Growth phenotypes of trk1Δtrk2Δ yeast expressing the ROMK1 variants.

Representative yeast viability assays with control strains and the ROMK1 variants in group 6 were performed on (A) solid and (B) liquid medium. (A) Ten-fold dilutions of overnight, saturated cultures of yeast were inoculated on medium as described in Fig 4. Images were taken after two days of incubation at 30°C. (B) Saturated yeast cultures were diluted to a starting OD600 of 0.2 with assay medium supplemented with 25mM KCl and grown at 30°C. OD600 readings were recorded and data were standardized as described in the Materials and Methods and in Fig 4. Data represent results from two independent experiments (n = 2–3 each), ± the range of the data. (C) Table summarizing the growth phenotypes of the six ROMK1 groups described in the text. The predicted consequence of each group is denoted. Growth phenotypes were obtained in a blinded fashion, compared to the growth of trk1Δ trk2Δ yeast expressing WT ROMK1, and the results are color-coded: Red denotes a severe growth defect, orange denotes a moderate growth defect, green denotes no growth defect (WT-like), and blue denotes a slight increase in growth compared to the WT control. These classifications were performed by visual inspection. For example, as shown in Fig 5A and 5B, the P265R variant exhibited levels of growth that matched the vector control (i.e., the errors overlapped in Fig 5B), and hence this mutation was designated “severe growth defect” in part (C). In contrast, the P265Y variant exhibited levels of growth that were intermediate to that of the vector control and “Wild-type” (Fig 5B). Hence, this mutation was designated “moderate growth defect” in part (C).