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

J Lipid Res. 2005 Aug;46(8):1678-91. doi: 10.1194/jlr.M500060-JLR200. Epub 2005 May 1.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 5'-Nucleotidase / analysis
  • Animals
  • Animals, Newborn
  • Ceramides / metabolism*
  • Cholesterol / metabolism*
  • Membrane Microdomains / chemistry*
  • Membrane Proteins / analysis
  • Mitochondrial Proton-Translocating ATPases / analysis
  • Proteomics
  • Rats
  • Rats, Sprague-Dawley

Substances

  • Ceramides
  • Membrane Proteins
  • Cholesterol
  • 5'-Nucleotidase
  • ATP synthase beta subunit, rat
  • Mitochondrial Proton-Translocating ATPases