Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition

Abstract

The failure of most non-ionic detergents to release patches of DRM (detergent-resistant membrane) at 37 degrees C undermines the claim that DRMs consist of lipid nanodomains that exist in an L(o) (liquid ordered) phase on the living cell surface. In the present study, we have shown that inclusion of cations (Mg(2+), K(+)) to mimic the intracellular environment stabilizes membranes during solubilization sufficiently to allow the isolation of DRMs at 37 degrees C, using either Triton X-100 or Brij 96. These DRMs are sensitive to chelation of cholesterol, maintain outside-out orientation of membrane glycoproteins, have prolonged (18 h) stability at 37 degrees C, and are vesicles or sheets up to 150-200 nm diameter. DRMs containing GPI (glycosylphosphatidylinositol)-anchored proteins PrP (prion protein) and Thy-1 can be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking on the neuronal surface. Thy-1, but not PrP, DRMs are associated with actin. EM (electron microscopy) immunohistochemistry shows most PrP, and some Thy-1, to be clustered on DRMs, again maintaining their organization on the neuronal surface. For DRMs labelled for either protein, the bulk of the surface of the DRM is not labelled, indicating that the GPI-anchored protein is a minor component of its lipid domain. These 37 degrees C DRMs thus have properties expected of raft membrane, yet pose more questions about how proteins are organized within these nanodomains.