Investigating Ca2+ Sensitivity in Yeast

The AtCCX4 cDNA was subcloned in the pYES2 vector (Thermo Fisher Scientific). The csg1, 2 mutants, reported to be sensitive to high Ca²⁺ (Beeler et al. 1994 (link)), were obtained through EUROSCARF (http://www.euroscarf.de). The genotypes and the accession numbers of the strains are [MATa, his3, leu2, ura3, met15, ade2, YPL057c::kanMX4] and Y02771 (for csg1), and [MATa, his3, leu2, ura3, met15, ade2, YBR036c::kanMX4] and Y03173 (for csg2), respectively. Transformation using the csg2 strain was performed and the transformants, which grew on Ura-selective medium containing 0.67% yeast nitrogen base (w/v: BD science, USA), 2% sucrose (w/v), complete supplement dropout-URA (770 mg/L: FORMEDIUM, UK), and 2% agar (w/v) were selected as reported previously (Horie et al. 2001 (link)). Ca²⁺ hypersensitive assays were performed as described previously (Yadav et al. 2015 (link)). Briefly, transformants selected for uracil prototrophy on synthetic complete (SC) medium were grown at 30 °C for 7 d on the modified SC medium without (NH₄)_sSO₄ (MP Biomedicals), supplemented with 2% (w/v) galactose, 5 g/L NH₄Cl, and 1 or 100 mM CaCl₂.