Reconstitution of yeast eIF3. (A) Comparison of reconstituted and native eIF3. Native eIF3 (3nat) copurifies with eIF5. Reconstituted eIF3 (3rec) comprises exclusively its five subunits. 3bcgi is a side product of the reconstitution of full eIF3 when Tif32 is added in lesser amounts compared with other subunits to promote the complex formation. All single recombinant subunits are shown on the gel to demonstrate the high degree of purity. (B) Size comparison between recombinant and native eIF3. Recombinant or native eIF3 were analyzed by size-exclusion chromatography (SEC). Recombinant eIF3 was prepared freshly by mixing five subunits in a nonstoichiometric ratio, therefore in addition to the peak for the full complex (between 9 and 10 mL), a second peak for Nip1-Prt1-Tif34-Tif35 complex (11–12 mL, corresponding to the third lane on A) and a third peak at ∼15 mL (corresponding to Tif34) were obtained. (C) Overlay of the SEC and MALS experiments for recombinant eIF3. The leftmost peak corresponds to recombinant eIF3. Molar mass distribution of the peak as calculated by MALS is indicated by a short line inside this peak and corresponds to a molecular weight of 340–354 kDa. The second peak corresponds to Nip1-Prt1-Tif34-Tif35 complex and corresponds to a molecular weight of 208–225 kDa. (D) Overlay of the SEC and MALS experiments for native eIF3. Molar mass distribution of the peak as calculated by MALS is indicated by a short line and corresponds to a molecular weight of 340–369 kDa. (E,F) Mass-spectrometry results for the eIF3rec (E) or eIF3nat (F). The upper panel is a zoom in the 10- to 120-kDa range of the spectrum for the control experiment without the cross-linker. The lower panel represents the whole spectrum for the cross-linked samples.
