Mass spectrometry reveals modularity and a complete subunit interaction map of the eukaryotic translation factor eIF3

Abstract

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.

Fig. 1.

MS of the intact eIF3 revealing a series of well resolved charge states consistent with the predominant species being the intact 13 subunit complex. Masses of the 13 subunits were confirmed in a separate proteomics analysis. The y axis is the relative intensity of the peaks. The inventory of subunits is shown with the radius of each subunit scaled according to mass. (Inset) High-energy MS spectrum of eIF3 from 150 mM AmAc solution, showing a second charge state series (blue star) resulting from the dissociation of eIF3j from the intact complex (purple star).

Fig. 2.

Mass spectra recorded after increasing the ionic strength from 250 mM AmAc (a) to 350 mM (b) and 500 mM (c). Series of subcomplexes, observed with decreasing m/z values as the ionic strength is increased, are assigned based on their masses, the observation of common subunit losses, and from tandem MS.

Fig. 3.

MS spectrum of the yeast eIF3 isolated by tagging subunit eIF3b. Charge state series are assigned on the basis of masses to subcomplexes eIF3i:g, eIF3b:g:i, and eIF3a:b. eIF3i is observed dissociating from the yeast complex at ≈m/z 3,000. (Inset) The interaction network for yeast eIF3 derived from seven subcomplexes observed by MS.

Fig. 4.

Model of the human eIF3 derived from 27 subcomplexes, IP data, and interactions identified in the yeast complex. The complex dissociates into distinct modules in response to changes in ionic strength; in this case, the spectrum shown was recorded at intermediate ionic strength (350 mM AmAc). Arrows denote additional interactions not readily represented in this model.

Fig. 5.

Proposed model for the eIF3:HCV IRES interaction. (a) Subunit organization colored according to signature domains contained within the various subunits. PCI-containing domains (green), MPN domains (red), and RNA recognition motifs (yellow) show direct interactions with the exception of eIF3m and eIF3a. Subunits with no common signature domains are shown in gray. Subunits that are affected by binding of HCV IRES are within the dashed line. The location of subunits satisfies the interaction network and is not indicative of their location within the EM density. (b) EM model of eIF3-IRES-40S complex showing the binding of IRES (yellow) to both eIF3 (green) and the 40S (blue).