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Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045
Published, JLR Papers in Press, May 1, 2005. DOI 10.1194/jlr.M500060-JLR200
1 To whom correspondence should be addressed. e-mail: dobrowsky{at}ku.edu
Addition of exogenous ceramide causes a significant displacement of cholesterol in lipid raft model membranes. However, whether ceramide-induced cholesterol displacement is sufficient to alter the protein composition of caveolin-enriched lipid raft membranes is unknown. Therefore, we examined whether increasing endogenous ceramide levels with bacterial sphingomyelinase (bSMase) depleted cholesterol and changed the protein composition of caveolin-enriched membranes (CEMs) isolated from immortalized Schwann cells. bSMase increased ceramide levels severalfold and decreased the cholesterol content of detergent-insoluble CEMs by 25–50% within 2 h. To examine the effect of ceramide on the protein composition of the CEMs, we performed a quantitative proteomic analysis using stable isotope labeling of cells in culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Although ceramide rapidly depleted lipid raft cholesterol, the levels of the cholesterol binding protein caveolin-1 (Cav-1) decreased by 25% only after 8 h. Importantly, replenishing the cells with cholesterol rapidly reversed the loss of Cav-1 from the CEMs. Ceramide-induced cholesterol depletion increased the association of 5'-nucleotidase and ATP synthase ß-subunit with the CEMs but had a minimal effect on changing the abundance of other lipid raft proteins, such as flotillin-1 and G-proteins.
These results suggest that the ceramide-induced loss of cholesterol from CEMs may contribute to altering the lipid raft proteome.
Abbreviations: bSMase, bacterial sphingomyelinase; Cav-1, caveolin-1; CEM, caveolin-enriched membrane; dFCS, dialyzed fetal calf serum; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MßCD, methyl-ß-cyclodextrin; SC, Schwann cell; SILAC, stable isotope labeling with amino acids in cell culture
Supplementary key words caveolae • proteomics • sphingolipid • Schwann cells • stable isotope labeling with amino acids in cell culture • SILAC
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