The Sap proteins are required for regulation of GCN4 translation via Gcn2. (A) Isogenic wild-type (WT; MLY41a), gcn2 (SCY51), sap185 sap190 (JRY29), and sap185 sap190 gcn2 (JRY48) mutant strains were grown, serially diluted, and spotted onto plates of YEPD with or without 20 nM rapamycin as indicated in Fig. 1. After 3 days incubation at 30°C, the plates were photographed. (B) Cultures of isogenic wild-type (MLY41) or sap185 sap190 (JRY29), only-SAP155 (sap4 sap185 sap190; JRY46), gcn2 (SCY51), sap185 sap190 gcn2 (JRY48), sap155 sap4 (SCY115), sap155 (SCY108), only-SAP190 (sap4 sap155 sap185; JRY44), no-SAP (sap4 sap155 sap185 sap190; JRY40), and sit4 (SCY94) mutant strains harboring the GCN4-lacZ reporter plasmid p180 were grown to exponential phase in SD-Ura medium. Cultures were treated with 100 nM rapamycin for 0, 60, and 180 min and analyzed for β-galactosidase activity. (C) Isogenic wild-type (MLY41a), only-SAP155 (sap4 sap185 sap190; JRY46), sap185 (JRY20), sap190 (JRY21), and sap185 sap190 (JRY29) strains were spotted onto YEPD medium with or without 30 mM 3-AT. After 3 days of incubation at 30°C, the plates were photographed.