Asymmetric and Symmetric Membrane Reconstitution by Detergent Elimination

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Abstract
The dissociation and reconstitution of the Semliki Forest virus membrane using the nonionic detergent octyl .beta.-D-glucoside was studied by sucrose density gradient centrifugation. The dissociation occurred in 3 stages: lysis at a free equilibrium octyl glucose concentration of 14-18 mM, solubilization at 18-20 mM and delipidation of the spike glycoproteins at the critical micellar concentration (22 mM) or higher. After solubilization the spike glycoproteins were present as soluble complexes with sedimentation coefficients of 19S and 6S. The 6S form probably corresponded to a glycoprotein monomer complexed to detergent, and the 19S form consisted of oligomeric detergent-protein complexes. The 2 forms were in slow equilibrium with each other. When the soluble spike protein complexes and egg lecithin solubilized with octyl glucoside were mixed and the octyl glucoside concentration lowered either by dialysis or by dilution, reconstitution occurred. Three types of products were obtained: vesicles with 30% of the spike protein facing inwards and 70% facing outwards, vesicles with virtually all (95%) of the spike proteins pointing outwards and small protein-rich soluble aggregates (Helenius et al., 1977). During reconstitution the symmetric vesicles were formed at 19 mM free equilibrium octyl glucoside by the association of the 6S protein complexes with the phospholipids, and the asymmetric vesicles were formed at 10-16 mM octyl glucoside when the 19S complexes associated with the lipids. Asymmetric membrane vesicles were also obtained when membrane penicillinase (EC 3.5.2.6) from B. licheniformis was reconstituted with egg lecithin using octyl glucoside. The penicillinase was oligomeric at the octyl glucoside concentration where the reconstitution occurred. Different mechanisms of reconstitution give rise to symmetric and asymmetric vesicles. The critical factor in determining the mechanism is the state of aggregation of the proteins at the octyl glucoside concentration where membranes begin to form from the solubilized lipids.