Asymmetric and symmetric membrane reconstitution by detergent elimination. Studies with Semliki-Forest-virus spike glycoprotein and penicillinase from the membrane of Bacillus licheniformis.

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 three stages: lysis at a free equilibrium octyl glucoside 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 19 S and 6 S. The 6-S form probably corresponded to a glycoprotein monomer complexed to detergent and the 19-S form consisted of oligomeric detergent-protein complexes. The two 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) J. Cell Biol. 75, 866]. It was demonstrated that during reconstitution the symmetric vesicles were formed at 19 mM free equilibrium octyl glucoside by the association of the 6-S protein complexes with the phospholipids, and the asymmetric vesicles were formed at 10--16 mM octyl glucoside when the 19-S complexes associated with the lipids. Asymmetric membrane vesicles were also obtained when membrane penicillinase from Bacillus licheniformis was reconstituted with egg lecithin using octyl glucoside. It could be shown that the penicillinase was oligomeric at the octyl glycoside concentration where the reconstitution occurred. The results demonstrate that different mechanisms of reconstitution give rise to the symmetric and the 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.