Gis4, a new component of the ion homeostasis system in the yeast Saccharomyces cerevisiae

Abstract

Gis4 is a new component of the system required for acquisition of salt tolerance in Saccharomyces cerevisiae. The gis4Delta mutant is sensitive to Na(+) and Li(+) ions but not to osmotic stress. Genetic evidence suggests that Gis4 mediates its function in salt tolerance, at least partly, together with the Snf1 protein kinase and in parallel with the calcineurin protein phosphatase. When exposed to salt stress, mutants lacking gis4Delta display a defect in maintaining low intracellular levels of Na(+) and Li(+) ions and exporting those ions from the cell. This defect is due to diminished expression of the ENA1 gene, which encodes the Na(+) and Li(+) export pump. The protein sequence of Gis4 is poorly conserved and does not reveal any hints to its molecular function. Gis4 is enriched at the cell surface, probably due to C-terminal farnesylation. The CAAX box at the C terminus is required for cell surface localization but does not seem to be strictly essential for the function of Gis4 in salt tolerance. Gis4 and Snf1 seem to share functions in the control of ion homeostasis and ENA1 expression but not in glucose derepression, the best known role of Snf1. Together with additional evidence that links Gis4 genetically and physically to Snf1, it appears that Gis4 may function in a pathway in which Snf1 plays a specific role in controlling ion homeostasis. Hence, it appears that the conserved Snf1 kinase plays roles in different pathways controlling nutrient as well as stress response.

FIG. 1.

Phenotypic analysis. Yeast strains were grown to an OD₆₀₀ of about 1.0 and adjusted to an OD₆₀₀ of exactly 1.0, and 5 μl was spotted in 1:10 dilution steps onto the indicated media. SNF1 encodes the Snf1 protein kinase in glucose derepression, which inactivates the transcriptional repressor Mig1. Hog1 is the mitogen-activated protein kinase in the HOG osmosensing pathway. CMP1 and CMP2 encode the catalytic subunits of the protein phosphatase calcineurin. Tpk1 and Tpk2 are catalytic subunits, Bcy1 is the regulatory (inhibitory) subunit of protein kinase A, and Pde2 encodes a cAMP phosphodiesterase. Hence, the tpk1Δ tpk2Δ mutant has reduced and the tpk1Δ tpk2Δ bcy1Δ and pde2Δ mutants have moderate, constitutive, and increased protein kinase A activity, respectively. Ena1 is the Na⁺/Li⁺ export ATPase. (A) The gis4Δ mutant is sensitive to Na⁺ and Li⁺, and this phenotype is aggravated by the calcineurin inhibitor FK506. (B and C) GIS4 genetically interacts with SNF1 and CMP1/CMP2. (D) Expression of ENA1 on a centromeric plasmid (i.e., one or few additional copies per cell) partially suppresses the growth defects of the gis4Δ and snf1Δ mutants on Na⁺ and Li⁺ media.

FIG. 2.

Mutants lacking Gis4 or Snf1 display increased content of Na⁺ and Li⁺ and a defect in exporting these ions from the cells. (A) Cells were incubated overnight at the indicated ion concentration, and then the cellular content of Na⁺ and Li⁺ was determined. (B) Efficiency of Na⁺ and Li⁺ efflux. Cells were incubated in the presence of 0.1 M NaCl or LiCl for 1.5 h. Then cells were washed and resuspended in assay buffer adjusted to pH 5.5 and supplemented with 50 mM KCl to trigger the efflux process.

FIG. 3.

Gene and protein expression is affected by deletion of GIS4. (A) Cells transformed with a plasmid expressing lacZ under the control of the ENA1 promoter were treated with 0.6 M NaCl, samples were taken at the time points indicated, and β-galactosidase activity was determined. (B) In a similar experiment, the Ena1 protein level was monitored by means of an Ena1-HA fusion and an anti-HA antibody in Western blot analysis. An unspecific, constitutive band hybridizing to the anti-HA antibody served as a loading control.

FIG. 4.

Sequence alignment of parts of the predicted Gis4 sequence from different yeasts (for complete alignment, see Fig. S1 in the supplemental material). The species are S. cerevisiae, Saccharomyces mikatae, Saccharomyces kurdiavezii, Saccharomyces bayanus, Saccharomyces casei, Candida glabrata, Kluyveromyces lactis, Kluyveromyces waltii, and Ashbya gossypii. Only conserved parts are shown. Colors indicate chemically related amino acids.

FIG. 5.

Localization of Gis4. (A) Cells were transformed with a centromeric plasmid expressing a fusion of GFP to the N terminus of Gis4 (GFP-Gis4). Expression is mediated by the MET25 promoter and kept low by supplementing the growth medium with methionine. The plasmid complements the Li⁺ sensitivity of the gis4Δ mutant and, hence, the fusion is functional. The fusion protein is mainly localized at the cell surface. (B) The putative farnesylation sequence at the Gis4 C terminus was mutated (C771A) in the GFP-Gis4 fusion plasmid. The resulting plasmid complements the gis4Δ mutant, although less well than wild-type GIS4. The GFP-Gis4C771A fusion protein is localized throughout the cytosol. (C) The C771A mutation was introduced into GIS4 (without GFP) expressed from its own promoter on a centromeric plasmid. Also, this plasmid partly complements the Li⁺ sensitivity of the gis4Δ mutant.

FIG. 6.

Gis4 plays only a minor, if any, role in glucose derepression. (A) Cells were spotted onto medium in 1:10 dilutions series as described for Fig. 1. The medium contained the indicated compounds as sole sources for carbon and energy. (B) Cells were shifted from 8% to 0.2% glucose, and invertase activity was measured after 5 h. Data are averages of results from three independent experiments. (C) Cells were shifted from 8% to 0.2% glucose, samples were taken at the time point indicated, and mRNA levels of SUC2 were monitored by RT-PCR.