FIGURE 6.

Evaluation of the impact of the newly identified WENDF motif on the Gadkin/AP-1γ-ear interaction in vitro. A, binding of purified His₆-tagged Gadkin WT (Δ51) and its indicated mutants to GST-γ-ear. Immobilized GST fusion proteins (25 μg) were incubated with 25 μg of recombinant His₆-tagged Gadkin (Δ51) and washed extensively. One-tenth of each sample was analyzed by SDS-PAGE followed by Coomassie Blue staining. 40% (1 μg) of His₆-tagged Gadkin (Δ51) was loaded onto a gel as standard. B, association of AP-1γ-ear with Gadkin AP-1-binding motifs mutants. 25 μg of GST-γ-ear or GST were incubated with 0.5 mg (2 μg/μl) of Triton X-100-solubilized lysate of HEK293 cells expressing Gadkin-eGFP WT or the indicated mutants. Samples were analyzed by SDS-PAGE and immunoblotting for eGFP and actin. C, GST-Gadkin (Δ51) or GST-NGLEWENDFVSAE was assayed in pulldown experiments for their ability to associate with AP-1γ from rat brain extract. Samples were analyzed by immunoblotting for AP-1γ or actin. 40 μg of the input material was loaded as the standard. D, results of an EXPASY Prosite search for proteins containing putative AP-1γ-binding motifs (WY)XX(DE)(WF): Sec6 (O60645 in SWISSPROT), Munc13-1 (Q9UPW8), Sorting nexin-18 (Q96RF0), and EpsinR (Q14677).