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. 2003 Apr 1;17(7):859–872. doi: 10.1101/gad.1069003

Figure 4.

Figure 4

The tap42-11 mutation diminishes the effects of rapamycin in promoting dephosphorylation of Ser 577 in GCN2 and increased phosphorylation of Ser 51 in eIF2α at 25°C. (A) Transformants of strains CY1077 (TAP42) and CY1078 (tap42-11) containing GCN2-FL on pDH101 (lanes 2–5), or CY1077 transformed with pCB149 containing GCN2-S577A-FL (lane 1), were grown on SC medium lacking uracil to early logarithmic phase at 25°C and either treated with 200 ng/mL rapamycin for 2 h or left untreated. WCE extracts were prepared and analyzed for phosphorylation of Ser 577 in FL-GCN2 as described in Figure 1. (B) Strains CY1077 (TAP42) and CY1078 (tap42-11) were cultured in SC medium and analyzed for eIF2α phosphorylation as described in Figure 1. The gcn2Δ strain ESY6053 was analyzed in parallel (lane 1). (C,D) Multiple experiments analogous to those described in A and B were carried out identically except that the rapamycin treatment was for 30 min or 4 h. The eIF2α-P/eIF2α and S577-P/FL-GCN2 ratios were calculated as described in Figure 2A and plotted against the hours of rapamycin treatment.