RPE65, the major retinal pigment epithelium microsomal membrane protein, associates with phospholipid liposomes.
The retinal pigment epithelium (RPE)-specific protein RPE65 is the major 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, in 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 microsomal membranes. To explore the possible involvement of interactions with phospholipids, an isotonic salt-soluble extract of RPE was incubated with phosphatidylcholine (PC)/phosphatidylserine (PS)/phosphatidylinositol liposomes and centrifuged to sediment the liposomes. RPE65 cosedimented with the liposome pellet. RPE65 also cosedimented with synthetic dipalmitoyl-, 1-palmitoyl, 2-docosahexaenoyl-PC or dipalmitoyl-PS liposomes. Incubation with 1 mM Ca2+ or 1 mM EGTA had no effect, indicating a Ca2+-independent association. A spectrophotometric assay showed that this interaction of RPE65 with phospholipid vesicles resulted in increased light scattering, consistent with phospholipid vesicle aggregation. Resonance energy transfer experiments showed that any putative aggregation occurred without subsequent vesicle fusion. This PC affinity was further confirmed by incubation of RPE extract with dimyristoyl-PC-immobilized artificial membrane (IAM.PC) matrix. The RPE65 selectively bound and was elutable with 2% detergent. This RPE65-phospholipid liposome association may explain the solubilization characteristics of RPE65 and may be related to the function of RPE65 and to its physical association with the RPE smooth endoplasmic reticulum.
Tsilou
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E
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hamel
C P
CP
Yu
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S
Redmond
T M
TM
D000818:Animals; D002417:Cattle; D004735:Energy Transfer; D005136:Eye Proteins / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000737:chemistry*; D010712:Phosphatidic Acids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins; D014461:Ultracentrifugation
D005136:Eye Proteins; D008081:Liposomes; D008565:Membrane Proteins; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; D011506:Proteins