The roles of three palmitoylation sites of RPE65 in its membrane association and isomerohydrolase activity.
OBJECTIVE
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.
Takahashi
Yusuke
Y
Department of Medicine Endocrinology, University of Oklahoma Health Sciences Center (OUHSC), Oklahoma City, Oklahoma, USA.
Moiseyev
Gennadiy
G
Chen
Ying
Y
Ma
Jian-xing
JX
D000409:Alanine / Q000235:genetics; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics; D005136:Eye Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010168:Palmitates / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D013347:Subcellular Fractions; D019745:cis-trans-Isomerases / Q000378:metabolism*; D000099178:Retinoid Isomerohydrolase
D000409:Alanine; D002352:Carrier Proteins; D003545:Cysteine; D005136:Eye Proteins; D010168:Palmitates; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase