Characterization
of S. cerevisiae mutants lacking the KHA1 gene.
Lydie Maresova,
Hana Sychrova
Dept. Membrane Transport, Inst. Physiology, CzAcadSci, Videnska 1083, Prague 4,
142 20, Czech Republic (lydie@biomed.cas.cz)
Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. Potassium ions are involved, among other things, in the regulation of cell volume and intracellular pH. In S. cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p. It is a putative plasma membrane K+/H+ antiporter. But in our experiments, yeast cells ena1-4 nha1 were highly sensitive to extracellular KCl and only very low K+ efflux was measured for them. We are discussing here the possibility of Kha1p being localised elsewhere from the plasma membrane or being expressed and/or activated under specific conditions only. Strains with their KHA1 gene disrupted generally behave the same on the standard YPD and YNB media as their parent strains. But we found that on minimal medium (AP), strains kha1 are slightly more sensitive to KCl and on YNB medium with KCl they acidify the medium with an obvious delay in comparison to the parent strain. We prepared both centromeric and multicopy plasmids with the KHA1 gene and various promoters. We tagged the gene with GFP to detect its localisation in the cell. S. cerevisiae cells with their own KHA1 disrupted were transformed with these plasmids and compared to control cells. This work was supported by grants GA CR 204/01/0272 and AVOZ 5011922.