Sorting of lens aquaporins and connexins into raft and nonraft bilayers: role of protein homo-oligomerization.
Two classes of channel-forming proteins in the eye lens, the water channel aquaporin-0 (AQP-0) and the connexins Cx46 and Cx50, are preferentially located in different regions of lens plasma membranes (1,2). Because these membranes contain high concentrations of cholesterol and sphingomyelin, as well as phospholipids such as phosphatidylcholine with unsaturated hydrocarbon chains, microdomains (rafts) form in these membranes. Here we test the hypothesis that sorting into lipid microdomains can play a role in the disposition of AQP-0 and the connexins in the plane of the membrane. For both crude membrane fractions and proteoliposomes composed of lens proteins in phosphatidylcholine/sphingomyelin/cholesterol lipid bilayers, detergent extraction experiments showed that the connexins were located primarily in detergent soluble membrane (DSM) fractions, whereas AQP-0 was found in both detergent resistant membrane and DSM fractions. Analysis of purified AQP-0 reconstituted in raft-containing bilayers showed that the microdomain location of AQP-0 depended on protein/lipid ratio. AQP-0 was located almost exclusively in DSMs at a 1:1200 AQP-0/lipid ratio, whereas approximately 50% of the protein was sequestered into detergent resistant membranes at a 1:100 ratio, where freeze-fracture experiments show that AQP-0 oligomerizes (3). Consistent with these detergent extraction results, confocal microscopy images showed that AQP-0 was sequestered into raft microdomains in the 1:100 protein/lipid membranes. Taken together these results indicate that AQP-0 and connexins can be segregated in the membrane by protein-lipid interactions as modified by AQP-0 homo-oligomerization.
Tong
Jihong
J
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA.
Briggs
Margaret M
MM
Mlaver
David
D
Vidal
Adriana
A
McIntosh
Thomas J
TJ
D000818:Animals; D020346:Aquaporins / Q000737:chemistry* / Q000378:metabolism; D001703:Biophysics / Q000379:methods; D002417:Cattle; D002784:Cholesterol / Q000737:chemistry; D017630:Connexins / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D005136:Eye Proteins / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods; D013109:Sphingomyelins / Q000737:chemistry; D000099264:Connexin 50
D020346:Aquaporins; D002784:Cholesterol; D017630:Connexins; D003902:Detergents; D005136:Eye Proteins; D008051:Lipid Bilayers; D013109:Sphingomyelins; D000099264:Connexin 50; C030328:aquaporin 0; C082917:GJA3 protein, human