Intracellular Na+ and K+ distribution in Debaryomyces hansenii. Cloning and expression in Saccharomyces cerevisiae of DhNHX1

Abstract

Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.

Growth at High pH and Sodium and Potassium Tolerance in Media above the Cytoplasmic pH Depend on ENA ATPases in Ustilago maydis

“…The inability of fungal cells to decrease the cytoplasmic Na ϩ concentration by accumulating it into the vacuole, as reported here for U. maydis, was previously reported for S. cerevisiae (39,46,55) and Debaryomyces hansenii (39). These three species grow normally with a rather high Na ϩ content, exhibiting low cytoplasmic Na ϩ toxicity.…”

“…These three species grow normally with a rather high Na ϩ content, exhibiting low cytoplasmic Na ϩ toxicity. In S. cerevisiae, an Na ϩ /K ϩ ratio of 1 is completely nontoxic (39), and in D. hansenii, the Na ϩ /K ϩ ratio can be as high as 4 without detrimental effects (39). Similarly, Hortaea werneckii and Aureobasidium pullulans show comparable K ϩ and Na ϩ contents when actively growing at 0.8 M NaCl (37).…”

“…A low Na ϩ /K ϩ ratio has to be maintained by Neurospora crassa because it stops growing when Na ϩ and K ϩ contents reach an Na ϩ /K ϩ ratio that is much lower than 1 (52 (Fig. 1), and S. cerevisiae (39) are not affected by cytoplasmic molar Na ϩ /K ϩ ratios of 4, 2, and 1, respectively. Therefore, because their ENA ATPases do not discriminate between Na ϩ and K ϩ , they provide protection against high concentrations of any of these cations (Fig.…”

Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants

“…This is a convenient and rapid method, but the main limitation of the procedure is that the results are too simplistic, since it usually considers only two important compartments where the cations are present in significant amounts; K + extracted after permeabilization is considered the cytoplasmic fraction, whereas the remaining amount is ascribed to the vacuole. After using these procedures, most of the investigations conclude that cations accumulate in the vacuolar fraction to significantly higher amounts than in the cytoplasmic fraction [13,15,16], which is in contradiction with what was previously published by Roomans and Sevéus [12].…”

“…However, possibly due to the fact that the technique was not readily available for many groups or maybe because those experiments were performed under non-physiological conditions (prior to analysis, yeast cells were suspended in water for 1 day), the fact is that this work did not have the impact and significance that it may have had. The only relatively widely used approach to investigate the question of subcellular cation localization in yeast is the use of substances such as cytochrome c [13][14][15], DEAE dextran [16] or digitonin [17] to permeabilize the plasma membrane. This is a convenient and rapid method, but the main limitation of the procedure is that the results are too simplistic, since it usually considers only two important compartments where the cations are present in significant amounts; K + extracted after permeabilization is considered the cytoplasmic fraction, whereas the remaining amount is ascribed to the vacuole.…”