Abstract
Absorption of a photon by a vertebrate opsin pigment induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical re-isomerization of the chromophore via an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway is Rpe65, a membrane-associated protein in the retinal pigment epithelium (RPE) with no predicted membrane-spanning segments. It has been suggested that Rpe65 is S-palmitoylated by lecithin:retinol acyl transferase (LRAT) on Cys(231), Cys(329), and Cys(330), and that this palmitoylation is required for isomerase activity and the association of Rpe65 with membranes. Here we show that the affinity of Rpe65 for membranes is similar in wild-type and lrat(-/-) mice. The isomerase activity of Rpe65 is also similar in both strains when all-trans-retinyl palmitate is used as substrate. With all-trans-retinol substrate, isomerase activity is present in wild-type but undetectable in RPE homogenates from lrat(-/-) mice. Substitution of Cys(231), Cys(329), and Cys(330) with Ser or Ala did not affect the affinity of Rpe65 for membranes. Further, these Cys residues are not palmitoylated in Rpe65 by mass spectrometric analysis. Global inhibition of protein palmitoylation by 2-bromopalmitate did not affect the solubility or isomerase activity of Rpe65. Finally, we show that soluble and membrane-associated Rpe65 possesses similar isomerase specific activities. These results indicate that LRAT is not required for isomerase activity beyond synthesis of retinyl-ester substrate, and that the association of Rpe65 with membranes is neither dependent upon LRAT nor the result of S-palmitoylation. The affinity of Rpe65 for membranes is probably an intrinsic feature of this protein.
Highlights
The retinoid isomerase was recently identified as an abundant 63-kDa protein in retinal pigment epithelium (RPE) cells called Rpe65 (8 –10)
This study investigated the role of lecithin:retinol acyl transferase (LRAT) on retinoid isomerization in RPE cells
It was recently suggested that the affinity of Rpe65 for membranes is modulated by reversible palmitoylation by LRAT [15]
Summary
Mice and Bovine Eyes—Mice with a knock-out mutation in the gene for LRAT [16] were generously provided by Krzysztof Palczewski. Proteins (50 ng to 5.0 g) from the homogenate and the identical volume of membrane-free supernatant were analyzed by immunoblot analysis using an antibody against bovine Rpe (see below). 500 ng to 12 g of proteins in the total cell homogenates and the identical volumes of the membrane-free supernatants were analyzed by immunoblotting with antisera against Rpe. Pellets from the final centrifugation were resuspended in 20 mM HEPES buffer containing 0.45 M NaCl and 0 –10 mM sodium cholate (as indicated), and incubated on ice for 15 min to extract Rpe from the membranes. Cell homogenates and membrane-free supernatants were prepared from these cells as described above, and identical fractions were analyzed by immunoblotting using anti- In Vitro LRAT Activity Assay—The activity of LRAT was sera against bovine Rpe and human Fyn kinase, as determined by monitoring the formation of all-trans-RE from described above. Representative chromatograms and UV spectra acquired for 11-cis-ROL are shown in Fig. 2, D and E
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