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 β-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. 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 β-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. Caveolae and lipid raft domains are distinct but related regions of the plasma membrane that are enriched in cholesterol, sphingomyelin, and glycosphingolipids (1Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Google Scholar, 2Pike L.J. Han X. Chung K.N. Gross R.W. Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis.Biochemistry. 2002; 41: 2075-2088Google Scholar). Whereas lipid raft domains are present in many, if not all, cells (3Masserini M. Palestini P. Pitto M. Glycolipid-enriched caveolae and caveolae-like domains in the nervous system.J. Neurochem. 1999; 73: 1-11Google Scholar), caveolae are specialized, morphologically distinct lipid raft domains that require the cell-specific expression of the protein caveolin-1 (Cav-1) (4Razani B. Combs T.P. Wang X.B. Frank P.G. Park D.S. Russell R.G. Li M. Tang B. Jelicks L.A. Scherer P.E. et al.Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities.J. Biol. Chem. 2002; 277: 8635-8647Google Scholar). The integrity of lipid rafts is very dependent upon the presence of cholesterol (5Brown D.A. London E. Structure and origin of ordered lipid domains in biologic membranes.J. Membr. Biol. 1998; 164: 103-114Google Scholar). Relative to phospholipids, sphingolipids are more hydrophobic, undergo more hydrogen bonding, and tend to cluster within cell membranes (6Massey J.B. Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers.Biochim. Biophys. Acta. 2001; 1510: 167-184Google Scholar). Moreover, the ability of cholesterol to pack tightly with saturated lipids is essential to promoting the liquid-ordered structure of lipid raft domains (6Massey J.B. Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers.Biochim. Biophys. Acta. 2001; 1510: 167-184Google Scholar). The loss of cholesterol from caveolae by treatment with cholesterol-sequestering agents, through increased sterol oxidation, or by inhibiting its de novo synthesis leads to a more disordered state and the loss of raft-associated proteins. Indeed, because Cav-1 is a cholesterol binding protein, it is often used to monitor the loss of caveolar integrity after cholesterol displacement or depletion. Recent reports have provided evidence that ceramide production in raft model membranes and astrocytes causes a significant displacement of cholesterol from lipid raft membranes (7London M. London E. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function.J. Biol. Chem. 2004; 279: 9997-10004Google Scholar, 8Ito J.I. Nagayasu Y. Yokoyama S. Cholesterol-sphingomyelin interaction in membrane and apolipoprotein-mediated cellular cholesterol efflux.J. Lipid Res. 2000; 41: 894-904Google Scholar). However, whether a ceramide-induced displacement of cholesterol can lead to changes in the protein composition of caveolin-enriched membranes (CEMs) is unknown. The recent development of stable isotope labeling of cells in culture (SILAC) (9Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Google Scholar) has enabled a quantitative, hypothesis-driven assessment of changes in a single protein (10Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat. Biotechnol. 2003; 21: 315-318Google Scholar) as well as the unbiased, quantitative characterization of large proteomes (11Everley P.A. Krijgsveld J. Zetter B.R. Gygi S.P. Quantitative cancer proteomics: stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research.Mol. Cell. Proteomics. 2004; 3: 729-735Google Scholar). The basic premise of SILAC is that two populations of cells are grown in medium containing amino acids or amino acid containing or a sufficient to the amino acid the cellular of the cells as a and the other is to Cell are and in a mass upon protein The may to for a or the can by matrix-assisted laser desorption ionization time-of-flight mass or ionization minimal in the ionization the containing a amino the of of the to the a quantitative assessment of changes in the abundance of the protein after immortalized Schwann cells with or and with bacterial sphingomyelinase (bSMase) to lipid raft and the cholesterol content and protein composition of membranes Ceramide production decreased the cholesterol content of CEMs, to membranes. Ceramide-induced cholesterol displacement with a 25% in the of reversed by cholesterol to bSMase treatment increased the association of 5′-nucleotidase with the CEMs but had effect on other lipid raft-associated proteins, such as and G-proteins. These results suggest that a ceramide-induced in cholesterol may contribute to altering the protein composition of lipid from 2 or and isolated as S. D. the of caveolin-1 expression in Schwann cells Biol. Chem. 2003; Scholar). cells are immortalized that by of S. J. D. of the in of immortalized Schwann Neurochem. Scholar) and a from D. of cells in that in and to with the that and The amino medium and and the of the amino acids not stable the medium with and by the of in the stable the medium with or by the of in on with and and The cells with a and CEMs isolated by as of with and of sphingomyelin to Biol. Chem. Scholar). The in a or for analysis of protein from the of the the to and the membrane with in acid to the membrane in and the of the used to for flotillin-1 The of the used to for Cav-1 using sphingomyelin with for The medium and the cells with and in for 2 treatment with bSMase for h. These with bSMase not in cell or in not CEMs as and the lipids from of the A of lipid and J. Scholar). The of sphingomyelin and ceramide production by and the of sphingomyelin and ceramide levels and the their in to cell Cell Biol. 2001; Scholar). To the effect of ceramide production on cholesterol of the used in the cholesterol as by the the cholesterol has a of To cholesterol levels of the CEMs, the cells for with a L.J. depletion and Biol. Chem. 1998; Scholar). of cholesterol in of and the lipid to of in of with The cell culture medium a cholesterol of that of with the sufficient to caveolar cholesterol to cells with for 2 to caveolar cells in medium containing or and as the and The cells with or bSMase for as and The protein in for using the binding and as the protein that the of for the protein for The in a mass and of CEMs and the the of the and The protein with of and the membranes by for The of the isolated CEMs by the with and the and in a The and as to for J. S. and M. of mass to Scholar). The on in a minimal of in A sufficient of to the and the The to a and the from the with acid in The with and the with acid in and of the and from by using or a The in the the mass and mass performed using the and after or analysis by the mass the and using the protein by mass used a mass of to and as a to and the presence of the that the of by the or using from results using of bSMase is a tool to cellular ceramide levels because the sphingomyelin on the of the plasma cells with and CEMs isolated from of bSMase the sphingomyelin that to the CEMs of the cells within had effect on sphingomyelin with the of sphingomyelin, bSMase increased the mass of ceramide in the CEMs from to These results that bSMase CEMs in sphingomyelinase sphingomyelin and ceramide levels in caveolin-enriched membranes (CEMs) isolated from cells. cells with for and with or bSMase for h. CEMs isolated from of protein, the lipids from and sphingomyelin cells with or bSMase for 2 h. The CEMs isolated from of protein, and ceramide in Ceramide levels to and are as from To examine the effect of ceramide on the cholesterol content of the CEMs, the cells with bSMase for 2 CEMs isolated from of protein, and the of cholesterol and ceramide bSMase increased the of ceramide in the CEMs from a of to The in ceramide with a loss of of cholesterol, a 25% from The in cholesterol from the CEMs not to in sterol because the of cholesterol present in the and cells. Indeed, results in show that of bSMase the loss of cholesterol from the CEMs in membranes and plasma membranes. Importantly, effect because cholesterol levels in the CEMs after with the for not but cholesterol is more in the CEMs suggest that ceramide may very the sterol from lipid raft production is with a in the cholesterol content of CEMs. of cells with or bSMase for 2 and the CEMs isolated from of of cholesterol and ceramide in are from two performed in of cells with or bSMase for h. CEMs as and the cholesterol content of cholesterol from treatment as 2 8 are from a single and are of in The of cholesterol present in the CEMs and membranes of and cells from the in production is with a in the cholesterol content of CEMs. of cells with or bSMase for 2 and the CEMs isolated from of of cholesterol and ceramide in are from two performed in of cells with or bSMase for h. CEMs as and the cholesterol content of cholesterol from treatment as 2 8 are from a single and are of in The of cholesterol present in the CEMs and membranes of and cells from the in cholesterol the loss of Cav-1 from CEMs after treatment with cells with or bSMase for the CEMs and of the to analysis for Cav-1 or flotillin-1 performed after the the mass cells for 8 with or bSMase and the for the in of the with CEMs and of the to Cav-1 by cholesterol the loss of Cav-1 from CEMs after treatment with cells with or bSMase for the CEMs and of the to analysis for Cav-1 or flotillin-1 performed after the the mass cells for 8 with or bSMase and the for the in of the with CEMs and of the to Cav-1 by SILAC a quantitative approach to whether the ceramide-induced loss of cholesterol sufficient to changes in the of the CEMs. A for accurate by SILAC is that of cells a amino acid its S.E. Foster L.J. Mann M. in quantitative 2003; Scholar). can as the cells undergo However, the presence of amino acids in can with the amino acid and lead to a of and that contribute to protein Although the of (9Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Google Scholar), cells require for that are by Y. of labeling for proteomics in cancer Res. 2004; 3: Scholar). SILAC analysis has not performed on or immortalized it to the ability of cells to in and amino acids their their cell and in medium containing but their with that of cells in in the or of the immortalized in medium containing with medium with not To that the a amino we used because it is and is a amino acid in proteins. grown in containing or for the cells and the protein in a mass and of the two as and and to and A of SILAC is that the of is by the presence of a of that in mass by the of in the and by a for the and of The presence of two in the results in a in the mass of the by The and and in the the isotope cluster of the These from the that the abundance of the isotope in the H. Mann M. The of Rev. Mol. Cell Biol. 2004; Scholar). that and by The mass of on the of the and of the of and of the and The provided of and the of the is the of is by the of to that by in the and by the presence of and for and the and not the and a of with the presence of as the amino of the Schwann cell proteome. grown in medium with or for Cell and in a mass upon protein and the by The and with and the by matrix-assisted laser desorption ionization time-of-flight mass The is from the mass of to show the presence of a from of and of the The the for by of the of the of the Schwann cell proteome. grown in medium with or for Cell and in a mass upon protein and the by The and with and the by matrix-assisted laser desorption ionization time-of-flight mass The is from the mass of to show the presence of a from of and of the The the for by of the of the To the minimal for of the we the of its grown in and 2 containing in the medium containing for the the cells and the by The and for the of the of the presence of two the mass of the by to the of a that is 2 in medium containing the of the to the that the of Importantly, in the of the the presence of a However, the abundance of not to that present in cells that has saturated the and that present in the medium is the of amino acid for protein the for of the and it after of in medium containing results from cells grown in or for on mass of the by because the of to of the proteome. grown to in medium with and in medium with The cells grown and cell on the The by the and and the by is in the with the abundance of the the of the of grown in medium with or Cell on the and in a mass upon protein and the by The and and the by the cells undergo the are to the of the of the amino The of to for the containing or of the are of the of grown in medium with or Cell on the and in a mass upon protein and the by The and and the by the cells undergo the are to the of the of the amino The of to for the containing or of the are To of the with the of to from containing or if the amino acid is the only for protein of the after (9Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Google Scholar). the undergo a in the is to to with the the of after in the medium is to that of using is that the isotope in containing only may the in is present in the and the of the of Therefore, to the quantitative of the it is to from the using isotope is performed using the tool of but can using other amino as To for by SILAC using we changes in of and that a of grown in or for and the to mass of to and of to and the by The of within of the with the of the a of However, upon of the of the that or in of the very with the Although the of the to in the of not to signal in the mass These suggest that we may changing by or Although we have not the of it may in from a to the signal from in the of the of by stable isotope labeling with amino acids in cell grown in medium with or for Cell and in upon protein to containing the of to The by the and and the by show for the of to The for is The for containing or in of analysis performed to the the are Although is the essential amino acid in proteins, its in SILAC can it is to isotope for containing a single may not present in the of by of proteins. and Ong S.E. Mann M. to and Cell. Proteomics. 2004; 3: Scholar), the of for protein labeling that by is by a the of for the to for from the of and the of protein the results a containing to a in it is Therefore, used in the cells are immortalized of that undergo with Although the cells to a sufficient within not upon in medium with for the of of the cells with or bSMase for and cell the in a mass CEMs isolated and the by and for analysis by Cav-1 is a cholesterol binding protein M. J. K. is a cholesterol binding Scholar) association with CEMs is decreased upon cellular cholesterol with cholesterol binding such as P.E. cholesterol and in rafts and Biophys. Acta. 2003; Scholar). a mass for is the presence of that and Indeed, on used for that treatment the association of caveolin-1 (Cav-1) with CEMs. mass of Cav-1 isolated from a of CEMs. The and two that not as The mass of the is and the the for is not The in the is from and the is from The cells with or bSMase for and Cav-1 by The mass the changes in the as of Cav-1 The for of the Cav-1 and are in the as results for the of used for protein protein by stable isotope labeling with amino acids in cell culture and matrix-assisted laser desorption ionization time-of-flight mass of of in cell protein for protein and containing not in the of used for or if a from the are for in a for protein and containing not in the of used for or if a from the are for the in caveolar cholesterol within 2 of bSMase in Cav-1 by SILAC analysis However, Cav-1 levels decreased by after of bSMase treatment To the results of the analysis for Cav-1 with the SILAC of the that bSMase treatment for 8 decreased Cav-1 by Importantly, replenishing cholesterol for after of treatment with reversed the loss of Cav-1 These results the that the loss of Cav-1 from the CEMs with the ceramide-induced cholesterol depletion. bSMase treatment not alter the of and undergo a in lipid rafts after cholesterol depletion by L.J. Mann M. quantitative proteomics of lipid rafts for 2003; Scholar). Indeed, the depleted from the CEMs after 2 of treatment with but by bSMase treatment the other with bSMase a in a raft protein L.J. Mann M. quantitative proteomics of lipid rafts for 2003; Scholar), and in the raft protein ATP P. K. Biol. 2004; proteomic analysis of the effect of bSMase treatment on in caveolin-enriched of bSMase synthase of only two for of only two for of only two for of only two for bacterial not are of the of in protein and are as of only two for in a bacterial not are of the of in protein and are as results show that treatment of immortalized with bSMase increased the ceramide content of the CEMs and with a 25–50% in the cholesterol of the CEMs within 2 h. the cholesterol content of the CEMs well that present in cells after 8 in the presence of results with the in plasma membrane cholesterol after treatment of astrocytes J.I. Nagayasu Y. Yokoyama S. Cholesterol-sphingomyelin interaction in membrane and apolipoprotein-mediated cellular cholesterol efflux.J. Lipid Res. 2000; 41: 894-904Google Scholar) or with bSMase M. S. M. of by in 2003; Scholar). to cholesterol levels within in the presence of bSMase in may the of M. S. M. of by in 2003; Scholar) in or cell-specific that cholesterol synthesis and with upon results from lipid raft model membranes. lipid of raft domains and disordered the of exogenous ceramide cholesterol from the raft domains (7London M. London E. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function.J. Biol. Chem. 2004; 279: 9997-10004Google Scholar). the loss of cholesterol from the CEMs well with in the cholesterol content of of a of plasma membrane and membranes. These with a that the loss of plasma membrane cholesterol after bSMase treatment is not to cholesterol Y. a for cellular on cholesterol in Lipid Res. 2003; Scholar). However, we not whether the displacement of cholesterol from the CEMs with and the production of not ceramide may cholesterol from lipid raft The interaction of cholesterol with sphingomyelin through hydrogen of the of cholesterol with the of Although structure is in the loss of the results in ceramide a very for Indeed, have that ceramides very with cholesterol and undergo and the of a of sphingomyelinase and ceramide in biologic 2002; Scholar). recent evidence that ceramide the of the present in sphingomyelin, not with cholesterol B. the of the for sphingomyelin/cholesterol interactions in a with ceramide J. Scholar). These the that the is for interactions B. the of the for sphingomyelin/cholesterol interactions in a with ceramide J. Scholar). ceramides from sphingomyelin the and acid is not sufficient to a as sphingomyelin is the production of a for with cholesterol, sphingomyelin levels to a Y. a for cellular on cholesterol in Lipid Res. 2003; Scholar). sphingomyelin to ceramide not the of ceramide in the raft These results the that cholesterol displacement is with increased ceramide production the loss of sphingomyelin Y. a for cellular on cholesterol in Lipid Res. 2003; Scholar). To the effect of ceramide-induced cholesterol depletion on the protein composition of the CEMs, we used a stable isotope labeling that a quantitative proteomic analysis two cell SILAC has used to (10Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat. Biotechnol. 2003; 21: 315-318Google Scholar, H. B. M. Pandey A. Mann M. of the Biol. Chem. 2002; 277: Scholar) and more quantitative of cellular proteomes Y. of labeling for proteomics in cancer Res. 2004; 3: Scholar, L.J. Mann M. quantitative proteomics of lipid rafts for 2003; Scholar). have the of approach using and immortalized the cellular with effect on cell and have used and to and have with of the amino acids the not a cell can to in cells with S.E. Kratchmarova I. Mann M. of in stable isotope labeling by amino acids in cell culture Res. 2003; Scholar). Although is not for by SILAC, we of to in or the immortalized The for by SILAC to in protein However, it that accurate of changes of is by a and is by as as Although the of and the of for M. A. Pandey A. A proteomic approach for of using stable isotope labeling in cell Chem. 2003; Scholar), it has that are in the as has E. H. The of in mass of Chem. 1999; Scholar). results the evidence that SILAC analysis is using or immortalized analysis and caveolar the of cell and of have as lipid raft upon their in association with CEMs after a depletion of cholesterol using L.J. Mann M. quantitative proteomics of lipid rafts for 2003; Scholar). of cholesterol depletion in only a in Cav-1 L.J. Mann M. quantitative proteomics of lipid rafts for 2003; Scholar). Although bSMase treatment decreased cholesterol levels by 25–50% within 2 the abundance of Cav-1 decreased by 25% only after 8 h. the other ceramide-induced cholesterol depletion with changes in the abundance of the β-subunit of ATP synthase and 5′-nucleotidase but had effect on altering the association of or with CEMs. it that a loss of 25–50% of the cholesterol content of the CEMs not changes in the protein content of the membranes. domains ordered in membranes and with cholesterol for association with rafts (7London M. London E. Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function.J. Biol. Chem. 2004; 279: 9997-10004Google Scholar), it is that ceramide may in for the loss of cholesterol and from the CEMs. The ability of Cav-1 to cholesterol may protein more to from the CEMs as ceramide displaces To examine we to the for the proteins, is in E. B. D. M. Schwann the development of 1999; Scholar), is to changes in cholesterol to and to lipid rafts The with caveolin-1 in of the plasma Biol. Chem. 2001; Scholar). However, the of the of from the or protein not of to a bSMase is a tool for increasing the of the in ceramide is well within a that is often by or cell loss of cholesterol from the CEMs, and its effect on the proteins, may of changes in domains after the of plasma membrane However, the lipid and protein composition of lipid rafts is not cell and may the of cholesterol protein displacement by ceramide that other S. A. H. J. K. ceramide and by in Biol. Chem. 1998; Scholar) that may contribute to the ceramide content and protein composition of lipid that ceramide production and the loss of cholesterol from lipid rafts contribute to changes in the protein composition of membrane we the of using SILAC in immortalized and to a quantitative proteomic approach to signal in cells. The of SILAC analysis in the and of changes in protein abundance and protein Mann M. of for Biol. Chem. 2004; 279: Scholar) a tool for the effect of ceramide on cellular or by from the the of and a J. The for and and D. for the immortalized