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Fig. 6. Complementation of salt sensitivity of the Saccharomyces cerevisiae ena1-4, nha1, nhx1 mutant by CG10806 and CG31052. YFP-tagged CG31052 and CG10806 were cloned into the yeast expression vector pYes2.1, allowing expression of the recombinant protein upon galactose induction. To confirm the expression and examine the subcellular localisation of the transformed constructs, cells were viewed by confocal microscopy after 6 hours of induction of protein expression. (A) CG10806-YFP shows expression at both the plasma membrane and vacuolar membrane. (B) CG31052-YFP shows expression at the vacuolar membrane. (C) A western blot of the expressed proteins using an anti-GFP antibody. (D,E) Rescue of exchanger double-mutant yeast by Drosophila CPA2 genes was assessed by serial tenfold dilutions in 200 mM NaCl (D) or 1 M KCl (E) and comparison with wild-type (NHA1 NHX1) or exchanger double-mutant (nha1 nhx1) cells. The control NHX1 NHA1 strain G19 was transformed with empty pYes2.1 and the nha1 nhx1 mutant strain AXT3 was transformed with empty pYes2.1 or with YFP-tagged KHA ORFs. After induction of protein expression for 6 hours, cultures were adjusted to OD₆₀₀=1 and further serial tenfold dilutions were made. Equal volumes were then spotted onto selective media containing either 200 mmol/l NaCl (D) or 1 mol/l KCl (E). Cultures were then allowed to grow for a further 3 days before imaging.

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