E. Tsilou et al., RPE65, THE MAJOR RETINAL-PIGMENT EPITHELIUM MICROSOMAL MEMBRANE-PROTEIN, ASSOCIATES WITH PHOSPHOLIPID LIPOSOMES, Archives of biochemistry and biophysics, 346(1), 1997, pp. 21-27
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.