J. Lipid Res.
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A more recent version of this article appeared on August 1, 2005

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J. Lipid Res., doi:10.1194/jlr.M500060-JLR200
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Submitted on February 16, 2005
Revised on April 12, 2005
Accepted on April 26, 2005

Ceramide displaces cholesterol from lipid raft membranes and decreases the association of the cholesterol binding protein caveolin-1

Cuijuan Yu, Michail Alterman, and Rick T. Dobrowsky

Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045

Corresponding Author: 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 elevating endogenous ceramide levels with bacterial sphingomyelinase depleted cholesterol and changed the protein composition of caveolin-enriched membranes (CEMs) isolated from immortalized Schwann cells. Bacterial sphingomyelinase increased ceramide levels several fold and decreased the cholesterol content of detergent-insoluble CEMs by 25-50% within 2 hrs. 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-time of flight mass spectrometry. Although, ceramide rapidly depleted lipid raft cholesterol, the levels of the cholesterol binding protein, caveolin-1, decreased by 25% only after 8 hrs. Importantly, replenishing the cells with cholesterol rapidly reversed the loss of caveolin-1 from the CEMs. Ceramide-induced cholesterol depletion increased the association of 5’-nucleotidase and ATP synthase b-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.


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