Clathrin-induced pH-dependent fusion of phosphatidylcholine vesicles.

Abstract

Interaction of clathrin coat protein with dioleoyl-phosphatidylcholine (DOPC) vesicles at pH 6.5 and below results in the formation of stable vesicle-clathrin complexes (Steer, C. J., Klausner, R. D., and Blumenthal, R. (1982) J. Biol. Chem. 257, 8533-8540). In this report we show by gel chromatography and sedimentation analysis that the interaction of clathrin coat protein with unilamellar dioleoyl phosphatidylcholine vesicles at pH = 6.0 results in the formation of larger structures. As shown by electron microscopy and an increase in trapped volume of both sucrose and inulin those larger structures represent fused bilayers. We examined the mixing of membrane lipid as a result of membrane fusion using resonance energy transfer between two fluorescent lipid probes incorporated into the same vesicle membrane. At a protein:lipid ratio of 1:500 there was 50% vesicle-vesicle fusion, at pH 6.0, as indicated by the change in efficiency of energy transfer between the fluorescent probes. Fusion was completed within 60 s. A number of other proteins (ovalbumin, rabbit IgG, trypsin, pronase, calmodulin, tubulin, synexin, bovine serum albumin) at 10-fold or higher concentrations, did not induce fusion of dioleoyl phosphatidylcholine vesicles, either at pH 7.4 or at pH 6.0. This system provides a model for pH-dependent and protein-mediated fusion of uncharged lipid bilayers.