Plasma membrane composition of Debaryomyces hansenii adapts to changes in pH and external salinity.

Debaryomyces hansenii is a marine yeast that has to cope with different stress situations. Since changes in membrane properties can play an important function in adaptation, we have examined the fluidity and lipid composition of purified plasma membranes of D. hansenii grown at different external pH values and salt concentrations. Growth at low pH caused an increase in the sterol-to-phospholipid ratio and a decrease in fatty acid unsaturation which was reflected in decreased fluidity of the plasma membrane. High levels of NaCl increased the sterol-to-phospholipid ratio and fatty acid unsaturation, but did not significantly affect fluidity. The sterol-to-phospholipid ratios obtained in D. hansenii grown under any of these conditions were similar to the ratios that have been reported for halophilic/halotolerant black yeasts, but much smaller than those observed in the model yeast Saccharomyces cerevisiae.

Characterization of DhKHA1, a gene coding for a putative Na(+) transporter from Debaryomyces hansenii.

The KHA1 gene from Debaryomyces hansenii has been identified and characterized by heterologous expression in Saccharomyces cerevisiae. The gene is orthologous to ScKHA1, previously reported in S. cerevisiae, and on the basis of the deduced amino acid sequence, DhKha1p can be classified as an Na(+)/H(+) transporter. Reverse transcriptase (RT)-PCR experiments indicated that the expression level of DhKHA1 was not dependent on high pH or on the presence of a high salt level in the growth medium. Overexpression of DhKHA1 in a salt-sensitive S. cerevisiae mutant (ena1-4 nha1 kha1) rendered cells specifically more tolerant to Na(+). In addition, internal K(+) and Na(+) measurements and experiments performed with green fluorescence protein (GFP)-tagged DhKha1p indicated the intracellular localization of this protein when expressed in S. cerevisiae.

Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters.

K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves.

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

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.

Mechanisms underlying the halotolerant way of Debaryomyces hansenii.

The yeast Debaryomyces hansenii is usually found in salty environments such as the sea and salted food. It is capable of accumulating sodium without being intoxicated even when potassium is present at low concentration in the environment. In addition, sodium improves growth and protects D. hansenii in the presence of additional stress factors such as high temperature and extreme pH. An array of advantageous factors, as compared with Saccharomyces cerevisiae, is putatively involved in the increased halotolerance of D. hansenii: glycerol, the main compatible solute, is kept inside the cell by an active glycerol-Na+ symporter; potassium uptake is not inhibited by sodium; sodium protein targets in D. hansenii seem to be more resistant. The whole genome of D. hansenii has been sequenced and is now available at http://cbi.labri.fr/Genolevures/ and, so far, no genes specifically responsible for the halotolerant behaviour of D. hansenii have been found.

On the role of Trk1 and Trk2 in Schizosaccharomyces pombe under different ion stress conditions.

Trk1 and Trk2 are the major K(+) transport systems in Schizosaccharomyces pombe. Both transporters individually seem to be able to cope with K(+) requirements of the cells under normal conditions, since only the double mutant shows defective K(+) transport and defective growth at limiting K(+) concentrations. We have studied in detail the role of SpTrk1 and SpTrk2 under different ion stress conditions. Results show that the strain with only Trk1 (trk1(+)) is less sensitive to Li(+) and to hygromycin B, it grows better at low K(+) and it survives longer in a medium without K(+) than the strain expressing only Trk2 (trk2(+)). We conclude that Trk1 contributes more efficiently than Trk2 to the performance of the fission yeast under ion stress conditions. In the wild type both trk1(+) and trk2(+) genes are expressed and probably collaborate for the performance of the cells.