FIG. 5.

Gir2 expression and amino acid control. (A) Overexpression of Gir2 leads to growth inhibition on 3-AT. Tenfold serial dilutions of cells harboring vector alone (p426-GAL1) or expressing GCN2(GI), GIR2, GIR2N, or GIR2C under the GAL1 promoter were spotted on plates containing either glucose or galactose, as indicated, in the presence of 10 mM 3-AT and incubated at 30°C. Similar strains that also express Gcn2 from a high-copy plasmid (hcGCN2; pAH15) were grown in the presence of galactose and 10 mM 3-AT. (B) Overexpression of hcGCN2 slightly perturbs the growth of starved cells. Tenfold serial dilutions of wild-type cells harboring hcGCN2 or vector alone in the absence (unstarved) or presence (starved) of 10 mM 3-AT. (C) Yeast strains (H1511) expressing plasmid-borne GST alone or GST-Gir2 and the isogenic gcn2Δ strain H2557 were grown to exponential phase and harvested, followed by immunoblot analysis with antibodies to eIF-2α and phosphorylated eIF-2α. Two different exposure times for phosphorylated eIF-2α are shown (two panels linked together by a gray bar on the right-hand side, with the upper panel showing short exposure and the lower panel showing long exposure). (D) Expression of GCN4-lacZ. Expression of GCN4 was determined in the strains indicated using a lacZ-based reported construct (p180), and the fold derepression was determined as the ratio of the wild-type values under nonstarvation conditions. For each strain, at least two independent transformants were assayed, in duplicate (gcn2Δ strain) or at least in triplicate, and the average values as well as the standard error values are shown in the graph.