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Y. Takahashi, G. Moiseyev, Y. Chen, J.–X. Ma; The Role of Palmitoylation Sites of RPE65 in Its Membrane Association and Isomerohydrolase Activity . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2040.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65 is a membrane–associated protein which is abundantly expressed in the retinal pigment epithelium (RPE). Recently, we and the other group reported that RPE65 is the isomerohydrolase in the retinoid visual cycle. Furthermore, it has been demonstrated that three cystein (Cys) residues, Cys231, Cys329 and Cys330, in RPE65 are palmitoylated, and the palmitoylations have been suggested to be responsible for its membrane association. The purposes of this study were to evaluate the role of the Cys residues in palmitoylation, membrane association and isomerohydrolase activity of RPE65.
The three palmitoylated Cys residues in RPE65 were replaced by alanine residue using site–directed mutagenesis. The wtRPE65, single, double and triple mutants of these Cys residues were expressed in 293A–LRAT, a cell line stably expressing human lecithin retinol acyltransferase (LRAT), using an adenovirus system. Their expression levels were examined by Western blot analysis and semi–quantified by densitometry. Their subcellular localizations were determined by subcellular fractionation and Western blot analysis. Their enzymatic activities were measured by in vitro isomerohydrolase activity assay, and the retinoid products analyzed by HPLC. Their palmitoylation status was examined by palmitoylation assay using [9, 10–, 3H] palmitic acid as the substrate.
Under the same culture conditions, some Cys mutants showed significantly reduced protein levels, compared to the wtRPE65. Similar to wtRPE65, however, all the three single Cys mutants were predominantly present in the membrane fraction. The total cell lysate expressing LRAT and wtRPE65 converted a significant amount of 11–cis retinol from all–trans retinol, demonstrating a robust isomerohydrolase activity. In contrast, all the single and double mutants of the Cys showed substantially reduced isomerohydrolase activities. The triple Cys mutant had no detectable enzymatic activity. The palmitoylation assay showed, however, this triple Cys mutant was still palmitoylated.
Mutation of any single Cys of the palmitoylation sites in RPE65 does not affect its membrane association, but reduces its isomerohydrolase activity. There are probably some other palmitoylation sites, in addition to these Cys residues. The reduced protein levels and enzymatic activities of these Cys mutants may be due to their improper folding and structural distortion.
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