Letter to the Editors Support for a proposed retinoid-processing protein complex in apical retinal pigment epithelium Vera L. Bonilha a, * , Sanjoy K. Bhattacharya a , Karen A. West a , John S. Crabb a , Jian Sun a , Mary E. Rayborn a , Maria Nawrot b , John C. Saari b , John W. Crabb a, * a Cole Eye Institute (i31) Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA b Departments of Ophthalmology and Biochemistry, University of Washington, Seattle, WA 98195, USA Received 31 March 2004; accepted in revised form 22 April 2004 Available online 5 June 2004 Abstract The interaction of cellular retinaldehyde-binding protein (CRALBP) with ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50) in retinal pigment epithelium (RPE) microsomes has led to the hypothesis that a retinoid-processing protein complex exists in apical RPE. Mouse RPE apical processes were isolated on wheat germ agglutinin-coated agarose beads. Proteomic analyses of the isolated apical RPE demonstrated the presence of CRALBP, EBP50, 11-cis-retinol dehydrogenase, cellular retinol-binding protein 1, and interphotoreceptor retinoid-binding protein. The results support the hypothesis that a visual cycle protein complex may serve in the localization and release of 11-cis-retinoid in the apical RPE. q 2004 Elsevier Ltd. All rights reserved. Keywords: retinoids; proteomics; retinal pigment epithelium; visual cycle A hypothesis for a retinoid-processing complex in retinal pigment epithelium (RPE) was recently proposed that may provide a mechanism for localizing 11-cis-retinal to the apical RPE plasma membrane for export for visual pigment regeneration (Nawrot et al., 2004). Direct inter- actions were demonstrated between cellular retinaldehyde- binding protein (CRALBP) and ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 and both CRALBP and EBP50 were shown by immunocytochemistry to be distributed throughout the apical processes of RPE and Mu ¨ller cells. CRALBP endogenously carries 11-cis-retinoid ligands and functions in the RPE as an acceptor of 11-cis- retinol in the isomerization step of the rod visual cycle (Saari et al., 2001) and as a substrate carrier for 11-cis- retinol dehydrogenase (RDH5) (Saari et al., 1994; Golov- leva et al., 2003). EBP50 is a PDZ domain protein (also known as sodium/hydrogen exchanger regulatory factor type 1 or NHERF-1) that interacts with ERM family members in multifunctional roles and, in non-visual tissues, is responsible for localization of target proteins to the apical plasma membrane of polarized epithelia (Bretscher et al., 2002; Shenolikar et al., 2002). Ezrin and EBP50 have previously been shown to distribute along the full length of the RPE apical processes (Bonilha et al., 1999; Bonilha, Rodriguez-Boulan 2001). Binding between actin fibers, ezrin, EBP50 and CRALBP may function in apical localization of 11-cis-retinoids to the RPE plasma mem- brane (Nawrot et al., 2004). As part of ongoing studies to probe the validity of this hypothesis, we report here unique mass spectrometric proteome analyses of RPE apical processes. Mouse RPE apical processes were isolated using a modification of a procedure originally reported by McLaughlin and co-workers that uses wheat germ agglutinin (WGA) conjugated beads to bind plasma membrane glycoproteins containing N-acetylglucosamine and sialic acid (Cooper et al., 1987). Mice were sacrificed, the eyes immediately enucleated and the anterior segments and vitreous removed. Eyecups were incubated in 320 U ml 21 hyaluronidase in Hank’s buf- fered solution for 1 hr at 378C then the neural retina was peeled away, exposing the RPE. While apical RPE 0014-4835/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. DOI:10.1016/j.exer.2004.04.001 Experimental Eye Research 79 (2004) 419–422 www.elsevier.com/locate/yexer * Corresponding authors. Dr John W. Crabb and Dr Vera Bonilha, Cole Eye Institute (i31) Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA. E-mail addresses: crabbj@ccf.org (J.W. Crabb), bonilhar@ccf.org (V.L. Bonilha).