Unexpected asymmetric distribution of cholesterol and phospholipids in equilibrium model membranes

Lipid compositional asymmetry across the leaflets of the plasma membrane is an ubiquitous feature in eukaryotic cells. How this asymmetry is maintained is thought to be primarily controlled by active transport of lipids between leaflets. This strategy is facilitated by the fact that long-tail phospholipids and sphingolipids diffuse through the lipid bilayer slowly-taking many hours or days. However, a lipid like cholesterol-which is the most abundant lipid in the plasma membrane of animal cells-has been harder to pinpoint in terms of its favored side. In this work we show that, when a saturated lipid is added to a mix of the unsaturated lipid palmitoyl-oleoyl-phosphatidylcholine (POPC) and cholesterol, both cholesterol and the long-tail phospholipids organize asymmetrically across the membrane's leaflets naturally. In these extruded unilamellar vesicles, most cholesterol as well as the saturated lipid-dipalmitoylphosphatidylcholine or sphingomyelin-segregated to the inner leaflet while POPC preferentially localized in the outer leaflet. This asymmetric arrangement generated a slight phospholipid number imbalance favoring the outer leaflet and thus opposite to where cholesterol and the saturated lipids preferentially partitioned. These results were obtained using magic-angle spinning nuclear magnetic resonance (MAS NMR) in combination with small-angle neutron scattering (SANS) using isotope labeling to differentiate lipid species. We suggest that sidedness in membranes can be driven by thermodynamic processes. In addition, our MAS NMR results show that the lower bound for cholesterol's flip-flop half-time at 45°C is 10 ms, which is at least two orders of magnitude slower than current MD simulations predict. This result stands in stark contrast to previous work that suggested that cholesterol's flip-flop half-time at 37°C has an upper bound of 10 ms.