Isolation of Plasma Membrane Lipid Rafts

Recently, lipid raft plasma membrane domains were identified in plants based on their insolubility with the detergent Triton X-100 (Berczi and Horvath, 2003; Mongrand et al., 2004; Borner et al., 2005). First results obtained using thin-layer chromatography revealed that both quantitative and qualitative differences exist between the lipid composition of plant plasma membranes isolated from etiolated bean hypocotyls and green Arabidopsis leaves (Berci and Horvath, 2003). Later, protocols for the preparation of Triton X-100 detergent-resistant membranes (DRMs) from Arabidopsis callus were developed by Borner et al., (2005). Further, a proteomics approach using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. Among them, eight glycosylphosphatidylinositol (GPI)-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins and proteins of the stomatin/prohibitin/hypersensitive response family, were identified, suggesting that the DRMs originated from PM domains. Further analysis has shown that PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. DRMs were prepared by low-temperature detergent extraction. According to Borner et al., (2005), membranes were re-suspended in cold TNE (25 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, pH 7.5)

containing 4-6 : 1 (detergent-to-protein, w/w) excess of Triton X-100 (no detergent was used in the control extractions). The final concentration of Triton X-100 was approximately 2%. Extractions were performed on ice with shaking at 100 rpm for 35 min. Extracts were adjusted to 1.8 M sucrose (Suc)/TNE by addition of 3 volumes of cold 2.4 M Suc/TNE. Extracts were overlaid with Suc step gradients 1.6-1.4-1.2-0.15 M and centrifuged at 240 000 g in a Beck-man SW50.1 rotor for 18 h at 4 °C. DRMs were visible as off-white to white bands near the 1.2/1.4 and 1.4/1.6 M interfaces. Control fractions had a grey-green tinge. Fractions of 1 mL (0.5 mL above and 0.5 mL below the centre of the bands) were collected to harvest the DRM fractions and control fractions. Membranes were diluted with 4 volumes of cold TNE and pelleted at 100 000 g for 2 h in a Beckman 50Ti rotor.

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