Figure 4.

The Egl-binding site on BicD is highly similar to, or the same as, the binding site for Rab6^GTP. (A,B) Representative data (top) and quantification (bottom) of truncation (A) and mutational (B) analysis of the features of BicD important for binding Egl. Egl^1–79 was translated in vitro in the presence of ³⁵S-methionine and incubated with His-tagged BicD proteins immobilized on a nickel affinity matrix. Mean values for Egl^1–79 binding (generated following correction for variation in BicD signal in each sample and normalized to values for Egl^1–79 binding to wild-type [WT] His∷BicD^656–745) derive from three experiments for each condition, except for wild type and L731A (six each). Error bars are SEM. (C) Immunoblots from a representative pull-down assay showing that the binding of His∷BicD^656–745 to recombinant GST∷Egl^1–79 immobilized on a glutathione sepharose matrix is reduced by increasing concentrations of Rab6^GTP. BicD^656–745 does not bind to GST alone. Rab6^GDP does not compete for Egl:BicD binding even at an eightfold molar excess to BicD (Supplemental Fig. S7C). Proteins were visualized with antibodies against His or GST.