Mg2+ deprivation elicits rapid Ca2+ uptake and activates Ca2+/calcineurin signaling in Saccharomyces cerevisiae

Abstract

To learn about the cellular processes involved in Mg(2+) homeostasis and the mechanisms allowing cells to cope with low Mg(2+) availability, we performed RNA expression-profiling experiments and followed changes in gene activity upon Mg(2+) depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg(2+) depletion are also induced by high Ca(2+) and/or alkalinization. Among the genes significantly up-regulated by Mg(2+) starvation, Ca(2+) stress, and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p (calcineurin-responsive zinc finger protein) signaling pathway. Similarly to Ca(2+) stress, Mg(2+) starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg(2+) starvation depends on extracellular Ca(2+). Using fluorescence resonance energy transfer microscopy, we demonstrate that removal of Mg(2+) results in an immediate increase in free cytoplasmic Ca(2+). This effect is dependent on external Ca(2+). The results presented indicate that Mg(2+) depletion in yeast cells leads to enhanced cellular Ca(2+) concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg(2+) depletion stress.

FIG. 1.

ENA1 and PHO89 transcripts are induced upon Mg²⁺ starvation. Strain JS034-4C was grown in synthetic medium containing 1 mM Mg²⁺ to an OD₆₀₀ of 0.5, washed twice with SD medium containing either 1 mM Mg²⁺ or lacking Mg²⁺, and then incubated in the same medium. Samples were drawn at the indicated time points, and total RNA was prepared. Twenty-five micrograms of total RNA was loaded per lane, and Northern blot analysis was performed using radiolabeled probes specific for ENA1, PHO89, and ACT1 as a loading control.

FIG. 2.

The transcription factor Crz1p is required for the induction of ENA1 and PHO89 and translocated to the nucleus upon Mg²⁺ starvation. (A) Levels of expression of ENA1 and PHO89 in wild-type (WT [BY4741]) (lanes 1 and 2) and crz1Δ (lanes 3 and 4) cells were compared under standard conditions (1 mM Mg²⁺, lanes 1 and 3) or 70 min of Mg²⁺ starvation (lanes 2 and 4). Northern blot analysis is shown. (B) Strain BY4741-CRZ1-GFP was grown at 28°C to log-phase in SD −Ura medium containing 1 mM MgCl₂ (a and d) and then washed and incubated for 10 min at 28°C in SD −Ura medium containing either no MgCl₂ (b and e) or 1 mM MgCl₂-200 mM CaCl₂-50 mM MES (pH 6) (c and f) before cells were harvested for microscopy and analyzed.

FIG. 3.

The transcriptional response to Mg²⁺ starvation is dependent on calcineurin. Expression of ENA1 and PHO89 upon Mg²⁺ starvation (70 min) was followed in cnb1Δ mutant cells and in wild-type (WT) cells in the presence or absence of the calcineurin inhibitor FK506 (1.25 μM), which was added to the culture 10 min prior to the washes. Northern blot analysis is shown.

FIG. 4.

crz1Δ and cnb1Δ cells are sensitive to low Mg²⁺. BY4741 wild-type (WT) and crz1Δ and cnb1Δ mutant cells were cultured in synthetic SD medium containing 1 mM Mg²⁺ overnight, washed three times in distilled H₂O, and then inoculated (OD₆₀₀ of 0.05) into synthetic SD medium containing 1 mM Mg²⁺ (left panel) or no Mg²⁺ (right panel). Cells were incubated at 28°C with shaking, and growth was followed by measuring the OD₆₀₀.

FIG. 5.

External Ca²⁺ is required for the induction of ENA1 and PHO89 upon Mg²⁺ starvation. Expression of ENA1 and PHO89 upon Mg²⁺ starvation (70 min) was followed in the presence or absence of the Ca²⁺ chelator EGTA (10 mM). Northern blot analysis is shown.

FIG. 6.

Cytoplasmic Ca²⁺ levels increase upon Mg²⁺ depletion in dependence of external Ca²⁺. Cytosolic free Ca²⁺ concentrations were analyzed upon Mg²⁺ depletion (left panel) or upon addition of 200 mM Ca²⁺ (right panel) using fluorescence resonance energy transfer microscopy.