Figure 7.
Inhibition of SNARE-mediated proteoliposome fusion by α-SNAP. Fusion was measured as an increase of NBD fluorescence by using a lipid dequenching assay. Donor liposomes were reconstituted with an N-terminally truncated version of syntaxin 1 (2 μM, residues 183–288) (A) or with a preformed complex of syntaxin 1 (same construct as before) and SNAP-25 (B). Acceptor liposomes contained 10 μM synaptobrevin 2. If syntaxin-containing liposomes were used as donor, the liposomes were combined and preincubated for 10 min at 30°C. Where indicated, solutions contained in addition α-SNAP, α-SNAPL294A, NSF, or ATPγS, and the reaction was started by addition of 10 μM soluble SNAP-25a. (t = 0, reference point for normalization of the signal). In B, donor liposomes contained a preformed syntaxin-SNAP-25 complex, and the reaction was started by mixing donor and acceptor liposomes.