Rpe65 Is a Retinyl Ester Binding Protein That Presents Insoluble Substrate to the Isomerase in Retinal Pigment Epithelial Cells

Abstract

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the alltrans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of ␤-carotene dioxygenase, appears to play a role in this pathway. Rpe65 ؊/؊ knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds alltrans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65 ؊/؊ mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.Light perception in vertebrates is mediated by a group of G protein-coupled receptors called the opsins. Most opsin pigments contain 11-cis-retinaldehyde (11cRAL) 1 as the light-absorbing chromophore. Absorption of a photon induces 11-cis to all-trans isomerization of the chromophore, resulting in the activated species, metarhodopsin II. After a brief period, metarhodopsin II decays to yield apo-rhodopsin and free alltrans-retinaldehyde (atRAL). Before light sensitivity of the pigment can be restored, the atRAL must be chemically re-isomerized to 11cRAL by a metabolic pathway called the visual cycle. Most steps in this pathway take place within cells of the retinal pigment epithelium (RPE) adjacent to the photoreceptors. The key step in this pathway is all-trans to 11-cis re-isomerization of the retinoid, which is catalyzed by an enzyme activity called isomerohydrolase (IMH). IMH has been shown to use fatty acyl esters of retinol as a substrate (1, 2), harnessing the energy of ester hydrolysis [⌬G ϭ Ϫ5 kcal/mol (3)] for the endothermic conversion of all-trans-retinol (atROL) to 11-cis-retinol (11cROL) (ϩ4.1 kcal/mol, Ref. 4). IMH has never been purified or cloned. Leber's congenital amaurosis (LCA) is a severe and relatively common autosomal recessive disease that results in blindness at birth. LCA is frequently caused by mutations in the RP...

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

“…We did not observe any effect of all-trans-retinol addition on the localization of LRAT, which remained ER-associated. Of note is that the T-REx-293 cells we used for these experiments do not express detectable levels of RPE65, a protein proposed in some studies to be a retinyl ester-binding/transport protein (42,43), yet we were still able to detect retinosome formation. The N Terminus of LRAT Is Not Conserved in All VertebratesAnalysis of the N-terminal domains of the known and putative vertebrate homologs of LRAT showed a low degree of conservation.…”

“…The RPE65 protein is currently the only protein known to associate with retinyl esters (42,43). It has been reported that RPE65 is involved in the isomerization reaction that produces 11-cis-retinol and that retinyl esters are the preferred substrates for this reaction (51)(52)(53).…”

Aberrant Metabolites in Mouse Models of Congenital Blinding Diseases: Formation and Storage of Retinyl Esters

“…This observation is puzzling when it is considered that RPE65 has been recently proposed to be an isomerohydrolase, the enzyme responsible for isomerization and hydrolysis of all-trans-retinyl esters to 11-cis-retinol (23-26). It should be noted that purified RPE65 has no isomerase activity (27). Further studies are required to understand the protein complex responsible for the retinoid isomerization in the RPE cells.…”

“…The accumulation of retinyl esters is clearly dependent on LRAT activity (Figure 2), and these esters accumulate in Rpe65−/− mice. RPE65 was also proposed to be a retinyl esterbinding protein (27,44,45), hinting of its possible role in this process. Most insightful was the observation made by Pepperberg et al that, in normal RPE, there is a substantial exchange of all-trans-retinol with the blood circulation, whereas in Rpe65−/−mice, despite the presence of abnormally high molar levels of RPE retinyl esters, the outward movement is inhibited (43).…”

“…RPE65 has been proposed to be the isomerase responsible for transformation of all-trans-retinyl esters to 11-cis-retinol (23)(24)(25)(26) (Figure 1). However, in a purified form, it has been reported that RPE65 does not possess the isomerase activity (27).…”