Cloning and characterization of two K+ transporters of Debaryomyces hansenii

Abstract

Two genes from the halotolerant yeast Debaryomyces hansenii were cloned, DhTRK1 and DhHAK1. These genes encode K⁺ transporters with sequence similarities to the TRK and HAK transporters from Debaryomyces occidentalis and Candida albicans. The DhHAK1p transporter was only expressed in K⁺-starved cells, as shown by Northern blot analysis. Both DhTRK1p and DhHAK1p were expressed in a trk1Δ trk2Δ mutant of Saccharomyces cerevisiae, unable to grow at low K⁺. This expression resulted in partial recovery of growth and ability to retain K⁺ at low concentrations. In liquid media, 0.5 M NaCl affected growth of these S. cerevisiae transformants as it does in D. hansenii, resulting in a much less deleterious effect than in wild-type S. cerevisiae. Kinetics of Rb⁺ uptake in the transformants suggest that DhTRK1p and DhHAK1p code for moderate-affinity K⁺ transporters exhibiting a sigmoid response against Rb⁺ concentration and presenting a deviation from classic Michaelis–Menten kinetics at low substrate concentrations. Rb⁺ uptake by the DhTRK1p transporter was stimulated by millimolar concentrations of Na⁺ at pH 4.5. The good performance of DhTRK1p in the presence of NaCl may be a key feature in the halotolerance of D. hansenii.