Abstract
Eukaryotic translation initiation factor, eIF2B, is a guanine nucleotide exchange factor (GEF) composed of five dissimilar subunits. eIF2B is important for regenerating GTP-bound eIF2 during the initiation process. This event is obligatory for eIF2 to bind initiator methionyl-tRNA, forming the ternary initiation complex. In the current investigation, deletion mutants of the catalytic subunit, eIF2Bϵ, were constructed to identify regions that are necessary for eIF2B catalytic activity and formation of the holoprotein. We used the baculovirus expression system to coexpress wild-type and truncated forms of the ϵ-subunit of mammalian eIF2B (eIF2Bϵ) with the other four subunits (α, β, γ, δ) of the protein in Sf9 cells. Removal of either the N- or the C-terminal conserved domains of eIF2Bϵ resulted in a significant loss of GEF activity and reduced or abolished interaction with the α-, γ- and δ-subunits of eIF2B. Removal of the C-terminal 552 amino acids of eIF2Bϵ markedly reduced its interaction with the β-subunit of eIF2 whereas loss of the N-terminal 431 amino acids did not. The results suggest that intact eIF2Bϵ is required for full catalytic activity and formation of the eIF2B holoprotein. In contrast, the C-terminal domain of eIF2Bϵ is sufficient alone for binding the β-subunit of its substrate, eIF2, in vitro.