PURPOSE. Retinoschisin (RS) is a retina-specific, secreted protein implicated in X-linked juvenile retinoschisis and essential for the structural and functional integrity of the retina. This biochemical characterization and ultrastructural localization of RS in intact murine retina was performed to further understanding of the molecular basis of its function. METHODS. Subcellular fractions and fractions enriched in photoreceptor inner and outer segments were prepared from mouse retina by differential or density gradient ultracentrifugation. Immunoblot analysis was used to assess the expression of RS in various subcellular compartments and its fractionation into soluble phase on treatment of retinal cell membranes with several solubilizing reagents. RS-lipid interactions were evaluated by a protein-lipid overlay assay that used wild-type and mutant forms of RS discoidin domain glutathione S-transferase (GST) fusion proteins. The subcellular localization of RS in mouse retina was visualized by pre-embedding immunogold electron microscopy. Ultrastructure was evaluated by transmission electron microscopy. RESULTS. RS was intimately associated with cell membranes of the retina. It was found to cluster on the outer leaflet of the plasma membrane of the photoreceptor inner segments, which synthesize and secrete it. It was released from the membrane at high pH, which is characteristic of a peripheral membrane protein. It was extracted from the membrane by the nonionic detergent NP-40, together with glycerophospholipids. Protein-lipid overlay assays indicated a preferential interaction between RS and anioic phospholipids. Extraction of RS from the membrane was inhibited by divalent cations. Photoreceptor inner segment morphology was markedly affected in RS-/y mice, which failed to express RS protein. CONCLUSIONS. RS in intact retina is a peripheral membrane protein. Although distributed over the two membrane faces, RS is associated primarily with the outer leaflet of the inner segment plasma membrane through anionic phospholipids and divalent cations. RS's localization in photoreceptors and its biochemical properties suggest a functional role locally, at the site of secretion and membrane adhesion, in maintaining the photoreceptor inner segment stability and architecture.
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience