FIG 5.

Expression of TgZnT restores Zn²⁺ tolerance to yeast. (A) Western blot analysis of lysates from parental and zrc1Δ::cot1Δ yeasts transformed with pYES2-TgZnT or pYES2 (empty vector) and grown in medium containing galactose for induction. Lysate from parental and zrc1Δ::cot1Δ yeasts transfected with pYES2-TgZnT shows labeling with anti-TgZnT. A portion of the Ponceau-stained membrane is shown as a loading control. (B) Representative example of agar plate growth assays (n = 3) showing that TgZnT expressing zrc1Δ::cot1Δ::pYES2-TgZnT mutants are capable of growth in medium supplemented with up to 300 μM ZnSO₄, in contrast to the zrc1Δ::cot1Δ::pYES2 mutant, which can grow only in medium supplemented with up to 100 μM ZnSO_4. (C) Representative growth curves from 3 liquid culture assays show that zrc1Δ::cot1Δ mutants (blue) expressing TgZnT have increased tolerance to growth in medium supplemented with 100 μM ZnSO₄, in contrast to zrc1Δ::cot1Δ mutants not expressing TgZnT (red). The parental cell lines transformed with pYES2 and pYES2-TgZnT are shown as controls (yellow and green curves, respectively). (D) Quantification of growth assay endpoints (65 h) showing a significant difference in the increased growth of zrc1Δ::cot1Δ mutants when complemented with TgZnT (n = 3). *, P > 0.05; n.s., not significant; A.U., absorbance units.