Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters

Abstract

The regulation of electrical membrane potential is a fundamental property of living cells. This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses. In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown. We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1 (YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H(+)-ATPase (Pma1) in response to glucose metabolism. These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation. Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine. A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent. Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium. Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters).

FIG. 1

sen1-1 and ptk2::TRP1 mutations confer tolerance to different toxic cations and sensitivity to acetic acid. Strain SKY697 (wt) and its derivatives AG149 (ptk2) and AG151 (sen1) were grown in liquid SD medium to saturation, and serial dilutions were dropped on SD plates with either NaCl (0.2 M), LiCl (40 mM), KCl (1.2 M), sorbitol (1.8 M), acetic acid (0.12 M), or tetramethylammonium chloride (TMA, 1 M) or on YPD plates with MnCl₂ (2 mM) or hygromycin B (HygB, 125 μg/ml), as indicated. Growth was recorded after 2 days in the absence of toxic cations and after 3 to 4 days in its presence.

FIG. 2

Effect of the sen1-1 mutation on the yeast Pma1 proton pump. Cells of strains SKY697 (wt) and AG151 (sen1 mutant) were grown in YPD, and the in vivo activity (A) and amount (B) of the Pma1 proton-pumping ATPase were determined as described in the text. (A) pH changes of yeast suspensions induced by glucose. Cells (50 mg) were suspended in 10 ml of assay buffer, and pH changes were recorded after addition of 200 μmol of glucose. Calibration with 400 nmol of HCl was made as indicated. (B) Immunological quantification of Pma1 in yeast. Total membrane protein (25 μg) from strains SKY697 (wild type [wt] for PTK2, lanes 1 and 2) and AG151 (sen1-1, lanes 3 and 4) was immunodetected with anti-Pma1 antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting. Lane 5 contains molecular size standards as indicated.

FIG. 3

sen1-1 mutation is a solo δ-LTR insertion at the PTK2 locus. (A) Scheme of the region of chromosome X between positions 545000 and 548000, containing the PTK2 locus and the site of insertion of a solo δ-LTR. The ORF spans positions 545475 to 547931 on the w-strand, and the 336-bp insertion is at position 545942. sp and asp correspond to the primers used for the PCR in panel B. (B) Products of the PCR performed with chromosomal DNA from strains SKY697 (wt) and AG151 (sen1-1 mutant) with the primers indicated in panel A. Lane M, size markers; their sizes (in base pairs) and that of the amplified bands are indicated at the right and at the left of the figure, respectively.

FIG. 4

Deletion of the inhibitory domain of Pma1 suppresses the growth phenotypes of the ptk2 mutation. Strains SKY697 (wild type for PTK2, column 1), AG149 (ptk2, column 2), AG205 (ptk2 with plasmid pRS496 expressing truncated Pma1, column 3) and AG224 (ptk2 pma1::URA3-GAL1 promoter-PMA1 with chromosomal ATPase under galactose control and plasmid pRS496 expressing truncated Pma1, column 4) were grown in liquid SD medium to saturation, and serial dilutions were dropped on SD plates with either NaCl (0.2 M) or LiCl (40 mM) as indicated. Growth was recorded after 2 or 4 days in the absence or presence of toxic cations, respectively.

FIG. 5

Effect of mutations of Pma1 Ser-899 on tolerance to hygromycin B and NaCl of Ptk2⁺ (upper panels) and Ptk2⁻ (lower panels) yeast cells. Wild-type yeast strain W303-1B (PMA1 PTK2) and derivatives with pma1-Ser899 and ptk2 mutations as indicated (strains FPY398, FPY402, FPY1434, FPY1500, and FPY1502) were grown in liquid medium to saturation and diluted 20-fold, and 3 μl was dropped on YPD plates with no addition or containing hygromycin B (50 μg/ml) or NaCl (1.2 M), as indicated. Growth was recorded after 4 days.

FIG. 6

Effect of gain and loss of function of YOR267c and PTK2 on tolerance of yeast cells to hygromycin B and NaCl. Wild-type yeast strain (wt, FPY1506) and derivatives with disruptions (Δ) of either yor267c (strain FPY1456) or ptk2 (FPY1459) were transformed with either empty plasmid (YEp352) or multicopy plasmids with YOR267c and PTK2 as indicated. Experimental conditions were as described in the legend to Fig. 5.

FIG. 7

Model for the role of Pma1 and Trk1,2 on yeast salt tolerance by modulation of the electrical membrane potential (ΔΨ), which determines the uptake of toxic cations by different voltage-sensitive transporters.