Separate domains in GCN1 for binding protein kinase GCN2 and ribosomes are required for GCN2 activation in amino acid‐starved cells

GCN2 stimulates GCN4 translation in amino acid‐starved cells by phosphorylating the α‐subunit of translation initiation factor 2. GCN2 function in vivo requires the GCN1/GCN20 complex, which binds to the N‐terminal domain of GCN2. A C‐terminal segment of GCN1 (residues 2052–2428) was found to be necessary and sufficient for binding GCN2 in vivo and in vitro . Overexpression of this fragment in wild‐type cells impaired association of GCN2 with native GCN1 and had a dominant Gcn − phenotype, dependent on Arg2259 in the GCN1 fragment. Substitution of Arg2259 with Ala in full‐length GCN1 abolished complex formation with native GCN2 and destroyed GCN1 regulatory function. Consistently, the Gcn − phenotype of gcn1‐R2259A , but not that of gcn1Δ , was suppressed by overexpressing GCN2. These findings prove that GCN2 binding to the C‐terminal domain of GCN1, dependent on Arg2259, is required for high level GCN2 function in vivo . GCN1 expression conferred sensitivity to paromomycin in a manner dependent on its ribosome binding domain, supporting the idea that GCN1 binds near the ribosomal acceptor site to promote GCN2 activation by uncharged tRNA.