Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes.
We have made a comprehensive and quantitative analysis of the lipid composition of caveolae from primary rat fat cells and compared the composition of plasma membrane inside and outside caveolae. We isolated caveolae from purified plasma membranes using ultrasonication in carbonate buffer to disrupt the membrane, or extraction with nonionic detergent, followed by density gradient ultracentrifugation. The carbonate-isolated caveolae fraction was further immunopurified using caveolin antibodies. Carbonate-isolated caveolae were enriched in cholesterol and sphingomyelin, and the concentration was three- and twofold higher, respectively, in caveolae compared to the surrounding plasma membrane. The concentration of glycerophospholipids was similar suggesting that glycerophospholipids constitute a constant core throughout the plasma membrane. The composition of detergent-insoluble fractions of the plasma membrane was very variable between preparations, but strongly enriched in sphingomyelin and depleted of glycerophospholipids compared to carbonate-isolated caveolae; indicating that detergent extraction is not a suitable technique for caveolae preparation. An average adipocyte caveola contained about 22 x 10(3) molecules of cholesterol, 7.5 x 10(3) of sphingomyelin and 23 x 10(3) of glycerophospholipid. The glycosphingolipid GD3 was highly enriched in caveolae, whereas GM3, GM1 and GD1a were present inside as well as outside the caveolae membrane. GD1b, GT1b, GM2, GQ1b, sulfatide and lactosylceramide sulfate were not detected in caveolae.
Ortegren
Unn
U
Department of Cell Biology and Diabetes Research Centre, Linköping University, Sweden.
Karlsson
Margareta
M
Blazic
Natascha
N
Blomqvist
Maria
M
Nystrom
Fredrik H
FH
Gustavsson
Johanna
J
Fredman
Pam
P
Strålfors
Peter
P
D017667:Adipocytes / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000032:analysis / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000032:analysis / Q000378:metabolism; D020404:Glycerophospholipids / Q000032:analysis / Q000378:metabolism; D006028:Glycosphingolipids / Q000032:analysis / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D050356:Lipid Metabolism; D008055:Lipids / Q000032:analysis; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D013109:Sphingomyelins / Q000032:analysis / Q000378:metabolism
D020404:Glycerophospholipids; D006028:Glycosphingolipids; D008051:Lipid Bilayers; D008055:Lipids; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D013109:Sphingomyelins; C513045:Tgoln2 protein, rat; C049312:nuclear pore protein p62; D002784:Cholesterol; D003576:Electron Transport Complex IV