Reconstitution of Synaptic SNAREs into Large Liposomes with Reduced Curvature Stress

Abstract

Liposomes constitute a convenient biochemical model system to investigate mechanistic aspects of the membrane fusion of synaptic vesicles. The proteins responsible for mediating fusion are the SNAREs that belong to a highly conserved family of transmembrane proteins. Reconstituting SNAREs into liposomes using detergents has become a common approach not only to understand how SNAREs work, but also how fusion is regulated by the vast array of accessory proteins present at the presynapse. However, a concern has been that the high curvature stress of the small liposomes (diameters of ~40 nm) frequently used in many studies renders them prone to spontaneous fusion, bringing into question whether the measurements obtained faithfully represent SNARE-mediated fusion. By systematically varying the detergent concentration and characterizing the SNARE-liposome size distributions by light scattering, we describe a detailed procedure to reconstitute SNAREs into large liposomes with considerably reduced curvature stress.