Fig. 7.

Sustained eIF2α phosphorylation in yeast cells expressing the KKaAa mutant of eIF2γ. (A) Derivatives of yeast strain YM53 expressing WT GCN2 and the indicated eIF2γ protein were grown in SD medium to log phase, incubated for 1 h in the presence or absence of 1 μg/mL SM, and then washed and incubated in fresh SD medium at 30 °C. After the indicated times (min), cells were lysed, and equivalent amounts of WCEs were subjected to SDS/PAGE followed by immunoblot analysis to detect eIF2α–P and total eIF2α. (B) The relative level of phosphorylated to total eIF2α in three independent experiments was determined by quantitative densitometry and normalized to the ratio obtained in the nonstarved WT control (lane 1).