Translation Initiation Factor eIF4G-1 Binds to eIF3 through the eIF3e Subunit

Abstract

AbstracteIF3 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 capdependent 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.Eukaryotic translation initiation involves numerous initiation factors (eIFs)2 that participate in recruitment of initiator tRNA and mRNA to the 40 S ribosomal subunit, recognition of the initiator AUG codon, and joining of the 40 S and 60 S ribosomal subunits, culminating in formation of the first peptide bond (1). The factors required for recruitment of mRNA include eIF3, eIF4A, eIF4B, eIF4E, eIF4G, eIF4H, and PABP. eIF4E and PABP bind the 5′ cap and 3′ poly(A) tract of mRNA, respectively, whereas eIF4A unwinds 5′-terminal secondary structure in an ATP-dependent process that also involves the RNA-binding proteins eIF4B and eIF4H. eIF4G forms specific complexes with eIF4E, eIF4A, and PABP, thereby linking the processes of cap recognition, poly(A) binding, and secondary structure melting. eIF4G in turn is recruited to the 40 S ribosomal subunit via binding to the multisubunit complex eIF3.* This work was supported by National Institutes of Health Grants GM20818 (to R. E. R.) and GM22135 (to J. W. B. H.).1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932. Tel.: 318-675-5161; Fax: 318-675-5180; E-mail: rrhoad@lsuhsc.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ...

Int6/eIF3e Promotes General Translation and Atf1 Abundance to Modulate Sty1 MAPK-dependent Stress Response in Fission Yeast

“…7, C and D) indicate that Int6/eIF3e is a general translation factor. This idea agrees with recent findings that its mammalian homologue, Int-6/eIF3e, is one of the six subunits constituting the functional core of eIF3 (5) and interacts with eIF4G to mediate mRNA binding to the 40 S subunit (40).…”

The Role of the Poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants

“…16,19,25 Although eIF4A binds the HEAT-1 and HEAT-2 domains of plant eIFiso4G and eIF4G, they differ in that the eIFiso4G HEAT-1 domain is sufficient to bind eIF4A whereas a short region immediately C-proximal to the HEAT-1 domain of eIF4G is needed in addition to the HEAT-1 domain, 12,13 consistent with observations made with animal eIF4G in which the HEAT-1 domain alone bound eIF4A with lower affinity than did a longer region of eIF4G. 26 In animal eIF4G, eIF3 binds to a 90 amino acid region just Cproximal to the HEAT-1 domain through its c, d, and e subunits 27,28 which was reported to bind in a cooperative manner with eIF4A 29 although this was not confirmed in a subsequent study. 28 Whether eIF3 binds to the corresponding region in plant eIF4G or eIFiso4G remains to be determined.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…The 43S PIC is directed to bind an mRNA at the 59 end. In mammalian cells, eIF4G binds the 43S complex via contacts with the c, d, and e subunits of eIF3 (Korneeva et al 2000;LeFebvre et al 2006;Villa et al 2013). Neither eIF3d, eIF3e, nor the eIF4G domain used by mammals is conserved in yeast.…”