Novel insights into the architecture and protein interaction network of yeast eIF3

Activity tests for recombinant eIF3. (A) Analysis of the binding of eIF3 to the 40S ribosomal subunit by native PAGE and Western blot. A band shift toward higher molecular weights upon mixing 40S subunit and eIF3^rec indicates the binding of eIF3 and 40S ribosome. (B) Luciferase assay performed with the cytoplasmic extracts of temperature-sensitive mutant cells treated under permissive (26°C) or nonpermissive (37°C) temperatures. To the cytoplasmic extracts, either recombinant (3rec) or native (3nat) eIF3 was added. In control experiments, only buffer was added. (C) SDS-PAGE analysis of cosedimentation experiment between eIF3^rec and 40S. Mixtures were subjected to ultracentrifugation, and the total content of pellets (P) and supernatants (S) were loaded onto the gel. Under the conditions of this experiment, the majority of 40S subunits are found in the pellet, while all eIF3^rec complexes remain in the supernatant. Upon mixing eIF3^rec (0.1 μM) with 40S (1 μM), the factor was mainly found in the pellet, indicating binding to the 40S subunit. Addition of Hcr1 (1 μM) enhanced the binding of eIF3^rec to the 40S. The leftmost and rightmost lanes show eIF3, which was used in this study and the marker, respectively.