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.

中文翻译：

---

胆固醇和磷脂在平衡模型膜中的意外不对称分布


质膜小叶的脂质组成不对称性是真核细胞中普遍存在的特征。这种不对称性的维持方式被认为主要由脂质在小叶之间的主动运输来控制。长尾磷脂和鞘脂通过脂质双层缓慢扩散这一事实促进了这种策略——需要数小时或数天。然而，像胆固醇这样的脂质——它是动物细胞质膜中最丰富的脂质——就其偏爱的一面而言，更难确定。在这项工作中，我们表明，当将饱和脂质添加到不饱和脂质棕榈酰-油酰-磷脂酰胆碱 （POPC） 和胆固醇的混合物中时，胆固醇和长尾磷脂都会自然地在膜的小叶上不对称地组织。在这些挤压的单层囊泡中，大多数胆固醇以及饱和脂质（二棕榈酰磷脂酰胆碱或鞘磷脂）分离到内小叶，而 POPC 优先位于外小叶中。这种不对称的排列产生了轻微的磷脂数量不平衡，有利于外小叶，因此与胆固醇和饱和脂质优先分配的位置相反。这些结果是使用魔角自旋核磁共振 （MAS NMR） 与小角中子散射 （SANS） 相结合获得的，使用同位素标记来区分脂质种类。我们认为膜的侧向性可以由热力学过程驱动。此外，我们的 MAS NMR 结果表明，胆固醇在 45°C 时触发器半衰期的下限为 10 毫秒，这比当前 MD 模拟预测的至少慢了两个数量级。 这一结果与之前的工作形成鲜明对比，以前的工作表明胆固醇在 37°C 时触发器的半衰期上限为 10 毫秒。