Kch1 family proteins mediate essential responses to endoplasmic reticulum stresses in the yeasts Saccharomyces cerevisiae and Candida albicans

Abstract

The activation of a high affinity Ca(2+) influx system (HACS) in the plasma membrane is required for survival of yeast cells exposed to natural or synthetic inhibitors of essential processes (secretory protein folding or sterol biosynthesis) in the endoplasmic reticulum (ER). The mechanisms linking ER stress to HACS activation are not known. Here we show that Kch1, a recently identified low affinity K(+) transporter in the plasma membrane of Saccharomyces cerevisiae, is up-regulated in response to several ER stressors and necessary for HACS activation. The activation of HACS required extracellular K(+) and was also dependent on the high affinity K(+) transporters Trk1 and Trk2. However, a paralog of Kch1 termed Kch2 was not expressed and not necessary for HACS activation in these conditions. The pathogenic yeast Candida albicans carries only one homolog of Kch1/Kch2, and homozygous knock-out mutants were similarly deficient in the activation of HACS during the responses to tunicamycin. However, the Kch1 homolog was not necessary for HACS activation or cell survival in response to several clinical antifungals (azoles, allylamines, echinocandins) that target the ER or cell wall. Thus, Kch1 family proteins represent a conserved linkage between HACS and only certain classes of ER stress in these yeasts.

Keywords: Antifungals; Calcineurin; Calcium Channels; Cell Death; ER Stress; Potassium Transport.

FIGURE 1.

Kch1 regulates HACS-dependent Ca²⁺ accumulation in response to tunicamycin. A and B, ⁴⁵Ca²⁺ uptake into cultures of S. cerevisiae strains that contain combinations of Kch1, Kch2, and Cch1 (strains K601, CS01, CS02, CS03, CS04) was measured after a 4-h incubation in SC-100 (10 mm KCl) medium in the presence or absence of 3 μg/ml tunicamycin plus 0.25 μg/ml FK506, as indicated. C, β-galactosidase activity of the above mentioned strains transformed with a CDRE-lacZ reporter gene (pAMS366) in SC medium in the presence of 2.5 μg/ml tunicamycin. Samples were collected at the indicated time points. D, wild type and kch1 mutants (K601 and CS02) were exposed to 2-fold dilutions of tunicamycin, incubated for 10 h, and stained with propidium iodide. Live and dead cells were counted by flow cytometry. In all panels, averages for three biological replicates (±S.D.) are shown, and significant differences between kch1 and KCH1 control cells are indicated (**; see “Experimental Procedures”).

FIGURE 2.

Altered expression and modification of Kch1 in response to tunicamycin and DTT. A and B, whole-cell lysates of cells expressing Kch2-Myc₁₃ (A) or Kch1-Myc₁₃ (A and B) were prepared after a 4-h exposure to tunicamycin (TM), DTT, α-factor, and/or FK506 as indicated. C, assays of β-galactosidase activity of wild type, kch1, and ire1 mutants (K601, CS03, DNY1048) transformed with a UPRE-lacZ reporter gene (pCZY1) in the presence or absence of tunicamycin or BAPTA after incubation for 3 or 3.5 h, respectively. Averages for three biological replicates (±S.D.) are shown, and significant differences between kch1 and KCH1 control cells are indicated (**; see “Experimental Procedures”).

FIGURE 3.

Differential regulation of HACS by K⁺ ions through Kch1 and the K⁺ transporters Trk1 and Trk2 in response to tunicamycin and DTT. A–D, K⁺ dependent ⁴⁵Ca²⁺ uptake was measured in wild type, kch1/2, trk1/2, and quadruple mutants (K601, CS03, CS08, CS11) after a 4-h incubation in SC-100 medium containing the indicated concentrations of KCl plus 2.5 μg/ml tunicamycin (TM) in the presence (A) or absence (B) of 0.25 μg/ml FK506 or DTT (C and D), respectively. Averages for four biological replicates (±S.E.) are shown, and significant differences between kch1 and KCH1 control cells are indicated (* or **; see “Experimental Procedures”).

FIGURE 4.

Defects in Ca²⁺ homeostasis result in a Kch1-dependent Ca²⁺ accumulation. A, ⁴⁵Ca²⁺ uptake into pmr1 mutant backgrounds containing combinations of Kch1 and Kch2 (CS39, CS40, CS41, CS42) after 4 h of log-phase growth in SC-100 medium (5 mm KCl) in the presence or absence of 0.1 μg/ml FK506. B, ⁴⁵Ca²⁺ uptake into wild type and kch1 mutants (K601, CS02) in YPD medium in the presence or absence of 200 μm TPEN and 1 μg/ml FK506 after incubation for 4 h. Averages for three biological replicates (±S.D.) are shown, and significant differences between kch1 and KCH1 control cells are indicated (**; see “Experimental Procedures”).

FIGURE 5.

Kch1 function is conserved in the pathogenic fungi C. albicans. A, ⁴⁵Ca²⁺ uptake into C. albicans wild type, kch1^−/−, cch1^−/−, and the quadruple mutant (SC5314, JLR48, CS126, CS135) in SC-100 media (10 mm KCl) in the presence or absence of 3 μg/ml tunicamycin and 1 μg/ml FK506 after a 4-h incubation. B, cell death measurements on log phase cultures of strains listed above in SC medium in the presence of indicated tunicamycin concentrations. Cells were stained with propidium iodide and measured by flow cytometry. Averages for three biological replicates (±S.D.) are shown, and significant differences between kch1 and KCH1 control cells are indicated (* or **; see “Experimental Procedures”).

FIGURE 6.

Ca²⁺ accumulation and cell death in Ca²⁺ pathway mutants in response to commonly used antifungals. A, C, and E, ⁴⁵Ca²⁺ uptake into strains listed in Fig. 5 in SC-100 medium (10 mm KCl) after exposure to 0.25 μg/ml miconazole (MIC) (A), 25 μg/ml terbinafine (TB) (C), or 25 μg/ml caspofungin (CAS) (E) in the presence or absence of 1 μg/ml FK506 after 4 h of incubation. Averages for three biological replicates (±S.D.) are shown, and significant differences between kch1 and KCH1 control cells are indicated (**; see “Experimental Procedures”). B, D, and F, cell death measurements on saturated cultures in SC media in the presence of indicated concentrations of miconazole (B), terbinafine (D), or caspofungin (F) in the presence or absence of 1 μg/ml FK506 after a 24-h incubation. Cells were stained with propidium iodide and measured by flow cytometry.