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

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,, (liquid ordered) phase oil the living cell surface. In the present study, we have shown that inclusion of cations (Mg2+, 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 tip to 150-200 nm diameter. DRMs containing GPI (glycosylphosphatidylinositol)-anchored proteins PrP (prion protein) and Thy-1 call be separated by immunoaffinity isolation, in keeping with their separate organization and trafficking oil the neuronal surface. Thy-1, but not PrR DRMs are associated with actin. EM (electron microscopy) immunohistochemistry shows most PrP, and some Thy-1, to be clustered oil DRMs, again maintaining their organization oil the neuronal Surface. For DRMs labelled for either protein, the bulk of the surface of the DRMs 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]low proteins are organized within these nanodomains.