Fluorescence-based evaluation of the partitioning of lipids and lipidated peptides into liquid-ordered lipid microdomains: a model for molecular partitioning into "lipid rafts".
A fluorescence-quenching assay is described that can directly monitor the relative extents of partitioning of different but structurally homologous fluorescent molecules into liquid-ordered (l(o)) domains in lipid vesicles exhibiting liquid-ordered/liquid-disordered (l(o)/l(d)) phase coexistence. Applying this assay to a series of bimane-labeled diacyl phospholipid probes in cholesterol-containing ternary lipid mixtures exhibiting l(o)/l(d) phase separation, we demonstrate that partitioning into l(o)-phase domains is negligible for diunsaturated species and greatest for long-chain disaturated species. These conclusions agree well with those derived from previous studies of the association of lipids and lipid-anchored molecules with l(o)-phase domains, using methods based on the isolation of a detergent-insoluble fraction from model or biological membranes at low temperatures. However, we also find that monounsaturated and shorter-chain saturated species partition into l(o) phases with significant, albeit modest affinities, and that the level of partitioning of these latter species into l(o)-phase domains is significantly underestimated (relative to that of their long-chain saturated counterparts) by the criterion of low-temperature detergent insolubility. Finally, applying the fluorescence-quenching method to a family of lipid-modified peptides, we demonstrate that the S-palmitoyl/S-isoprenyl dual-lipidation motif found in proteins such as H- and N-ras and yeast Ste18p does not promote significant association with l(o) domains in l(o)/l(d)-phase-separated bilayers.
Wang
T Y
TY
Department of Biochemistry, McGill University, Montréal, Québec H3G 1Y6, Canada.
Leventis
R
R
Silvius
J R
JR
D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D002794:Choline / Q000737:chemistry; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D008074:Lipoproteins / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013113:Spin Labels
D001643:Bridged Bicyclo Compounds; D003497:Cyclic N-Oxides; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D008074:Lipoproteins; D009842:Oligopeptides; D010713:Phosphatidylcholines; D013113:Spin Labels; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine; D002794:Choline; C003959:TEMPO