EFFLUX OF SODIUM IONS BY A Na⁺/H⁺-ANTIPORTER DURING SALT STRESS IN THE SALT-TOLERANT YEAST ZYGOSACCHAROMYCES ROUXII

Abstract

The flux of Na⁺, Cl^- and H⁺ ions and the accumulation of glycerol in cells of a salt-tolerant yeast, Zygosaccharomyces rouxii, was examined during the initial stages of salt stress. Although the intracellular accumulation of glycerol that was required for osmoregulation began immediately after exposure of the cells to salt stress, rapid influx of Na⁺ and Cl^- ions into cells also occurred. These ions were subsequently extruded from the cells and the intracellular levels of both ions were maintained much lower than those in the external medium. Furthermore, a rapid efflux of protons was induced by activation of the plasma membrane ATPase (PM- ATPase) immediately after the start of salt stress and then the influx (re-influx) of protons into cells occurred. The addition of a specific inhibitor of PM-ATPase, diethylstilbestrol (DES), and of inhibitors of the energy-generating system, namely sodium azide, antimycin A and dinitrophenol, inhibited the intracellular accumulation of glycerol and activation of PM-ATPase, and they retarded the rapid efflux of protons and the extrusion of Na⁺ and Cl^- ions. The re-influx of protons into cells was inhibited by amphotericin B, probably because of the destruction of the structure of the plasma membrane. In mutants in which rapid activation of PM-ATPase was not observed immediately after the start of salt stress, the efflux of protons was markedly retarded and extrusion of Na⁺ and Cl^- ions was also suppressed during salt stress. Amiloride, an inhibitor of the Na⁺/H⁺-antiporter, inhibited the re-influx of protons and the efflux of Na⁺ and Cl^- ions, but this inhibitor did not inhibit the accumulation of glycerol, activation of PM-ATPase or the rapid efflux of protons. These results suggest that the Na⁺/H⁺-antiporter may play a significant role in extrusion of the ions that enter cells during the initial stages of salt stress.