Fig. 5. Synaptotagmin is important for efficient binding of AP-2 subunits to synaptic membranes. (A) µ2- and α-adaptin are the major membrane-binding subunits of AP-2. Carbonate-washed LP2 membranes (0.1 mg/ml) were incubated with ³⁵S-labeled in vitro translated α-, β2-, µ2- or σ2-adaptins for 10 min at 37°C. Membranes were re-isolated through a sucrose cushion, washed and resuspendedin sample buffer. Samples were analyzed by SDS–PAGE and autoradiography. 50% STD = half of the total amount of radiolabeled protein added to the assay. (B) Synprint inhibits the recruitment of both µ2- and α-adaptin to synaptic LP2 membranes. Carbonate-washed LP2 membranes (40 µg/ml) were incubated with ³⁵S-labeled in vitro translated µ2- or α-adaptin in the presence or absence of 0.3 mg/ml synprint and processed as described in (A). 25% STD = 1/4 of the total amount of radiolabeled protein added to the assay. (C) A double point mutation within subdomain B of µ2 reduces the interaction with synaptic LP2 membranes. Carbonate-washed LP2 membranes(40 µg/ml) were incubated with ³⁵S-labeled in vitro translated wild-type or mutant (Y344A, K354A) µ2-adaptin and analyzed by SDS–PAGE, staining with Coomassie Blue and autoradiography. 50% STD = half of the total amount of radiolabeled protein added to the assay.