Role of LRAT on the retinoid isomerase activity and membrane association of Rpe65.
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.
Jin
Minghao
M
Jules Stein Eye Institute, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095, USA.
Yuan
Quan
Q
Li
Songhua
S
Travis
Gabriel H
GH
D000042:Absorption; D000217:Acyltransferases / Q000502:physiology*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D008027:Light; D013058:Mass Spectrometry; D051379:Mice; D010168:Palmitates / Q000737:chemistry; D019308:Palmitic Acid / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D019745:cis-trans-Isomerases / Q000378:metabolism*; D000099178:Retinoid Isomerohydrolase
D000217:Acyltransferases; D002352:Carrier Proteins; D003545:Cysteine; D005136:Eye Proteins; D010168:Palmitates; D019308:Palmitic Acid; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C022776:2-bromopalmitate; C056942:lecithin-retinol acyltransferase