Reconstitution of SNARE proteins

Membrane scaffold protein (MSP) for 13 nm^{9 (link)} and 50 nm^{28 (link)} NDs, the maltose sensor^{29 (link)}, neuronal (rat syb2, syntaxin-1A and SNAP-25B) and yeast (Snc2p, Sso1p and Sec9c (residues 401-651)) SNAREs, were purified as described previously^{12 (link)}. T-SNARE complexes bearing truncated SNAP-25B (corresponding to residues 1-197 and residues 1-186) were also prepared and studied; the former truncation mimics cleavage by botulinum neurotoxin A^{30 (link)}. To prepare t-SNARE vesicles, lipids (10% PE, 15% PS and 75% PC) and the t-SNARE heterodimer were incubated with the respective cargoes and 2% OG on ice for 30 min. Detergent was removed by addition of Biobeads (Bio-Rad) (1/3 volume) followed by gentle shaking (4°C, overnight). The mixture was extruded through 0.2 μM filter and the t-SNARE vesicles were purified by passing through a PD10 column (5 ml) equilibrated in reconstitution buffer (25 mM HEPES, pH 7.5, 100 mM KCl, 1 mM DTT). Finally, purified t-SNARE vesicles were dialyzed against reconstitution buffer (4°C, overnight). Reconstitution of syb2 into 13 nm NDs was performed as described^{9 (link)}. For reconstitution of syb2 into 50 nm NDs, the MSP/lipid ratio was 2:4000. To incorporate different copy numbers of syb2 into 50 nm NDs, the following MSP/syb2 ratios were used: 2:2 (ND3), 2:4 (ND5) and 2:10 (ND7). The reconstituted NDs were incubated with Ni²⁺-NTA resin to remove syb2-free NDs. NDs containing syb2 were eluted by reconstitution buffer with 0.4M imidazole. The NDs were further purified via sucrose density gradient centrifugation^{31 (link)}, followed by dialysis against reconstitution buffer (4°C, overnight). The copy number of syb2 per ND refers to the total number of syb2 molecules, not the number of copies per face of the ND.