Figure 6.

A-SNAP binds to the SNARE-motif of synatxin 1. (A) A-SNAP binding requires syntaxin 1 but not SNAP-25 or synaptobrevin. Membrane sheets were incubated for 5 min with 2 μM of either the wt- or mutant form of α-SNAP. Where indicated, solutions contained in addition 2 μM purified light chains of either BoNT/C1 cleaving syntaxin 1, BoNT/C1mut (inactive form of BoNT/C1 carrying the mutation E230A), BoNT/E cleaving SNAP-25, or TeNT cleaving synaptobrevin. After brief washing, membrane sheets were processed for immunostaining and analyzed as shown in Figure 3, C and D). Values are given as mean ± SEM (n = 3–4 independent experiments, with 70–120 individual [mean = 106] membrane sheets analyzed for each experiment). (B) Antibodies directed against the SNARE-motif of syntaxin inhibit binding of α-SNAP. Membrane sheets were incubated for 15 min with anti-syntaxin 1 antibodies, washed twice with PBS and followed by 5-min incubation with 2 μM recombinant wild-type α-SNAP. The sheets were then washed, fixed, and immunolabeled for α-SNAP. The antibodies used for preincubation were R31 (polyclonal rabbit antiserum recognizing both the N-terminal domain and the SNARE motif) and HPC1 and Cl 78.3 (independently raised monoclonal antibodies specific for the N-terminal Habc-domain). For the detection of α-SNAP, we used either a monoclonal (Cl 77.2, left) or a polyclonal rabbit antibody (R34, right). In all experiments, fluorescence values were normalized to the immunoreactivity of membrane-bound, recombinant α-SNAP without prior anti-syntaxin 1 antibody treatment. Values are given as mean ± SEM (n = 6–7 independent experiments, with a minimum of 10–144 individual membrane sheets analyzed for each experiment).