Ceramide displaces cholesterol from lipid rafts and decreases the association of the cholesterol binding protein caveolin-1.
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 beta-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.
Yu
Cuijuan
C
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045, USA.
Alterman
Michail
M
Dobrowsky
Rick T
RT
D015720:5'-Nucleotidase / Q000032:analysis; D000818:Animals; D000831:Animals, Newborn; D002518:Ceramides / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000032:analysis; D025261:Mitochondrial Proton-Translocating ATPases / Q000032:analysis; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley
D002518:Ceramides; D008565:Membrane Proteins; D002784:Cholesterol; D015720:5'-Nucleotidase; C497342:ATP synthase beta subunit, rat; D025261:Mitochondrial Proton-Translocating ATPases