KCS1 and VIP1, the genes encoding yeast phosphoinositol pyrophosphate synthases, are required for Ca2+-mediated response to dimethylsulfoxide (DMSO)

Abstract

Dimethylsulfoxide (DMSO) is widely used as a solvent or as a carrier when screening for biologic activity of various chemicals, but results need to be interpreted carefully due to its intrinsic toxicity. DMSO has been previously observed to impair the growth of yeast cells defective in calcium movement across cellular membranes and in phosphoinositol pyrophosphate synthases. Here, we set out to investigate the Ca²⁺-mediated response to DMSO in Saccharomyces cerevisiae. The cell exposure to DMSO was signaled by a two-phase cytosolic Ca²⁺ wave that was dependent on Mid1, a subunit of the Cch1/Mid1 Ca²⁺ channel located at the plasma membrane. While the vacuolar Ca²⁺ channel Trpy1 also contributed by releasing Ca²⁺ from the vacuole, the immediate cell response to DMSO exposure depended on the external Ca²⁺ imported into the cell through Cch1/Mid1. A chemogenomic screen previously performed on a collection of yeast knockout mutants identified the two phosphoinositol pyrophosphate synthases Kcs1 and Vip1 as determinants for yeast tolerance to DMSO. Deletion of KCS1 or VIP1 genes suppressed the DMSO-induced Ca²⁺ response, suggesting that both Ca²⁺ and phosphoinositol pyrophosphate signaling contribute to cell adaptation under DMSO stress.

Keywords: Saccharomyces cerevisiae; aequorin; calcium; dimethylsulfoxide; inositol pyrophosphate synthase.

Fig. 1

Effect of dimethyl sulfoxide (DMSO) on yeast growth. (A) Effect of DMSO concentration on growth of wild‐type cells. (B) Growth of DMSO‐sensitive mutants. Yeast cells (wild‐type and knockout mutants) were inoculated from exponentially growing YPD cultures in SD liquid medium (2 × 10⁵ cells·mL⁻¹ initially) in the presence of various concentrations of DMSO and incubated with shaking (200 r.p.m.) at 30 °C. Cell density was determined spectrophotometrically at 600 nm (OD₆₀₀). Relative growth was determined as described in the Materials and methods section 24 h after the addition of DMSO and expressed as mean ± SEM (n = 3). Two‐way ANOVA with Bonferroni post‐test showing the level of significance when comparing each mutant with wild‐type strain exposed to the same concentration of DMSO. *P < 0.05; **P < 0.01.

Fig. 2

Dimethyl sulfoxide (DMSO) exposure induces the increase of cytosolic Ca²⁺ ([Ca²⁺]_cyt). Wild‐type (WT) cells expressing reconstituted aequorin were pregrown in SD‐Ura and subjected to DMSO stress as described in the Materials and methods section. [Ca²⁺]_cyt‐dependent aequorin luminescence was recorded on samples of approximately 10⁷ cells (OD₆₀₀ = 1). The arrows indicate the moment when the chemicals were added to induce stress. (A) Ca²⁺‐dependent luminescence of wild‐type exposed to DMSO. (B) Inhibition of Ca²⁺‐dependent luminescence by Ca²⁺ chelator BAPTA (5 mm final concentration). BAPTA was added 45 s prior to DMSO exposure (5% final concentration). (C) Dimethylformamide (DMF) does not induce Ca²⁺‐mediated luminescence. The curve shows the response to 5% DMF (final concentration) and no Ca²⁺‐dependen response could be recorded for other lower or higher concentrations. The luminescence traces represent the mean ± SEM from three independent transformants. RLU, relative luminescence units.

Fig. 3

Ca²⁺‐dependent response to dimethyl sulfoxide (DMSO) of cells with defects in Ca²⁺ channels. Wild‐type (WT) and isogenic knockout mutants cch1Δ, mid1Δ, or trpy1Δ expressing reconstituted aequorin were pregrown in SD‐Ura and subjected to DMSO stress as described in the Materials and methods section. [Ca²⁺]_cyt‐dependent aequorin luminescence was recorded on samples of approximately 10⁷ cells (OD₆₀₀ = 1). The arrows indicate the moment when DMSO was added to achieve a 5% final concentration. Ca²⁺‐dependent luminescence of: (A) wild‐type, (B) cch1Δ, (C) mid1Δ, or (D) trpy1Δ exposed to 5% DMSO. Blue line in D: response of trpy1Δ cells pretreated with 5 mm BAPTA 30 s prior to DMSO exposure. The luminescence traces represent the mean ± SEM from three independent transformants. RLU, relative luminescence units.

Fig. 4

Ca²⁺‐dependent response of cells defective in inositol pyrophosphates (PP‐IPs) synthesis. Wild‐type (WT) and isogenic knockout mutants kcs1Δ or vip1Δ expressing reconstituted aequorin were pregrown in SD‐Ura and subjected to various stresses as described in the Materials and methods section. [Ca²⁺]_cyt‐dependent aequorin luminescence was recorded on samples of approximately 10⁷ cells (OD₆₀₀ = 1). The arrows indicate the moment when the stressor was added. (A) Ca²⁺‐dependent luminescence of kcs1Δ exposed to 5% DMSO. (B) Ca²⁺‐dependent luminescence of vip1Δ exposed to 5% DMSO. (C) Effect of alkaline stress (10 mm KOH, final concentration, pH approximately 8.5) on Ca²⁺‐dependent luminescence of WT, kcs1Δ, and vip1Δ cells. (D) Effect of oxidative stress (5 mm H₂O₂, final concentration) on Ca²⁺‐dependent luminescence of WT, kcs1Δ, and vip1Δ cells. The luminescence traces represent the mean ± SEM from three independent transformants. RLU, relative luminescence units.

Fig. 5

Effect of KCS1 or VIP1 overexpression on yeast tolerance to dimethyl sulfoxide (DMSO). WT, cch1Δ, and mid1Δ cells transformed with pYX212 (empty vector), pYX212‐KCS1 (expressing KCS1 from TPI promoter) or pYX212‐VIP1 (expressing VIP1 from TPI promoter) were inoculated from exponentially growing cultures in SD‐Ura liquid medium to an initial density of 2 × 10⁵ cells·mL⁻¹ in the presence of various concentrations of DMSO. Cell density was determined spectrophotometrically at 600 nm (OD₆₀₀). Relative growth was determined as described in the Materials and methods section 24 h after the addition of DMSO and expressed as mean ± SEM (n = 3). Two‐way ANOVA with Bonferroni posttest indicated the level of significance when comparing each overexpressing mutant with the cells harboring the empty vector at the same concentration of DMSO. *P < 0.05; **P < 0.01; ***P < 0.001.