Translation initiation factor eIF4G-1 binds to eIF3 through the eIF3e subunit.
eIF3 in mammals is the largest translation initiation factor ( approximately 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 eIF2alpha from complexes sedimenting at approximately 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.
LeFebvre
Aaron K
AK
Department of Biochemistry and Molecular Biology and Research Core Facility, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA.
Korneeva
Nadejda L
NL
Trutschl
Marjan
M
Cvek
Urska
U
Duzan
Roy D
RD
Bradley
Christopher A
CA
Hershey
John W B
JW
Rhoads
Robert E
RE
D000465:Algorithms; D000818:Animals; D002474:Cell-Free System; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry*; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D039621:Eukaryotic Initiation Factor-3; D039603:Eukaryotic Initiation Factor-4G; D011994:Recombinant Proteins