Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells - Methyl-beta-cyclodextrin does not affectcell surface transport of a GPI-anchored protein

Recent data suggest that membrane microdomains or rafts that are rich in sp hingolipids and cholesterol are important in signal transduction and membra ne trafficking. Two models of raft structure have been proposed. One propos es a unique role for glycosphingolipids (GSL), suggesting that GSL-head-gro up interactions are essential in raft formation, The other model suggests t hat close packing of the long saturated acyl chains found on both GSL and s phingomyelin plays a key role and helps these lipids form liquid-ordered ph ase domains in the presence of cholesterol, To distinguish between these mo dels, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficie nt derivative, GM-95, Rafts were isolated from cell lysates as detergent-re sistant membranes (DRMs). The two cell lines had very similar DRM protein p rofiles, The yield of DRM protein was 2-fold higher in the parental than th e mutant line, possibly reflecting cytoskeletal differences. The same amoun t of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compen sated for the lack of GSL, DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene, Methyl-beta-cy clodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylino sitol-anchored protein and residual cholesterol showed the same distributio n between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored prot ein was delivered to the cell surface at similar rates in the two lines, ev en after cholesterol depletion with methyl-beta-cyclodextrin. me conclude t hat GSL are not essential for the formation of rafts and do not play a majo r role in determining their properties.