Association of GCN1-GCN20 regulatory complex with the N-terminus of elF2 alpha kinase GCN2 is required for GCN2 activation

Stimulation of GCN4 mRNA translation due to phosphorylation of the a-subuni t of initiation factor 2 (eIF2) by its specific kinase, GCN2, requires bind ing of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like domain in GCN2. GCN2 function in vivo also requires GCN1 and GCN20, but it was unknow n whether these latter proteins act directly to promote the stimulation of GCN2 by uncharged tRNA, We found that the GCN1-GCN20 complex physically int eracts with GCN2, binding to the N-terminus of the protein, Overexpression of N-terminal GCN2 segments had a dominant-negative phenotype that correlat ed with their ability to interact with GCN1-GCN20 and impede association be tween GCN1 and native GCN2, Consistently, this Gcn(-) phenotype was suppres sed by overexpressing GCN2, GCN1-GCN20 or tRNA(His). The requirement for GC N1 was also reduced by overexpressing tRNA(His) in a gcn1 Delta strain. We conclude that binding of GCN1-GCN20 to GCN2 is required for its activation by uncharged tRNA, The homologous N-terminus of Drosophila GCN2 interacted with yeast GCN1-GCN20 and had a dominant Gcn(-) phenotype, suggesting evolu tionary conservation of this interaction.