title: Identification of a novel palmitylation site essential for membrane association and isomerohydrolase activity of RPE65.
abstract: RPE65 is a membrane-associated protein abundantly expressed in the retinal pigment epithelium, which converts all-trans-retinyl ester to 11-cis-retinol, a key step in the retinoid visual cycle. Although three cysteine residues (Cys-231, Cys-329, and Cys-330) were identified to be palmitylated in RPE65, recent studies showed that a triple mutant, with all three Cys replaced by an alanine residue, was still palmitylated and remained membrane-associated, suggesting that there are other yet to be identified palmitylated Cys residues in RPE65. Here we mapped the entire RPE65 using mass spectrometry analysis and demonstrated that a trypsin-digested RPE65 fragment (residues 98-118), which contains two Cys residues (Cys-106 and Cys-112), was singly palmitylated in both native bovine and recombinant human RPE65. To determine whether Cys-106 or Cys-112 is the palmitylation site, these Cys were separately replaced by alanine. Mass spectrometry analysis of purified wild-type RPE65 and C106A and C112A mutants showed that mutation of Cys-106 did not affect the palmitylation status of the fragment 98-118, whereas mutation of Cys-112 abolished palmitylation in this fragment. Subcellular fractionation and immunocytochemistry analyses both showed that mutation of Cys-112 dissociated RPE65 from the membrane, whereas the C106A mutant remained associated with the membrane. In vitro isomerohydrolase activity assay showed that C106A has an intact enzymatic activity similar to that of wtRPE65, whereas C112A lost its enzymatic activity. These results indicate that the newly identified Cys-112 palmitylation site is essential for the membrane association and activity of RPE65.
authors.affiliation: Department of Medicine Endocrinology, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takahashi
authors.affiliation: Department of Medicine Endocrinology, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Gennadiy
authors.initials: G
authors.lastname: Moiseyev
authors.affiliation: Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425.
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Ablonczy
authors.affiliation: Department of Medicine Endocrinology, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Ying
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425.
authors.forename: Rosalie K
authors.initials: RK
authors.lastname: Crouch
authors.affiliation: Department of Medicine Endocrinology, Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104. Electronic address: jian-xing-ma@ouhsc.edu.
authors.forename: Jian-Xing
authors.initials: JX
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases / Q000378:metabolism*; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019308:Palmitic Acid / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
chemical_list: D002352:Carrier Proteins; D005136:Eye Proteins; D006867:Hydrolases; D019308:Palmitic Acid; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase