Fluorescence-based evaluation of the partitioning of lipids and lipidated peptides into liquid-ordered lipid microdomains: a model for molecular partitioning into "lipid rafts"

Biophys J. 2000 Aug;79(2):919-33. doi: 10.1016/S0006-3495(00)76347-8.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Bridged Bicyclo Compounds / chemistry
  • Cholesterol / chemistry
  • Choline / chemistry
  • Cyclic N-Oxides
  • Fluorescent Dyes
  • Kinetics
  • Lipid Bilayers / chemistry*
  • Lipoproteins / chemistry*
  • Oligopeptides / chemistry*
  • Phosphatidylcholines / chemistry*
  • Spectrometry, Fluorescence / methods
  • Spin Labels

Substances

  • Bridged Bicyclo Compounds
  • Cyclic N-Oxides
  • Fluorescent Dyes
  • Lipid Bilayers
  • Lipoproteins
  • Oligopeptides
  • Phosphatidylcholines
  • Spin Labels
  • Cholesterol
  • 1,2-oleoylphosphatidylcholine
  • Choline
  • TEMPO