title: Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. methyl-beta-cyclodextrin does not affect cell surface transport of a GPI-anchored protein.
abstract: Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-beta-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-beta-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.
authors.affiliation: Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Ostermeyer
authors.forename: B T
authors.initials: BT
authors.lastname: Beckrich
authors.forename: K A
authors.initials: KA
authors.lastname: Ivarson
authors.forename: K E
authors.initials: KE
authors.lastname: Grove
authors.forename: D A
authors.initials: DA
authors.lastname: Brown
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005260:Female; D006028:Glycosphingolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D008560:Membrane Fluidity; D008563:Membrane Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014407:Tumor Cells, Cultured; D047392:beta-Cyclodextrins
chemical_list: D003505:Cyclodextrins; D003902:Detergents; D006028:Glycosphingolipids; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D008563:Membrane Lipids; D008565:Membrane Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D000469:Alkaline Phosphatase