Liposome and Nanotube reactions
Liposomes were made as described before52. Briefly, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was purchased from Avanti Polar Lipids. DOPS dissolved in chloroform was dried under a steady stream of nitrogen and dried under vacuum for an hour. The dried lipid film was resuspended in n-hexane and dried again under nitrogen. The resulting lipid film was dried under vacuum for 4 hours and was finally resuspended in 20 mM HEPES pH 7.5 and 150 mM KCl. The same protocol was followed for making nanotubes, where the mixture contained 60% D-galactosyl-ß-1,1′ N-nervonoyl-D-erythro-sphingosine (Galactosyl Ceramide), 30% DOPS and 10% Ni2+-NTA DOGS (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] Nickel Salt).
For assembly reactions of DRP1G362D over lipid, 0.5-2μM protein was incubated with liposomes or nanotubes for an hour and dialyzed against the assembly buffer without detergent.