Preparation of PM-mimic Acceptor Vesicles

PM-mimic acceptor vesicles with reconstituted syntaxin-1A and SNAP-25A were generated as described previously (Diao et al., 2013 (link); Kyoung et al., 2013 (link); Lai et al., 2017 (link), Leitz et al., 2024a , Leitz et al., 2024b ). Briefly, a lipid film of Porcine Total Brain Extract (Avanti Polar Lipids, 131101C), 3% PIP2 (Avanti Polar Lipids, 840046X), 1% Biotin PE (Avanti Polar Lipids, 870285P) and 1% DAG (Avanti Polar Lipids, 800811C) was generated by combining the lipids followed by evaporation under argon. Lipid films were allowed to dry overnight under a vacuum. PM vesicles were reconstituted with syntaxin-1A and SNAP-25A in a VB containing 50 mM of sulforhodamine B (Thermo Fisher Scientific). The protein-lipid mixture was then added to a 5-mL CL-4B (Millipore Sigma GE17-01501-01) column. 200 μL fractions were collected and analyzed on an SDS PAGE gel with Coomassie stain to assess which fractions had visible bands for both syntaxin-1A and SNAP25. Select fractions were pooled and dialyzed overnight at 4 °C against 2 L of VB with Bio-Beads SM-2 resin (BioRad, 1523920). Vesicle size and homogeneity were determined via dynamic light scattering (DLS) using a DynaPro NanoStar (Wyatt Technologies) (Supplementary Fig. 3).