Fig. 5.

Stonin 2 directly binds to the α-adaptin ear and to the tyrosine motif-binding domain of μ2. (A) ³⁵S-labeled AP-2 subunits synthesized by coupled transcription/translation in vitro were incubated with GST-stonin 2NT or GST. Affinity-purified material was analyzed by SDS/PAGE and PhosphorImager analysis. (B) Affinity purification from rat brain by using the α-ear domain of AP-2 fused to GST in the presence or absence of the indicated peptides (500 μM). Samples were analyzed by immunoblotting for Eps15, stonin 2, auxilin, heat shock cognate protein 70, and CLCs. (C) Binding of purified μ2 (amino acids 157–435) to GST-WVxF or single-point mutants thereof. Samples were separated by SDS/PAGE and were analyzed by Coomassie blue staining. (D) Purified wild-type μ2 (amino acids 157–435) or a W421A single-point mutant was incubated with GST-WVSF peptide or GST alone. Binding of μ2 (amino acids 157–435) to an immobilized peptide harboring the tyrosine-based sorting signal of TGN38 (YQRL) was assayed as a control.