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Y. Takahashi, G. Moiseyev, Z. Ablonczy, Y. Chen, R. K. Crouch, J.-X. Ma; Identification of Novel Palmitylation Site in RPE65 Essential for Its Membrane Association and Isomerohydrolase Activity. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3520.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65, isomerohydrolase, is a membrane-associated protein which is abundantly expressed in the retinal pigment epithelium (RPE) and catalyzes a key reaction of all-trans retinyl ester to 11-cis retinol in the retinoid visual cycle. It has been reported that three cysteine (Cys) residues, Cys231, Cys329 and Cys330, in RPE65 are palmitylated, and the palmitylations have been suggested to be responsible for its membrane association. However, we recently showed that a triple Cys mutant with all of the three Cys replaced by alanine residues was still palmitylated and was still associated with the membrane. This result suggests that RPE65 possesses other yet to be identified palmitylated Cys residues responsible for its membrane association. The purpose of this study was to identify novel palmitylation sites, and to examine their impacts on the membrane association and isomerohydrolase activity of RPE65.
The candidate Cys residues in RPE65 were replaced by alanine using site-directed mutagenesis. The wtRPE65 and the mutants were expressed in 293A-LRAT, a cell line stably expressing human lecithin retinol acyltransferase (LRAT). Their expression levels were examined by Western blot analysis and semi-quantified by densitometry. Their membrane association was determined by subcellular fractionation and Western blot analysis. Their enzymatic activities were measured by an in vitro isomerohydrolase assay, and the retinoid products were analyzed by HPLC.
Candidate Cys residues were selected based on mass-spectral data on the wtRPE65 and the Cys mutants were generated. Under the same culture conditions, most single Cys mutants showed protein levels similar to that of wtRPE65. Most of the Cys mutants were predominantly present in the membrane fraction, similar to wtRPE65. In 293A-LRAT cells, these Cys mutants and wtRPE65 showed robust isomerohydrolase activities. In contrast, one of the mutants was almost completely dissociated from the membrane. Moreover, this Cys mutation abolished the isomerohydrolase activity of RPE65. Mass-spectrometry identified this peptide fragment containing palmitylated Cys residue.
Our present data showed that a single Cys residue is potentially pamitylated and responsible for the membrane association and essential for the isomerohydrolase activity of RPE65.
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