Internalization of Interphotoreceptor Retinoid-Binding Protein by the Xenopus Retinal Pigment Epithelium LISA L. CUNNINGHAM 1 AND FEDERICO GONZALEZ-FERNANDEZ 2 * 1 Virginia Merrill Bloedel Hearing Research Center and Department of Otolaryngology- HNS, University of Washington, Seattle, Washington 98195 2 Departments of Ophthalmology, Pathology, and Biochemistry, State University of New York and Medical Research Service, Veterans Affairs Medical Center, Buffalo, New York 14215 ABSTRACT Xenopus rods and cones secrete into the interphotoreceptor matrix (IPM) a 124-kDa glycoprotein termed interphotoreceptor retinoid-binding protein (IRBP; Hessler et al. [1996] J. Comp. Neurol. 367:329 –341). IRBP is confined to the IPM, being too large to diffuse through the zonulae adherentes between adjacent photoreceptor and Mu ¨ ller cells. Despite this physical entrapment within the subretinal space, IRBP is rapidly cleared from the IPM by an unknown mechanism. Immunohistochemistry and immunoelectron microscopy were used to localize IRBP in intact and detached retina-retinal pigment epithelium (RPE) eye- cups. The effects of light, dark, and time of day on the compartmentalization of IRBP were characterized by quantitative Western blot analysis and by immunoprecipitation of IRBP labeled in vivo by intraocular injection of [ 35 S]methionine. Immunohistochemistry showed that the apparent intercellular IRBP in both the RPE and the photoreceptors is resistant to saline extraction, in contrast to that in the IPM. In the RPE, IRBP was associated with matrix material within phagosomes and endosomes. The IPM, RPE, and retina contained 75%, 18%, and 7% of the total IRBP in the eye, respectively. The IPM and RPE contain 130  14 pmoles and 34  4 pmoles of IRBP, respectively. The amounts of IRBP in the RPE at middark and midlight were the same. Furthermore, the in vivo uptake of [ 35 S]methionine- labeled IRBP was light independent. Our studies suggest that IRBP is not strictly confined to the subretinal space but rather that significant amounts are present intracellularly, particularly within the RPE, which does not synthesize IRBP. Furthermore, IRBP secreted by the photoreceptors is taken up from the IPM mainly through a light-independent endocytic pathway separate from outer segment phagocytosis. The role of RPE endocytosis should be explored in relation to the function of IRBP. J. Comp. Neurol. 466:331–342, 2003. © 2003 Wiley-Liss, Inc. Indexing terms: interphotoreceptor retinoid-binding protein; interphotoreceptor matrix; protein turnover; retina; retinoid-binding proteins; vitamin A It is generally thought that interphotoreceptor retinoid- binding protein (IRBP) is restricted to the interphotore- ceptor matrix (IPM), the extracellular material that fills the subretinal space separating the neural retina from the retinal pigment epithelium (RPE; Adler and Martin, 1982; Liou et al., 1982; Pfeffer et al., 1983; Saari et al., 1984). In most, but not all, vertebrates (Rajendran et al., 1996), IRBP is secreted into the IPM only by the photoreceptors (Hollyfield et al., 1985b). For example, in Xenopus, mRNA in situ hybridization studies have shown that IRBP is synthesized uniquely by the rods and cones, but not the RPE or Mu ¨ ller cells, the other two cell types bordering the subretinal compartment (Hessler et al., 1996). Grant sponsor: National Institutes of Health; Grant number: EY09412 (F.G.-F.); Grant sponsor: Wyeth-Ayerst Laboratories Scholarship through the Business and Professional Women’s Foundation (L.L.C.). *Correspondence to: Federico Gonzalez-Fernandez, Medical Research Service, Veterans Affairs Medical Center, Buffalo, NY 14215. E-mail: fg23@buffalo.edu Received 6 March 2003; Revised 2 April 2003; Accepted 5 June 2003 DOI 10.1002/cne.10861 Published online the week of September 29, 2003 in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 466:331–342 (2003) © 2003 WILEY-LISS, INC.