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

Eukaryotic initiation factor 5 (eIF5), a monomeric protein of 49 kDa in mammals (9, 10, 21) and 46 kDa in the yeast Saccharomyces cerevisiae (5, 6), plays an essential role in the initiation of protein synthesis. Following scanning of mRNA by the 40S preinitiation complex (40S–eIF3–Met-tRNAf–eIF2–GTP) and positioning of the initiator Met-tRNAf at the AUG codon of the mRNA to form the 40S initiation complex (eIF3–40S–AUG–Met-tRNAf–eIF2–GTP), the initiation factor eIF5 interacts with the 40S initiation complex to effect the hydrolysis of ribosome-bound GTP. Hydrolysis of GTP causes the release of eIF2-GDP, Pi, and eIF3 from the 40S initiation complex, which is essential for the subsequent joining of the 60S ribosomal subunit to the 40S complex to form a functional 80S initiation complex (80S–mRNA–Met-tRNAf) that is active in peptidyl transfer (for reviews, see references 16, 18, and 19). eIF5-dependent GTP hydrolysis has also been shown to play an important role in the selection of the AUG start codon (15). An interesting feature of the derived amino acid sequence of mammalian (rat and human) and yeast eIF5 proteins (26) is the presence of sequence motifs at the N-terminal region of eIF5 that have weak homology to characteristic domains present in proteins belonging to the GTPase superfamily (3). However, unlike these proteins, eIF5 neither binds nor hydrolyzes free GTP or GTP bound to the (Met-tRNAf–eIF2–GTP) ternary complex in the absence of 40S ribosomal subunits (4, 7). eIF5 promotes GTP hydrolysis only when the nucleotide is bound to eIF2 in the 40S initiation complex (Met-tRNAf–eIF2–GTP–eIF3–40S–AUG) (4, 7). These results suggest that eIF5 must interact with one or more components of the 40S initiation complex to cause hydrolysis of GTP. In agreement with this hypothesis, we observed that mammalian eIF5 forms a complex with mammalian eIF2 (7), a component of the 40S initiation complex, and that eIF5-eIF2 complex formation occurs through the β subunit of eIF2 (11). Complex formation between eIF5 and Nip1p subunit of eIF3 has also been reported (1, 2). In the case of eIF5 and eIF2β interaction, deletion studies have shown that the N-terminal region of eIF2β binds eIF5 and that the conserved stretch of lysine residues in this region plays an important role in this interaction (11). Similar interaction between yeast eIF5 and yeast eIF2β was also reported from this laboratory (11) and later by others (1), indicating that the interaction domains of eIF5 and the β subunit of eIF2 are conserved through evolution. In later studies, Asano et al. (1) observed that a bipartite motif at the C-terminal region of yeast eIF5 containing conserved aromatic and acidic residues is required for binding to both eIF2β and the Nip1p subunit of eIF3. However, the important question remained as to whether the interaction of eIF5 with eIF2β is required for eIF5-dependent GTP hydrolysis and is thus a key molecular interaction in the translation initiation pathway. In the work presented here, we demonstrate that mammalian eIF5 interacts with mammalian eIF2β through a conserved C-terminal region. We have carried out a systematic mutational analysis of conserved residues in the C-terminal eIF2β-binding region of rat eIF5 to generate mutants which are defective in binding to eIF2β but are active in binding to Nip1p. We show that these mutants are also defective in eIF5-dependent GTP hydrolysis and consequently in 80S initiation complex formation as well as in in vitro protein synthesis. Furthermore, whereas mammalian eIF5 can functionally substitute for the homologous yeast protein in vivo in yeast cells (17), the mutant eIF5 proteins that are defective in binding to eIF2β are unable to complement a genetic disruption in the chromosomal copy of the TIF5 gene in vivo. Taken together, our results suggest that the interaction between eIF5 and the β subunit of eIF2 is required for eIF5-dependent hydrolysis of GTP during translation initiation and consequently is essential for overall protein synthesis.