Intracellular sequestration of sodium by a novel Na⁺/H⁺ exchanger in yeast is enhanced by mutations in the plasma membrane H⁺-ATPase. Insights into mechanisms of sodium tolerance

Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, which is required for modulation of Na⁺ uptake and efflux mechanisms. Five Na⁺-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H⁺-ATPase. One mutant, pma1-α4, which has the single amino acid change Glu³⁶⁷ → Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na⁺ tolerance. After exposure to Na⁺ in the culture medium, ²²Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity. Efflux of ²²Na from intact cells was relatively unchanged in the pma1 mutant. However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na⁺ within a slowly exchanging pool. We show that NHX1, a novel gene homologous to the mammalian NHE family of Na⁺/H⁺ exchangers, is required for Na⁺ sequestration in yeast and contributes to the Na⁺-tolerant phenotype of pma1-α4.