Fig. 8. Characterization of the tolerance to toxic cations mediated by YEp-HAL3 and ppz1,2 in the trk1,2 background. (A) The strains indicated (W303-1A trk1::LEU2 trk2::HIS3 [YPGE15], W303-1A trk1::LEU2 trk2::HIS3 [YPGE15-HAL3] and W303-1A trk1::LEU2 trk2::HIS3 ppz1::URA3 ppz2::TRP1) were grown to saturation in selective media supplemented with 0.2 M KCl, serially diluted in sterile water and spotted on to YPD plates containing the indicated amount of cations. Images were taken after 2–3 days (top panel) or 6 days (lower panel) of incubation at 28°C. Identical results were obtained from at least three independent colonies in three separate experiments. (B) The strains indicated were grown to OD₆₆₀ = 0.5 in YPD media containing 0.8 M NaCl. Cells were washed extensively with 1.2 M sorbitol, 10 mM MgCl₂, lysed by boiling and after centrifugation to remove cellular debris, internal ion concentrations were determined by HPLC analysis, as described. Similar results were observed in two separate experiments.