The N-terminal lysine residue-rich domain II and the 340–430 amino acid segment of eukaryotic initiation factor 2-associated glycoprotein p67 are the binding sites for the γ-subunit of eIF2

Eukaryotic initiation factor 2 (eIF2)-associated glycoprotein, p67, plays an important role in protecting eIF2α from phosphorylation by eIF2α-specific kinases. To understand the molecular details of interaction between p67 and the subunits of eIF2, we applied several biochemical and mutational analyses to identify interacting domains within p67 and eIF2γ. These studies were combined with functional in vivo and in vitro assays to address the importance of the interactions between p67 and eIF2γ in eIF2α phosphorylation. Studies from yeast two-hybrid assays show that p67 interacts strongly with eIF2γ, relatively weakly with eIF2α, and no interaction with eIF2β. Further mutational analyses provided evidence that the N-terminal lysine-rich domain II and the 340–430 amino acid segment of p67 interact strongly with the C-terminal 409–472 amino acid segment of eIF2γ. GST pull-down assays show that the interaction between p67 and eIF2γ is direct. From co-immunoprecipitation studies, we find that the interaction between p67 and eIF2γ could not only be detected in mammalian cells growing in growth medium, it could also be detected in transiently transfected cells with expression plasmids encoding p67 and eIF2γ. However, this interaction could not be detected in p67 mutants lacking lysine-rich domain II and the 340–430 amino acid segment. We also find a very good correlation between p67 binding to eIF2γ and the protection of eIF2α from phosphorylation. Altogether, our data provide genetic evidence for the interaction between p67 and eIF2γ and that this interaction modulates the phosphorylation of eIF2α.