5 mutants is limited by potassium. Cells from wild-type yeast (W303-1A; squares), hal4 hal5 mutant cells (SKY637; circles) and the trk1 trk2 mutant (WΔ3; triangles) were grown overnight in SD containing 0.2 M KCl and the required supplements, washed, and resuspended at time zero in the same medium without (open symbols) or with (solid symbols) 0.2 M KCl. Growth was recorded by measurement of the absorbance at 660 nm (OD 660) at the indicated times.
FIG. 6
FIG. 6
Rubidium uptake, an indicator of Trk1-Trk2 activity, is modulated by the gene dosage of HAL4 and HAL5. Cells from wild-type yeast (W303-1A; squares), wild-type yeast transformed with YEp351-HAL5 (JM76; crosses), the hal4 hal5 mutant (SKY637; circles), and the trk1 trk2 mutant (WΔ3; triangles) were grown to exponential phase in SD containing 0.2 M KCl and the required supplements, washed, and incubated for 4 h in potassium starvation medium. After further washing, the uptake of 0.2 mM 86Rb was determined at the indicated times as described in Materials and Methods.
FIG. 7
FIG. 7
Methylammonium uptake, an indicator of membrane potential, is modulated by the gene dosage of HAL4 and HAL5. Strains, symbols, culture treatments, and transport procedures were as described in the legend to Fig. 6, except that 0.2 mM [14C]methylammonium was used as the transported substrate instead of 86Rb.
FIG. 8
FIG. 8
Similar sensitivities to toxic cations of hal4 hal5 and trk1 trk2 mutants. Strain W303-1A (lane 1) and its derivatives the ena1-4 mutant (SKY697) (lane 2), the hal4 hal5 mutant (SKY637) (lane 3), and the trk1 trk2 mutant (WΔ3) (lane 4) were spotted on YPD plates containing the indicated concentrations of toxic cations or acidic buffer, and growth was monitored after 2 days. Hyg B, hygromycin B; TMA, tetramethylammonium.
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