Interaction of the sperm adhesive protein, bindin, with phospholipid vesicles. II. Bindin induces the fusion of mixed-phase vesicles that contain phosphatidylcholine and phosphatidylserine in vitro

摘要

Bindin from sea urchin sperm associates with gel-phase phospholipid bilayers (Glabe, C. G., 1985, J. Cell Biol., 100:794-799). Bindin also interacts with phospholipid vesicles containing both gel-phase and fluid-phase domains and thereby induces their aggregation. Association of bindin with vesicles containing gel-phase domains of dipalmitoylphosphatidylcholine (DPPC) and fluid-phase domains of brain phosphatidylserine (PS) was found to result in the fusion of the vesicles. After incubation with bindin, these mixed-phase vesicles were much larger as determined by gel filtration chromatography and electron microscopic observations of negatively stained samples. The average diameter of the vesicles after incubation was 190 +/- 109 nm compared with 39 +/- 20 nm for vesicles incubated in the absence of bindin. Resonance energy transfer studies also indicated that bindin induces the fusion of vesicle bilayers. Two fluorescent probes (NBD-PE and Rh- PE) were incorporated into the membrane of mixed-phase DPPC:PS vesicles at a density of 0.5 mol%, where efficient energy transfer occurs between the probes. The efficiency of energy transfer was proportional to the concentration of the fluorescence energy acceptor in the bilayer. The fluorescent vesicles were mixed with an excess of unlabeled target vesicles to quantify fusion. After bindin addition, there was a significant decrease in the efficiency of energy transfer compared with controls incubated in the absence of bindin. Although bindin induced the fusion of vesicles in the absence of calcium, the rate of fusion in the presence of 2 mM calcium was three-fourfold higher. In the presence of calcium, approximately half of the vesicles in the population had fused with another vesicle after incubation with bindin for 20 min. Bindin did not induce the fusion of gel-phase DPPC vesicles or mixed-phase vesicles of DPPC and dioleoylphosphatidylcholine, which suggests that the fusagenic activity of bindin requires specific phospholipids. Electron microscopic observations of DPPC:PS vesicles incubated in the presence of bindin suggest that the outer leaflets of bindin-aggregated vesicles are in close apposition. This is believed to be an important initial event for membrane fusion. These observations suggest that bindin may play a dual role in fertilization: Bindin mediates the attachment of sperm to glycoconjugate receptors of the egg surface and may also participate in the fusion of the sperm and egg plasma membranes.