Figure 5.
Both Srb10 and Pho85 contribute to Gcn4 turnover in vivo. (A) Gcn4myc9 stability under normal and overproduced conditions. Wild-type (YC19, YC23) and srb10Δ (YC21, YC24) cells grown in YP-raffinose medium were transferred either to synthetic galactose medium (top two panels) or synthetic dextrose medium (bottom two panels) for 1 h prior to being subjected to pulse-chase analysis as described for Figure 4A. Quantitation of the results is shown on the right. The arrowhead denotes a posttranslationally modified form of Gcn4myc9 that appeared rapidly in SRB10 cells, but was only slowly generated in srb10Δ strains. Note that the biphasic degradation observed for GAL–GCN4myc9 strains was likely owing to the carry-over synthesis of Gcn4myc9 during the first 5 min of chase. (B) Gcn4 stability in wild-type (YC19), srb10Δ (YC21), pho85Δ (YC59), and srb10Δ pho85Δ (YC61) cells was evaluated by pulse-chase analysis (see Fig. 4A legend). Quantitation is shown on the right. In two independent experiments, the half-life of Gcn4 in the various strains varied by less than 2 min.