FIGURE 5.

Binary complexes between SNAP-25 and syntaxin, or substitution of SNAP-25 with SNAP-23, are unable to dock liposomes. (A) Docking of liposomes containing N-terminally truncated syntaxin (syx_183-288) in comparison with liposomes containing synaptobrevin. SNAP-25 was either added in solution (left), or the coverslips were preincubated with SNAP-25 for 30 min, followed by washing. (B) Docking of synaptobrevin liposomes in the presence of full-length SNAP-25, the BoNT/A fragment of SNAP-25 (SN25_1-197), the BoNT/E-fragment of SNAP-25 (SN25_1-180), or SNAP-23. (For A and B, n = 2 and bars indicate range of values.) (C) Formation of ternary SNARE complexes (TC) between SNAP-25 and SNAP23 (SN) in solution, monitored by the appearance of high-molecular-weight bands in SDS-PAGE, indicating SDS-resistance typical of SNARE complexes. (D) Fusion of liposomes containing synaptobrevin and a truncated variant of syntaxin 1 (syx_183-288) mediated by SNAP-25 or SNAP-23. Fusion was monitored using a standard dequenching assay (see Materials and Methods). All binding experiments were normalized to the binding of synaptobrevin-containing liposomes in the presence of SNAP-25 under standard assay conditions.)