FIGURE 3.

Aup1 affects Rtg3 phosphorylation during growth on lactate. A, wild type (wt) (DJY8) and aup1Δ (DJY9) cells expressing Rtg3-HA were grown in SD medium supplemented with 0.2% glutamate at 26 °C to an A₆₀₀ of 0.5 and challenged with 50 mm 3-AT or 0.2 μg/ml rapamycin as indicated. Cells were incubated for 30 min, and 20 μg of protein extracted from each sample were processed for Western blotting. B, cells expressing Rtg3-HA (DJY8) were grown in synthetic lactate medium supplemented with 0.2% glutamate. At each time point, 10 A₆₀₀ units of cells were sampled, and 20 μg of protein from each sample were analyzed by immunoblotting. The arrow denotes the migration distance of the hyperphosphorylated form in each experiment, whereas asterisks denote the faster migrating (hypophosphorylated) species of Rtg3-HA.