Mutational Analysis of Mammalian Translation Initiation Factor 5 (eIF5): Role of Interaction between the β Subunit of eIF2 and eIF5 in eIF5 Function In Vitro and In Vivo

Abstract

ABSTRACT Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40S initiation complex (40S–eIF3–AUG–Met-tRNA f –eIF2–GTP) to promote the hydrolysis of ribosome-bound GTP. eIF5 also forms a complex with eIF2 by interacting with the β subunit of eIF2. In this work, we have used a mutational approach to investigate the importance of eIF5-eIF2β interaction in eIF5 function. Binding analyses with recombinant rat eIF5 deletion mutants identified the C terminus of eIF5 as the eIF2β-binding region. Alanine substitution mutagenesis at sites within this region defined several conserved glutamic acid residues in a bipartite motif as critical for eIF5 function. The E346A,E347A and E384A,E385A double-point mutations each caused a severe defect in the binding of eIF5 to eIF2β but not to eIF3-Nip1p, while a eIF5 hexamutant (E345A,E346A,E347A,E384A,E385A,E386A) showed negligible binding to eIF2β. These mutants were also severely defective in eIF5-dependent GTP hydrolysis, in 80S initiation complex formation, and in the ability to stimulate translation of mRNAs in an eIF5-dependent yeast cell-free translation system. Furthermore, unlike wild-type rat eIF5, which can functionally substitute for yeast eIF5 in complementing in vivo a genetic disruption of the chromosomal copy of the TIF5 gene, the eIF5 double-point mutants allowed only slow growth of this Δ TIF5 yeast strain, while the eIF5 hexamutant was unable to support cell growth and viability of this strain. These findings suggest that eIF5-eIF2β interaction plays an essential role in eIF5 function in eukaryotic cells.