Status of caveolin-1 in various membrane domains of the bovine lens.
Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25-30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells.
Cenedella
Richard J
RJ
Department of Biochemistry, A.T. Still University of Health Sciences, Kirksville, MO 63501, USA. rcenedella@atsu.edu
Sexton
Patricia S
PS
Brako
Lawrence
L
Lo
Woo-Kuen
WK
Jacob
Robert F
RF
D000818:Animals; D002417:Cattle; D051242:Caveolin 1 / Q000032:analysis*; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D004847:Epithelial Cells / Q000737:chemistry; D005136:Eye Proteins / Q000032:analysis*; D005455:Fluorescent Antibody Technique / Q000379:methods; D007903:Lens Capsule, Crystalline / Q000737:chemistry; D007904:Lens Cortex, Crystalline / Q000737:chemistry; D007908:Lens, Crystalline / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry; D013109:Sphingomyelins / Q000032:analysis
D051242:Caveolin 1; D005136:Eye Proteins; D013109:Sphingomyelins