Budding yeast GCN1 binds the GI domain to activate the eIF2 alpha kinase GCN2

When starved for a single amino acid, the budding yeast Saccharomyces cerev isiae activates the eukaryotic initiation factor 2 alpha (eIF2 alpha) kinas e GCN2 in a GCN1-dependent manner. Phosphorylated eIF2 alpha inhibits gener al translation but selectively derepresses the synthesis of the transcripti on factor GCN4, which leads to coordinated induction of genes involved in b iosynthesis of various amino acids, a phenomenon called general control res ponse. We recently demonstrated that this response requires binding of GCN1 to the GI domain occurring at the N terminus of GCN2 (Kubota, H,, Sakaki, Y., and Ito, T. (2000) J. Biol. Chem. 275, 20243-20246), Here we provide th e first evidence for the involvement of GCN1-GCN2 interaction in activation of GCN2 per se. We identified a C-terminal segment of GCN1 sufficient to b ind the GI domain and used a novel dual bait two-hybrid method to identify mutations rendering GCN1 incapable of interacting with GCN2, The yeast bear ing such an allele, gcn1-F2291L, fails to display derepression of GCN4 tran slation and hence general control response, as does a GI domain mutant, gcn 2-Y74A,, defective in association with GCN1. Furthermore, we demonstrated t hat phosphorylation of eIF2 alpha is impaired in both mutants. Since GCN2 i s the sole eIF2 alpha kinase in yeast, these findings indicate a critical r ole of GCN1-GCN2 interaction in activation of the kinase in vivo.