Fig. 4: Sugar phosphate binding by the α subunit enhances eIF2B decamer formation.
a–d eIF2B complex assembly from WT and eIF2BαE198K HEK293T lysates treated with ISRIB (blue) or F6P (green) was monitored by sucrose gradient centrifugation. Fractions from the sucrose gradient were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. eIF3a was used as an internal control. Western blot data in a, c are quantified in b, d, respectively. Data shown are representative of 2–3 replicates of each experiment. Bands were normalized by the total intensity of each subunit in its respective gradient. Dashed red lines demark the boundary of the decameric eIF2B peak. WT eIF2B forms a decamer in the presence of both ISRIB and F6P. By contrast, eIF2BαE198K complexes respond to ISRIB but not F6P. e GDP release t1/2 in a GEF assay using lysates from WT or eIF2BαE198K cells. WT lysate activity is stimulated by both ISRIB and F6P, whereas eIF2BαE198K lysate does not respond to F6P. Bars are mean ± standard deviation of n = 3 independent experiments of 3 technical replicates each. Statistical significance was tested by one-way ANOVA with Tukey’s multiple testing correction.
