LOSS OF PLASMA MEMBRANE LIPID ASYMMETRY CAN INDUCE ORDERED DOMAIN (RAFT) FORMATION

In some cases lipids in one leaflet of an asymmetric artificial lipid vesicle can suppress formation of ordered lipid domains (rafts) in the opposing leaflet. Whether suppression of domain formation might occur in plasma membranes was studied using plasma membrane vesicles (PMVs) from RBL-2H3 cells. Membrane domain formation and order was assessed by FRET and fluorescence anisotropy. Ordered domains in PMV prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ~37{degrees}C, while ordered domains in symmetric vesicles formed from extracted PMV lipids were stable to 55{degrees}C, indicating that stability of ordered domains was substantially less in intact PMV. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting that intact PMV have some degree of lipid asymmetry. This was confirmed by annexin binding showing that NEM PMV are much more asymmetric than PMV formed by dithiothreitol/paraformaldehyde treatment. Stabilization of ordered domain formation, and increased membrane order at low temperature was also observed after detergent solubilization of PMV followed by membrane reconstitution via dilution from detergent, which also should destroy asymmetry, even though membrane proteins remained associated with the reconstituted vesicles. Similar changes in domain formation and membrane order were observed after detergent reconstitution of artificial asymmetric lipid vesicles. PMV ordered domain stability was not increased by digesting peripheral domains of PMV proteins with proteinase K. We conclude loss of PMV lipid asymmetry can induce ordered domain formation. Dynamic control of asymmetry may regulate ordered domain formation in plasma membranes.