Abstract
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.
Highlights
Spontaneous insertion of integral membrane proteins across lipid bilayers is unlikely from a thermodynamic point of view [1]
Permeabilization was shown to be a very transient phenomenon in pure lipid systems, while it is long lived in cells [19]. 31P NMR spectroscopy showed that the polar heads of phospholipid bilayers were transiently tilted during the electric pulse with very fast relaxation [20]
Localized perturbation of the polar head group region of phospholipids, which supports the transient permeabilization of lipid bilayers, allows spontaneous trans-insertion of glycophorin across the lipid bilayers in the case of multilamellar vesicles (MLVs)1 [8]
Summary
Chemicals—DPPC (reference P5911), calcein (reference C1075), and glycophorin A (reference G5017) were purchased from Sigma. 1 ml of 150 mM NaCl, 10 mM Tris-HCl (pH 7.4) was added, and the hydrated lipid samples were incubated for 10 min in buffer at 55 °C, i.e. above the phospholipid Tm (41 Ϯ 1 °C) to ensure formation of intact vesicles. The samples were bubbled above the Tm by a gentle stream of N2 to remove ethanol They were washed three times with 50 mM NaCl, 10 mM Tris-HCl (pH 7.4) at room temperature by 15-min centrifugations at 12,000 ϫ g using a Sorvall centrifuge equipped with a Sorvall SS34 rotor. After the pulses, the vesicles were washed three times with Hepes buffer (pH 7.4) by centrifugation at 12,000 ϫ g for 5 min at room temperature. This methodology gives access to a true quantitative, highly sensitive observation of electroinsertion
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