Specific interaction of eukaryocytic translation initiation factor 5 (eIF5) with the β-subunit of eIF2

Abstract

Eukaryotic translocation initiation factor 5 (eIF5) interacts with the 40 S initiation complex (40 S · mRNA · eIF3 · Met-tRNA(f) · eIF2 · GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5- interacting proteins in crude initiation factor preparations. We observed that eIF5 specifically interacted with the β subunit of initiation factor eIF2. No other initiation factors including the γ subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian or yeast eIF2. Binding analysis with human eIF2β deletion mutants expressed in Escherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2β. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bounds as Met-tRNA(f) · eIF2 · GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the β subunit of eIF2 may be critical for the hydrolysis of GTP during translation initiation.