Mutational analysis of Yih1 and IMPACT reveals amino acids required for Gcn2 inhibition

In response to amino acid starvation, the protein kinase Gcn2 phosphorylates the eukaryotic translation initiation factor eIF2α, allowing cells to adapt to adverse conditions. Gcn2 function requires direct binding to effector protein Gcn1 via the Gcn2 RWD-domain. The orthologues yeast Yih1 and mammalian IMPACT also contain an RWD-domain that can bind Gcn1, thereby impairing the Gcn2-Gcn1 interaction. In yeast, overexpressed Yih1/IMPACT impairs eIF2α phosphorylation, visible by reduced growth under starvation conditions. We found that Yih1 D102A and D108A substitutions each revert this defect, suggesting that Yih1-mediated Gcn2 inhibition is impaired. Similar effects were found for at least the D111A substitution in IMPACT. The respective amino acids are located in a common helix, suggesting this helix is a conserved determinant for Gcn1 binding.