Fig. 5.

The N-terminal segment of GAC1 functionally replaces the N-terminal extension of eIF2γ. (A) Schematic diagram of eIF2γ mutants and fusion proteins. The GAC1 element contains residues 1–93; the Lim domain contains residues 1–56 of Xlim-1. (B) Derivatives of yeast strain YM53 expressing the indicated eIF2γ protein and WT GCN2 were grown in SD medium to log phase, incubated for 1 h in the presence or absence of 1 μg/mL SM, and then equivalent amounts of WCEs were subjected to SDS/PAGE followed by immunoblot analysis to detect eIF2α–P, total eIF2α, GCN2, and GLC7-myc. The relative level of phosphorylated to total eIF2α was determined by quantitative densitometry and normalized to the ratio obtained in the nonstarved WT control (lane 1). (C) Yeast transformants described in B were grown to saturation in SD medium, and 4-µL volumes of serial dilutions (optical density; OD₆₀₀ = 1.0, 0.1, 0.01, 0.001, and 0.0001) were spotted on SD medium or SD medium supplemented with 1 μg/mL SM and incubated 3 d at 30 °C.