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The trk1؉ gene has been proposed as a component of the K ؉ influx system in the fission yeast Schizosaccharomyces pombe. Previous work from our laboratories revealed that trk1 mutants do not show significantly altered content or influx of K ؉ , although they are more sensitive to Na ؉ . Genome database searches revealed that S. pombe encodes a putative gene (designated here trk2 ؉ ) that shows significant identity to trk1 ؉ . We have analyzed the characteristics of potassium influx in S. pombe by using trk1 trk2 mutants. Unlike budding yeast, fission yeast displays a biphasic transport kinetics. trk2 mutants do not show altered K ؉ transport and exhibit only a slightly reduced Na ؉ tolerance. However, trk1 trk2 double mutants fail to grow at low K ؉ concentrations and show a dramatic decrease in Rb ؉ influx, as a result of loss of the high-affinity transport component. Furthermore, trk1 trk2 cells are very sensitive to Na ؉ , as would be expected for a strain showing defective potassium transport. When trk1 trk2 cells are maintained in K ؉ -free medium, the potassium content remains higher than that of the wild type or trk single mutants. In addition, the trk1 trk2 strain displays increased sensitivity to hygromycin B. These results are consistent with a hyperpolarized state of the plasma membrane. An additional phenotype of cells lacking both Trk components is a failure to grow at acidic pH. In conclusion, the Trk1 and Trk2 proteins define the major K ؉ transport system in fission yeast, and in contrast to what is known for budding yeast, the presence of any of these two proteins is sufficient to allow growth at normal potassium levels.

A screening for multicopy suppressors of the G 1 /S blockage of a conditional sit4 hal3 mutant yielded the NHA1 gene, encoding a Na ؉ ,K ؉ /H ؉ antiporter, composed of a transmembrane domain and a large carboxyl-terminal tail, which has been related to cation detoxification processes. Expression of either the powerful Saccharomyces cerevisiae Ena1 Na ؉ /H ؉ -ATPase or the Schizosaccharomyces pombe Sod2 Na ؉ /H ؉ antiporter, although increasing tolerance to sodium, was unable to mimic the Nha1 function in the cell cycle. Mutation of the conserved Asp residues Asp 266 -Asp 267 selectively abolished Na ؉ efflux without modifying K ؉ efflux and did not affect the capacity of Nha1 to relieve the G 1 blockage. Mutagenesis analysis revealed that the region near the carboxyl-terminal end of Nha1 comprising residues 800 -948 is dispensable for sodium detoxification but necessary for transport of K ؉ cations. Therefore, this portion of the protein contains structural elements that selectively modulate Nha1 antiporter functions. This region is also required for Nha1 to function in the cell cycle. However, expression of the closely related Cnh1 antiporter from Candida albicans, which also contains a long carboxyl-terminal extension, although allowing efficient K ؉ transport does not relieve cell cycle blockage. This indicates that although the determinants for Nha1-mediated regulation of potassium transport and the cell cycle map very closely in the protein, most probably the function of Nha1 on cell cycle is independent of its ability to extrude potassium cations.

Debaryomyces hansenii is an osmotolerant and halotolerant yeast of increasing interest for fundamental and applied research. In this work, we have performed a first study on the effect of oxidative stress on the performance of this yeast. We have used Saccharomyces cerevisiae as a well-known reference yeast. We show that D. hansenii is much more susceptible than S. cerevisiae to cadmium chloride, hydrogen peroxide or 1,4-dithiothreitol. These substances induced the formation of reactive oxygen species (ROS) in both yeasts, the amounts measured being significantly higher in the case of D. hansenii. We also show that NaCl exerted a protective effect against oxidative stress in Debaryomyces, but that this was not the case in Saccharomyces because sodium protected that yeast only when toxicity was induced with cadmium. On the basis of the present results, we raised the hypothesis that the sensitivity to oxidative stress in D. hansenii is related to the high amounts of ROS formed in that yeast and that observations such as low glutathione amounts, low basal superoxide dismutase and peroxidase activities, decrease in ATP levels produced in the presence of ROS inducers and high cadmium accumulation are determinants directly or indirectly involved in the sensitivity process.

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