Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3.
The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us to establish unambiguously its stoichiometry and its overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Dissociation takes place as a function of ionic strength to form three stable modules eIF3(c:d:e:l:k), eIF3(f:h:m), and eIF3(a:b:i:g). These modules are linked by interactions between subunits eIF3b:c and eIF3c:h. We confirmed our interaction map with the homologous yeast eIF3 complex that contains the five core subunits found in the human eIF3 and supplemented our data with results from immunoprecipitation. These results, together with the 27 subcomplexes identified with increasing ionic strength, enable us to define a comprehensive interaction map for this 800-kDa species. Our interaction map allows comparison of free eIF3 with that bound to the hepatitis C virus internal ribosome entry site (HCV-IRES) RNA. We also compare our eIF3 interaction map with related complexes, containing evolutionarily conserved protein domains, and reveal the location of subunits containing RNA recognition motifs proximal to the decoding center of the 40S subunit of the ribosome.
Zhou
Min
M
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Sandercock
Alan M
AM
Fraser
Christopher S
CS
Ridlova
Gabriela
G
Stephens
Elaine
E
Schenauer
Matthew R
MR
Yokoi-Fong
Theresa
T
Barsky
Daniel
D
Leary
Julie A
JA
Hershey
John W
JW
Doudna
Jennifer A
JA
Robinson
Carol V
CV
D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D053719:Tandem Mass Spectrometry / Q000379:methods*
D039621:Eukaryotic Initiation Factor-3