eIF3 in mammals is the largest translation initiation factor (∼800 kDa) and is composed of 13 nonidentical subunits designated eIF3a-m. The role of mammalian eIF3 in assembly of the 48 S complex occurs through high affinity binding to eIF4G. Interactions of eIF4G with eIF4E, eIF4A, eIF3, poly(A)-binding protein, and Mnk1/2 have been mapped to discrete domains on eIF4G, and conversely, the eIF4G-binding sites on all but one of these ligands have been determined. The only eIF4G ligand for which this has not been determined is eIF3. In this study, we have sought to identify the mammalian eIF3 subunit(s) that directly interact(s) with eIF4G. Established procedures for detecting protein-protein interactions gave ambiguous results. However, binding of partially proteolyzed HeLa eIF3 to the eIF3-binding domain of human eIF4G-1, followed by high throughput analysis of mass spectrometric data with a novel peptide matching algorithm, identified a single subunit, eIF3e (p48/Int-6). In addition, recombinant FLAG-eIF3e specifically competed with HeLa eIF3 for binding to eIF4G in vitro. Adding FLAG-eIF3e to a cell-free translation system (i) inhibited protein synthesis, (ii) caused a shift of mRNA from heavy to light polysomes, (iii) inhibited cap-dependent translation more severely than translation dependent on the HCV or CSFV internal ribosome entry sites, which do not require eIF4G, and (iv) caused a dramatic loss of eIF4G and eIF2α from complexes sedimenting at ∼40 S. These data suggest a specific, direct, and functional interaction of eIF3e with eIF4G during the process of cap-dependent translation initiation, although they do not rule out participation of other eIF3 subunits.
This work was supported by National Institutes of Health Grants GM20818 (to R. E. R.) and GM22135 (to J. W. B. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
