Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
Department of Biochemistry, Weill Cornell Medical College, New York, New York.
Traffic. 2018 Mar;19(3):198-214. doi: 10.1111/tra.12545.
Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.
双层脂质不对称性是真核细胞膜（PM）的基本特征。尽管 PM 磷脂的不对称性已有详细记录，但 PM 甾醇（如哺乳动物胆固醇和酵母麦角固醇）的跨双层分布尚不可靠。我们现在报告说，甾醇在酵母 PM 中呈不对称分布，大部分（80%）位于细胞质小叶中。通过利用甾醇营养缺陷型 hem1Δ 酵母菌株，我们获得了细胞，其中内源性麦角固醇被定量取代为脱氢麦角固醇（DHE），这是一种密切相关的荧光甾醇，在体内可有效地替代麦角固醇。使用荧光分光光度法和显微镜，我们发现当含有 DHE 的细胞暴露于膜不可渗透的碰撞淬灭剂（带荧光标记的磷脂酰胆碱和三硝基苯磺酸）时，只有不到 20%的 DHE 荧光被淬灭。只有在细胞被玻璃珠裂解或反复冻融破坏后，才能看到有效的淬灭，从而使淬灭剂能够进入细胞内部。淬灭的程度不受降低细胞内 ATP 水平、崩溃 PM 电化学梯度或影响肌动蛋白细胞骨架的处理影响。然而，在缺乏磷脂翻转酶的细胞中改变 PM 磷脂的不对称性会导致甾醇的跨双层分布更加对称。同样，当用 myriocin 处理细胞以降低 PM 鞘脂水平时，可观察到 DHE 的可淬灭池增加。我们推断，甾醇占 PM 双层脂质内层的45%，这一结果需要对 PM 脂质双层的结构模型进行修正。
