Ion Tolerance of Saccharomyces cerevisiae Lacking the Ca2+/CaM-Dependent Phosphatase (Calcineurin) Is Improved by Mutations in URE2 or PMA1

Abstract

Calcineurin is a conserved, Ca2+/CaM-stimulated protein phosphatase required for Ca2+-dependent signaling in many cell types. In yeast, calcineurin is essential for growth in high concentrations of Na+, Li+, Mn2+, and OH−, and for maintaining viability during prolonged treatment with mating pheromone. In contrast, the growth of calcineurin-mutant yeast is better than that of wild-type cells in the presence of high concentrations of Ca2+. We identified mutations that suppress multiple growth defects of calcineurin-deficient yeast (cnb1Δ or cna1Δ cna2Δ). Mutations in URE2 suppress the sensitivity of calcineurin mutants to Na+, Li+, and Mn2+, and increase their survival during treatment with mating pheromone. ure2 mutations require both the transcription factor Gln3p and the Na+ ATPase Pmr2p to confer Na+ and Li+ tolerance. Mutations in PMA1, which encodes the yeast plasma membrane H+-ATPase, also suppress many growth defects of calcineurin mutants. pma1 mutants display growth phenotypes that are opposite to those of calcineurin mutants; they are resistant to Na+, Li+, and Mn2+, and sensitive to Ca2+. We also show that calcineurin mutants are sensitive to aminoglycoside antibiotics such as hygromycin B while pma1 mutants are more resistant than wild type. Furthermore, pma1 and calcineurin mutations have antagonistic effects on intracellular [Na+] and [Ca2+]. Finally, we show that yeast expressing a constitutively active allele of calcineurin display pma1-like phenotypes, and that membranes from these yeast have decreased levels of Pma1p activity. These studies further characterize the roles that URE2 and PMA1 play in regulating intracellular ion homeostasis.

Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane

“…The plasma membrane potential is a major determinant of toxic cation tolerance. Mutations in the electrogenic H + ‐ATPase, Pma1p, lead to increased Na + resistance, partly because of the reduced Na + uptake linked to a reduction in the membrane potential (Nass et al ., 1997; Withee et al ., 1998). pma1 mutants having reduced Pma1p activity exhibit increased tolerance to hygromycin B (McCusker et al ., 1987), as a consequence of their membrane potential defect (Perlin et al ., 1988).…”

“…Deletion of both genes, PMP1 and PMP2 , significantly decreases the rate of ATP hydrolysis of Pma1p, the plasma membrane H + ‐ATPase (Navarre et al ., 1994). Recently, mutations in Pma1p were reported to be Na + resistant partly by reducing Na + uptake, but also by up‐regulating the activity of an intracellular Na + /H + exchanger (Nass et al ., 1997; Withee et al ., 1998).…”

Overexpression profiling reveals cellular requirements in context of genetic backgrounds and environments

“…2J ). Since Pma1 and Pmr1 are also involved in calcium homeostasis 30,31 ( Fig. 2B ), we speculated that cells may have also evolved to compensate for calcium deficiency under our conditions through these mutations.…”

The eukaryotic response regulator Skn7p regulates calcineurin signaling through stabilization of Crz1p

“…To identify possible interactions between calcineurin and Skn7p with respect to cell wall stress, we tested their effects on hygromycin B sensitivity. Several mutants that have defects in cell wall synthesis or structure exhibit sensitivity to this drug (Dean, 1995; Lussier et al ., 1997), and calcineurin and skn7 mutants are both sensitive to hygromycin B (Withee et al ., 1998; S.Li and J.S.Fassler, in preparation) (Figure 3B). Notably, the skn7Δcnb1Δ strain is significantly more sensitive than either single mutant (Figure 3B).…”

“…In the budding yeast Saccharomyces cerevisiae , calcineurin allows the cell to adapt to environmental stress. Mutants lacking the genes encoding either the calcineurin catalytic subunits CNA1 and CNA2 (Cyert et al ., 1991; Y.Liu et al ., 1991), or the regulatory subunit CNB1 (Kuno et al ., 1991; Cyert and Thorner, 1992) are viable under normal growth conditions, but die in the presence of high concentrations of different ions including Mn 2+ , Na + , Li + and OH − (Nakamura et al ., 1993; Mendoza et al ., 1994; Farcasanu et al ., 1995; Pozos et al ., 1996), as well as in the presence of the cationic aminoglycoside hygromycin B (Withee et al ., 1998). Calcineurin mutants also lose viability during prolonged exposure to mating pheromone (Moser et al ., 1996; Withee et al ., 1997).…”