Apparent coordination of plasma membrane component synthesis in the lens.

PURPOSE This study explores the order of assembly of the lens fiber cell plasma membrane. Because the lens must synthesize most of its membrane components, our approach was to map directly the spatial distribution of cholesterol, fatty acid (reflecting phospholipid), and the main intrinsic protein, MP26, synthesis in the lens and, thereby, determine the extent to which membrane component synthesis was coordinated during fiber cell elongation. METHODS Young rat lenses were incubated with either tritiated water as the substrate for cholesterol and fatty acid synthesis or tritiated leucine as the substrate for MP26 synthesis. We developed a simple technique for uniformly dissolving the decapsulated lens into small fractions by incubating the lens with gentle stirring in sodium dodecyl sulfate-containing buffer. Based on the protein content of each fraction and available information on the radial distribution of protein in the young rat lens, each fraction was equated to a specific percentage of the lens radius. Cholesterol was precipitated from each fraction by digitonin; fatty acids were extracted and isolated by thin-layer chromatography. The MP26 was recovered both by immunoprecipitation from each fraction with anti-MP26 polyclonal antibody and from sodium dodecyl sulfate polyacrylamide gel electrophoresis gels of intact crude membrane, which was isolated from lens fractions by dissolving the lens in a urea containing-buffer. RESULTS The spatial distribution of incorporation of cholesterol, fatty acid, and MP26 was virtually superimposable, with essentially all the incorporation occurring in the outer 10% of the lens radius and peak incorporation occurring in approximately the outer 3-6% of the radius. CONCLUSIONS These results indicate that the synthesis of lens membrane components is highly coordinated and imply that the plasma membrane accumulates constant proportions of cholesterol, phospholipid, and MP26 throughout the course of fiber cell elongation.