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The Roles of Three Palmitoylation Sites of RPE65 in Its Membrane Association and Isomerohydrolase Activity

From Departments of Medicine Endocrinology and Cell Biology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, Oklahoma.

PURPOSE. RPE65, a membrane-associated protein predominantly expressed in the retinal pigment epithelium, is the isomerohydrolase in the retinoid visual cycle. Three cysteine (Cys) residues, Cys231, Cys329, and Cys330, in RPE65 have been shown to be palmitoylated and have been suggested to be responsible for its membrane association. The purpose of this study was to define the role of these Cys residues in palmitoylation, membrane association, and isomerohydrolase activity of RPE65.

METHODS. The three Cys residues in RPE65 were replaced by Alanine (Ala) with site-directed mutagenesis. The mutant protein levels and subcellular localizations were determined by Western blot analysis and subcellular fractionation, respectively. Their enzymatic activities were evaluated with the in vitro isomerohydrolase activity assay. Palmitoylation of the mutants was examined by labeling of the protein with [³H]-labeled palmitic acid.

RESULTS. Mutation of any single residue of these three Cys significantly reduced protein levels of RPE65. Similar to wild-type RPE65, however, all three single Cys mutants were predominantly present in the membrane fraction. Mutations of any one or two of these Cys substantially weakened the isomerohydrolase activity of RPE65, whereas mutations of all three Cys (triple mutant) completely abolished the enzymatic activity. However, this triple Cys mutant was still palmitoylated and associated with the membrane, although at a reduced level.

CONCLUSIONS. There are additional yet to be identified palmitoylation sites in RPE65. The structural distortions induced by the Cys mutations may be responsible for the mislocalization and decreased isomerohydrolase activities of RPE65.

This article has been cited by other articles:

				Y. Takahashi, G. Moiseyev, Z. Ablonczy, Y. Chen, R. K. Crouch, and J.-x. Ma Identification of a Novel Palmitylation Site Essential for Membrane Association and Isomerohydrolase Activity of RPE65 J. Biol. Chem., January 30, 2009; 284(5): 3211 - 3218. [Abstract] [Full Text] [PDF]