Rod outer segment disc membranes are capable of fusion

The retinal rod outer segment (ROS) is maintained at a constant length through the formation of new discs and the phagocytosis of old discs by the pigment epithelium. The ROS is composed of approximately 50 mol% of phosphatidylethanolamine (PE), an unusually high PE content for biologic membranes. Because this lipid is highly fusogenic, due to its low head group hydration, the ability of ROS disc membranes to fuse with PE large unilamellar vesicles (LUV) was examined. The initial rates of fusion of discs with LUV were measured by following the relief of self-quenching of octadecylrhodamine B chloride (R18)-labeled disc membranes. Fusion was initiated by reducing the pH of the mixture to 7.2. The initial rates of fusion of disc membranes with transphosphatidylated PE (trans-PE LUV) were measured as a function of temperature. The ROS discs fused readily with these LUV. Fusion was confirmed by density-gradient centrifugation. The initial rates of fusion increased with increasing temperature. Subsequently, the initial rates of fusion between disc membranes and disc lipid vesicles were examined using the R18 mixing assay. Fusion of the two membranes was confirmed by sucrose density-gradient centrifugation. Calcium and EGTA had no significant effect on disc membrane-trans-PE LUV fusion or on disc membrane-disc lipid vesicle membrane fusion. Papain proteolysis of the disc membranes enhanced initial rates of fusion between disc membrane and PE LUV but inhibited disc membrane-disc lipid vesicle fusion.