title: Rpe65 isomerase associates with membranes through an electrostatic interaction with acidic phospholipid headgroups.
abstract: Opsins are light-sensitive pigments in the vertebrate retina, comprising a G protein-coupled receptor and an 11-cis-retinaldehyde chromophore. Absorption of a photon by an opsin pigment induces isomerization of its chromophore to all-trans-retinaldehyde. After a brief period of activation, opsin releases all-trans-retinaldehyde and becomes insensitive to light. Restoration of light sensitivity to the apo-opsin involves the conversion of all-trans-retinaldehyde back to 11-cis-retinaldehyde via an enzyme pathway called the visual cycle. The critical isomerization step in this pathway is catalyzed by Rpe65. Rpe65 is strongly associated with membranes but contains no membrane-spanning segments. It was previously suggested that the affinity of Rpe65 for membranes is due to palmitoylation of one or more Cys residues. In this study, we re-examined this hypothesis. By two independent strategies involving mass spectrometry, we show that Rpe65 is not palmitoylated nor does it appear to undergo other post-translational modifications at significant stoichiometry. Instead, we show that Rpe65 binds the acidic phospholipids, phosphatidylserine, phosphatidylglycerol, and cardiolipin, but not phosphatidic acid. No binding of Rpe65 to basic phospholipids or neutral lipids was observed. The affinity of Rpe65 to acidic phospholipids was strongly pH-dependent, suggesting an electrostatic interaction of basic residues in Rpe65 with negatively charged phospholipid headgroups. Binding of Rpe65 to liposomes containing phosphatidylserine or phosphatidylglycerol, but not the basic or neutral phospholipids, allowed the enzyme to extract its insoluble substrate, all-trans-retinyl palmitate, from the lipid bilayer for synthesis of 11-cis-retinol. The interaction of Rpe65 with acidic phospholipids is therefore biologically relevant.
authors.affiliation: Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 90095, USA.
authors.forename: Quan
authors.initials: Q
authors.lastname: Yuan
authors.forename: Joanna J
authors.initials: JJ
authors.lastname: Kaylor
authors.forename: Anh
authors.initials: A
authors.lastname: Miu
authors.forename: Sara
authors.initials: S
authors.lastname: Bassilian
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: Gabriel H
authors.initials: GH
authors.lastname: Travis
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D055355:Opsins / Q000235:genetics / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology; D012172:Retinaldehyde / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
chemical_list: D002352:Carrier Proteins; D005136:Eye Proteins; D008051:Lipid Bilayers; D055355:Opsins; D010743:Phospholipids; D019308:Palmitic Acid; D012172:Retinaldehyde