RPE65, THE MAJOR RETINAL-PIGMENT EPITHELIUM MICROSOMAL MEMBRANE-PROTEIN, ASSOCIATES WITH PHOSPHOLIPID LIPOSOMES

The retinal pigment epithelium (RPE)-specific protein RPE65 is the maj or protein of the RPE microsomal membrane fraction, Though RPE65 lacks transmembrane domains or signal peptide, detergents are required for its maximally effective solubilization in isotonic buffers, However, i n 0.75-1.0 M KCl, RPE65 is as soluble without detergent, indicating a peripheral membrane association, We wished to understand why this non- membrane-inserted protein was so closely associated with RPE microsoma l membranes, To explore the possible involvement of interactions with phospholipids, an isotonic salt-soluble extract of RPE was incubated w ith phosphatidylcholine (PC)/phosphatidylserine (PS)/phosphatidylinosi tol liposomes and centrifuged to sediment the liposomes. RPE65 cosedim ented with the liposome pellet, RPE65 also cosedimented with synthetic dipalmitoyl-, 1-palmitoyl, 2-docosahexaenoyl-PC or dipalmitoyl-PS lip osomes. Incubation with 1 mM Ca2+ or 1 mM EGTA had no effect, indicati ng a Ca2+-independent association. A spectrophotometric assay showed t hat this interaction of RPE65 with phospholipid vesicles resulted in i ncreased light scattering, consistent with phospholipid vesicle aggreg ation, Resonance energy transfer experiments showed that any putative aggregation occurred without subsequent vesicle fusion, This PC affini ty was further confirmed by incubation of RPE extract with dimyristoyl -PC-immobilized artificial membrane (IAM.PC) matrix, The RPE65 selecti vely bound and was elutable with 2% detergent. This RPE65-phospholipid liposome association may explain the solubilization characteristics o f RPE65 and may be related to the function of RPE65 and to its physica l association with the RPE smooth endoplasmic reticulum. (C) 1997 Acad emic Press.