Fig. 3

Phenotypic analysis of OsSIP-expressing cells of the S. cerevisiae BY4742 Δfps1 mutant. The wild-type BY4742 or BY4742Dfps1 mutant transformed with the pRS426met25::GFP vector was used as the positive and negative control, respectively. Expression of the recombinant OsSIP1-GFP or OsSIP2-GFP (GFP fusion at the C-terminus) fusion protein was confirmed by fluorescence imaging (A) and Western blot using an antibody against the GFP-tag (B). Growth assays were performed on SD-Ura (pH: 5.5) plates with different concentrations of the KCl, sorbitol and H₂O₂ (C-E). Ammonia permeation experiments were performed on plates in Mes- or Mops-buffered (20 mM, pH 5.5) YNB-glucose medium containing different concentrations of methylamine (MeA) with 0.1% proline added as a nitrogen source (F). The different yeast lines were grown in the appropriate liquid medium until the OD₆₀₀ was 1.0, then serially diluted 10-fold and spotted onto different plates. Plates were cultured at 30 °C for 3–5 days and then imaged