Caveolar structure and protein sorting are maintained in NIH 3T3 cells independent of glycosphingolipid depletion

Glycosphingolipids have been proposed to be critical components of clustere d lipids within cell membranes that serve as rafts for the attachment and s orting of proteins to the cell membrane. Density gradient centrifugation wa s used to isolate and to ascertain the lipid composition of caveolin-enrich ed membranes. These membranes demonstrated a significant enrichment of sphi ngolipids and cholesterol containing up to 20 and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of gl ucosylceramide synthase, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propano l, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When MH 3T3 cells were depleted of th eir glucosylceramide based glycosphingolipid mass, the caveolar structure r emained intact as determined by electron microscopy and confocal microscopy . The caveolar proteins caveolin and annexin II sorted normally to caveolae , as determined by immunoblotting and confocal microscopy. When the GPI-lin ked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depl etion. These observations suggest that protein sorting to caveolae in fibro blasts occurs independently of glycosphingolipid synthesis. (C) 2000 Academ ic Press.