EFFECTS OF CHOLESTEROL DEPLETION BY CYCLODEXTRIN ON THE SPHINGOLIPID MICRODOMAINS OF THE PLASMA-MEMBRANE

Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored protei ns are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resis tant membranes) in isopycnic density gradients. Here the effects of me thyl-beta-cyclodextrin (MBCD), a specific cholesterol-binding agent th at neither binds nor inserts into the plasma membrane, were investigat ed on the sphingolipid microdomains of lymphocytes. MBCD released subs tantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, an d also other surface proteins including CD45, and intracellular Lck an d Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59 , and CD44, but only a negligible amount of caveolin. Most MBCD-releas ed Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 r eleased by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X- 100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Trit on X-100-isolated detergent-resistant membranes with MBCD extracted mo st of the cholesterol without affecting the buoyant properties of Thy- 1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2 ) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid micro domains in both vesicular and non-vesicular form.