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RPE65, the major retinal pigment epithelium microsomal membrane protein, associates with phospholipid liposomes
1Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract:
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.
Insights
Retinal pigment epithelium (RPE) protein RPE65 associates with phospholipids, explaining its membrane association. This interaction influences RPE65
Area of Science:
- Biochemistry
- Cell Biology
- Ophthalmology
Background:
- The retinal pigment epithelium (RPE)-specific protein RPE65 is a major component of RPE microsomal membranes.
- RPE65 lacks transmembrane domains but exhibits peripheral membrane association, requiring detergents for solubilization in isotonic buffers.
- Its close association with RPE microsomal membranes, despite not being membrane-inserted, prompted investigation into its interaction mechanisms.
Purpose of the Study:
- To elucidate the mechanism behind the tight association of RPE65 with RPE microsomal membranes.
- To explore the potential involvement of phospholipid interactions in RPE65's membrane association.
- To understand the functional implications of RPE65's interaction with phospholipids.
Main Methods:
- Incubation of RPE extract with various phosphatidylcholine (PC)/phosphatidylserine (PS)/phosphatidylinositol liposomes.
- Centrifugation to sediment liposomes and assessment of RPE65 cosedimentation.
- Spectrophotometric assays and resonance energy transfer experiments to analyze RPE65-phospholipid interactions.
- Binding studies using dimyristoyl-PC-immobilized artificial membrane (IAM.PC) matrix.
Main Results:
- RPE65 cosedimented with liposomes, indicating an association with phospholipids.
- The interaction was Ca2+-independent.
- Spectrophotometry revealed phospholipid vesicle aggregation upon RPE65 interaction, without vesicle fusion.
- RPE65 selectively bound to IAM.PC and was elutable with detergent.
Conclusions:
- RPE65 exhibits a direct affinity for phospholipids, explaining its peripheral membrane association and solubilization characteristics.
- This RPE65-phospholipid interaction may be crucial for its function within the RPE.
- The findings suggest a functional link between RPE65, phospholipids, and its association with the RPE smooth endoplasmic reticulum.
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