Electroinsertion of glycophorin A in interdigitation-fusion giant unilamellar lipid vesicles.
Previously we demonstrated that transmembrane back insertion of glycophorin A, a solubilizable intrinsic protein, can be obtained in dipalmitoylphosphatidylcholine multilamellar vesicles, MLVs, by electropulsation (Raffy, S., and Teissié, J. (1995) Eur. J. Biochem. 230, 722-732). Here we report that transmembrane back insertion of protein is obtained by electropulsion of unilamellar giant vesicles, termed interdigitation-fusion vesicles (IFVs), which are better membrane models than MLVs due to their unilamellarity. Electropulsation promotes a field-dependent local permeabilization of the lipid layer, as shown by the associated leakage of entrapped calcein. Glycophorin insertion is assayed by immunofluorescence. Electroinsertion is obtained by pulsing the vesicle/protein mixture. Glycophorin insertion is observed under more drastic electrical conditions than needed for permeabilization. Direct observation of glycophorin insertion in the vesicles under a microscope shows a localized process in agreement with the theoretical prediction. A quantitative evaluation of the immunofluorescence pattern shows that insertion was higher on one side of the vesicle than on the other. This suggests that an electrophoretic movement of the solubilized glycophorin could take place during electropulsation. Insertion of glycophorin, a prefolded intrinsic protein, is then obtained in the lipid bilayer brought transiently to the electropermeabilized state.
Raffy
S
S
Institut de Pharmacologie et de Biologie Structurale du CNRS, 118 rte de Narbonne, F-31062 Toulouse Cedex, France.
Teissié
J
J
D015060:1,2-Dipalmitoylphosphatidylcholine; D000817:Anilino Naphthalenesulfonates; D018274:Electroporation; D005456:Fluorescent Dyes; D006021:Glycophorins / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D010539:Permeability
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006021:Glycophorins; D008051:Lipid Bilayers; D015060:1,2-Dipalmitoylphosphatidylcholine; C027132:1-anilino-8-naphthalenesulfonate