Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada.
Keenan Research Centre, St. Michael's Hospital, Toronto, Canada.
Elife. 2018 Nov 13;7:e38493. doi: 10.7554/eLife.38493.
The plasma membrane in mammalian cells is rich in cholesterol, but how the cholesterol is partitioned between the two leaflets of the plasma membrane remains a matter of debate. Recently, Liu et al. used domain 4 (D4) of perfringolysin O as a cholesterol sensor to argue that cholesterol is mostly in the exofacial leaflet (Liu et al., 2017). This conclusion was made by interpreting D4 binding in live cells using in vitro calibrations with liposomes. However, liposomes may be unfaithful in mimicking the plasma membrane, as we demonstrate here. Also, D4 binding is highly sensitive to the presence of cytosolic proteins. In addition, we find that a D4 variant, which requires >35 mol% cholesterol to bind to liposomes in vitro, does in fact bind to the cytoplasmic leaflet of the plasma membrane in a cholesterol-dependent manner. Thus, we believe, based on the current evidence, that it is unlikely that there is a significantly higher proportion of cholesterol in the exofacial leaflet of the plasma membrane compared to the cytosolic leaflet.
哺乳动物细胞的质膜富含胆固醇，但胆固醇如何分配到质膜的两个叶中仍然存在争议。最近，Liu 等人使用产气荚膜梭菌溶细胞素的结构域 4（D4）作为胆固醇传感器，认为胆固醇主要位于质膜的外叶（Liu 等人，2017）。这一结论是通过使用体外脂质体校准来解释 D4 在活细胞中的结合得出的。然而，正如我们在这里所证明的，脂质体可能不能忠实地模拟质膜。此外，D4 结合对细胞质蛋白的存在非常敏感。此外，我们发现 D4 的一种变体，其在体外需要 >35mol%的胆固醇才能与脂质体结合，但实际上以胆固醇依赖性的方式与质膜的细胞质叶结合。因此，基于目前的证据，我们认为与细胞质叶相比，质膜的外叶中胆固醇的比例不太可能显著更高。
