ELECTROINSERTION OF GLYCOPHORIN-A IN INTERDIGITATION-FUSION GIANT UNILAMELLAR LIPID VESICLES

Previously we demonstrated that transmembrane back insertion of glycop horin A, a solubilizable intrinsic protein, can be obtained in dipalmi toylphosphatidylcholine multilamellar vesicles, MLVs, by electropulsat ion (Raffy, S., and Teissie, J. (1995) Eur. J. Biochem. 230, 722-732). Here we report that transmembrane back insertion of protein is obtain ed by electropulsion of unilamellar giant vesicles, termed interdigita tion-fusion vesicles (IFVs), which are better membrane models than MLV s due to their unilamellarity. Electropulsation promotes a field-depen dent local permeabilization of the lipid layer, as shown by the associ ated leakage of entrapped calcein, Glycophorin insertion is assayed by immunofluorescence. Electroinsertion is obtained by pulsing the vesic le/protein mixture. Glycophorin insertion is observed under more drast ic electrical conditions than needed for permeabilization, Direct obse rvation of glycophorin insertion in the vesicles under a microscope sh ows 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. Th is suggests that an electrophoretic movement of the solubilized glycop horin could take place during electropulsation, Insertion of glycophor in, a prefolded intrinsic protein, is then obtained in the lipid bilay er brought transiently to the electropermeabilized state.