Rapid transmembrane diffusion of ceramide and dihydroceramide spin-labelled analogues in the liquid ordered phase

Pohl, Andreas; López‐Montero, Iván; Rouvière, Florent; Giusti, Fabrice; Devaux, Philippe F.

doi:10.1080/09687680902733815

Cited by 17 publications

(14 citation statements)

References 55 publications

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“…From a simple physiochemical perspective, it is hard to explain the discrepancy in fl ip-fl op behavior among these three types of ceramide. Our results suggest that ceramide fl ip-fl op in a liquid-disordered bilayer is faster than in a liquid-ordered bilayer, although we still fi nd a half time of 30 s in the liquid-ordered phase, which is faster than the experimental value ( 24 ).…”

Section: Flip-fl Opcontrasting

confidence: 67%

“…The much less polar headgroups of cholesterol, diacylglycerol, and ceramide suggest these lipids can translocate bilayers on a much faster timescale than phospholipids. Experimental evidence supports this hypothesis (20)(21)(22)(23)(24)(25)(26)(27), although the rates vary signifi cantly depending on the experimental conditions, often by orders of magnitude. These differences may be due to uncertainties with the interpretation of the complex experiments, but it seems more likely that they can be explained by a significant effect of differences in the structure and dynamics of the membranes themselves on the kinetics and thermodynamics of lipid fl ip-fl op.…”

Section: Methodsmentioning

confidence: 96%

“…The half time for unlabeled C-16 ceramide fl ip-fl op was estimated to be < 1 min in egg PC giant unilamellar vesicles ( 25 ). Subsequently, the same group showed that the half time for spin-labeled ceramide fl ip-fl op was < 1 min in both a liquid-disordered (egg PC) large unilamellar vesicle and a liquid-ordered (20:20:60 mol ratio of SM:POPC:CHOL) large unilamellar vesicle ( 24 ). This was in contrast to glucosylceramide and galactosylceramide, which both had signifi cantly reduced fl ip-fl op rates in bilayers with higher cholesterol concentrations ( 24 ).…”

Section: Flip-fl Opmentioning

confidence: 99%

“…Subsequently, the same group showed that the half time for spin-labeled ceramide fl ip-fl op was < 1 min in both a liquid-disordered (egg PC) large unilamellar vesicle and a liquid-ordered (20:20:60 mol ratio of SM:POPC:CHOL) large unilamellar vesicle ( 24 ). This was in contrast to glucosylceramide and galactosylceramide, which both had signifi cantly reduced fl ip-fl op rates in bilayers with higher cholesterol concentrations ( 24 ). From a simple physiochemical perspective, it is hard to explain the discrepancy in fl ip-fl op behavior among these three types of ceramide.…”

Section: Flip-fl Opmentioning

confidence: 99%

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Molecular simulation of rapid translocation of cholesterol, diacylglycerol, and ceramide in model raft and nonraft membranes

Bennett

Tieleman

2012

Journal of Lipid Research

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sorting of lipids and proteins and the role of lipid rafts in vivo remains controversial ( 4,(6)(7)(8). The microscopic structure of mixed membranes is a complex problem because the local concentration of individual lipids affects the phase and structure of the membrane, which in turn affect the local concentration of these lipids. In a model system, it has been shown that both C18:0 ceramide and dipalmitoylglycerol can displace cholesterol from liquid-ordered domains, and form larger and more stable domains than cholesterol-enriched ordered domains ( 9 ). Both ceramide ( 10 ) and diacylglycerol ( 11 ) are nonlamellar forming lipids, which are important for cellular processes, such as pore formation, vesicle fusion, and budding, as well as membrane protein function ( 12 ). Currently we lack a molecular level physical and thermodynamic understanding of lipid-lipid interactions in biological membranes.Ceramide is a key metabolic intermediate for sphingolipids with an amide backbone ( 13 ), as diacylglycerol is for glycerol-derived phospholipids ( 14 ). In cells, there are many variants of both ceramide ( 13 ) and diacylglycerol ( 15 ), including short and long tails, saturated and unsaturated tails, and minor changes in backbone structure. Both ceramide and diacylglycerol are lipid second messengers that play important roles in many signaling pathways. The mechanism of lipid second messengers can involve specifi c lipid-protein interactions or more general membrane altering effects, such as vesicle budding and domain formation or stabilization. A well-studied example of a specifi c lipid-protein mechanism is the breakdown of phosphoinositol or phosphatidylcholine by a phospholipase into diacylglycerol, which then binds to the C1 domain of protein kinase C ( 14 ). The formation of larger and more sta-

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Section: Flip-fl Opcontrasting

confidence: 67%

Section: Methodsmentioning

confidence: 96%

Section: Flip-fl Opmentioning

confidence: 99%

Section: Flip-fl Opmentioning

confidence: 99%

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Molecular simulation of rapid translocation of cholesterol, diacylglycerol, and ceramide in model raft and nonraft membranes

Bennett

Tieleman

2012

Journal of Lipid Research

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“…However, although likely, it is not yet proven that the resulting ceramide is released into the cytoplasmic leaflet of the ER membrane. Natural and spin-labeled ceramides could rapidly flip-flop in liquid ordered giant unilamellar vesicles [12,13], whereby the relevance of data obtained with these highly artificial systems to natural membranes remains to be demonstrated. In particular, the flip rate of C26:0-containing ceramides, typical of yeast and presumed to be concentrated in rafts, i.e.…”

Section: Introductionmentioning

confidence: 99%

Membrane topology of yeast alkaline ceramidase YPC1

Ramachandra

Conzelmann

2013

Biochemical Journal

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Condensed title: Topology of yeast alkaline ceramidase YPC1Key words: YPC1, ceramidase, topology, cysteine accessibility, CREST superfamily Abbreviations: DHS, dihydrosphingosine; DTR, dual topology reporter; DTT, dithiothreitol; FOA, 5'-fluoroorotic acid; IPC, inositolphosphorylceramide; LCB, long chain base; MIPC, mannosyl-IPC; M(IP) 2 C, inositolphosphoryl-MIPC; NEM, N-ethylmaleimide; PEG-mal, PEG-5000-maleimide; PHS, phytosphingosine; RT, room temperature; SCAM TM , substituted cysteine accessibility method for TM determination; TM, transmembrane helix; TNBS, trinitrobenzene sulfonic acid; UBI-mal, ubiquitin-maleimide; VLCFA, very long chain fatty acid; wt, wild type. SynopsisYpc1p and Ydc1p are alkaline ceramide hydrolases, which reside in the ER. Ypc1p can catalyze the reverse reaction, i.e. the condensation of free fatty acids with phytosphingosine or dihydrosphingosine and overexpression of YPC1 or YDC1 can provide enough ceramide synthesis as to rescue the viability of cells lacking the normal acyl-CoA-dependent ceramide synthases. To better understand the coexistence of acyl-CoA dependent ceramide synthases and ceramidases in the ER we investigated the membrane topology of Ypc1p by probing cysteine accessibility of natural and substituted cysteines with membrane non-permeating mass-tagged probes. The N-and C-terminal ends of Ypc1p are oriented towards the lumen and cytosol, respectively. Two of the 5 natural cysteines, Cys27 and Cys219, are essential for enzymatic activity and form a disulfide bridge. The data allow inferring that all amino acids of Ypc1p that are conserved in the pfam PF05875 ceramidase motif and the CREST superfamily are located in or near the ER lumen. Microsomal assays using a lysine-specific reagent show that the reverse ceramidase activity can only be blocked when the reagent has access to Ypc1p from the lumenal side. Overall the data suggest that the active site of Ypc1p resides at the lumenal side of the ER membrane. IntroductionThe mature sphingolipids of Saccharomyces cerevisiae, namely the inositolphosphorylceramides (IPCs), mannosyl-IPCs (MIPCs) and inositolphosphoryl-MIPCs (M(IP) 2 Cs) are major components of the yeast plasma membrane. Moreover, as in higher eukaryotes, the metabolic intermediates of sphingolipid metabolism have been advocated as signaling molecules in many cell biological phenomena and responses (for review see [1]). Ceramide is a central metabolic intermediate (Fig. 1), which can result from breakdown of IPCs by Isc1p [2] or can be synthesized from acyl-CoA and long chain bases (LCBs) by ceramide synthases consisting of Lag1p or Lac1p in complex with (Fig. 1). Yeast Lag1p and Lac1p are functionally redundant but concomitant deletion of LAG1 and LAC1 causes a significant growth defect in the genetic background of W303 cells and is lethal in the YPK9 background [3,4,8]. Insertion of glycosylation sites and factor Xa protease sites into Lag1p and Lac1p, the catalytically active subunits of the yeast ceramide synthase, generated a topological model compris...

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