Abstract
Purpose: :
Retinal pigment epithelium specific protein-65 kDa (Rpe65) is the isomerohydrolase in RPE cells. Rpe65 strongly associates with membranes although it contains no transmembrane segments. Palmitoylation of three Cys residues in Rpe65 was previously reported to mediate membrane association of Rpe65. However, later studies showed that none of these residues are palmitoylated. The purpose of the current work is to determine the mechanism for membrane association of Rpe65.
Methods: :
RPE microsomes were prepared from freshly slaughtered cow eyes. After digestion with proteases and reaction with deuterated and non-deuterated alkylating agents, Rpe65 in these microsome samples was analyzed by liquid chromatography (LC) and mass spectrometry (MS) with collision-induced fragmentation (MS/MS). Bovine Rpe65 was incubated with membrane strips containing various immobilized neutral and charged lipids to test for binding. The isomerase activity of Rpe65 in RPE homogenates and microsomes was determined using all trans retinol (all trans ROL) and all trans retinyl palmitate (all trans RP) substrates in the presence of phospholipid liposomes.
Results: :
By LC-MS and MS/MS analysis we identified proteolytic peptides containing each of the 12 Cys residues in Rpe65. None were palmitoylated or otherwise modified to any significant extent. Further, high-resolution MS analysis of intact Rpe65 from bovine RPE homogenates suggested that Rpe65 does not undergo any form of post-translational modification. However, we show that Rpe65 has high affinity for negatively charged phospholipids including phosphatidylglycerol (PG), cardiolipin and phosphatidylserine (PS), but not positively charged phospholipids such as phosphatidylethanolamine (PE) or phosphatidylcholine (PC). The association of Rpe65 with membranes is pH dependent. The presence of PG- or PS-containing liposomes inhibited Rpe65 enzyme activity using all trans ROL as a substrate precursor. Addition of all trans RP substrate in PS- or PG-containing liposomes dramatically increased isomerase activity, whereas addition of this substrate in PE- or PC-containing liposomes had no on Rpe65 activity.
Conclusions: :
By isotopic MS analysis, none of the Cys residues in Rpe65 are significantly palmitoylated. The strong interaction of Rpe65 with membranes appears to be mediated by electrostatic interactions between basic residues and acidic phospholipid headgroups. This interaction may involve a Lys-rich region on the predicted surface of Rpe65.
Keywords: retinal pigment epithelium • protein structure/function