Sterols are important lipid components of the plasma membrane (PM) in eukaryotic cells, but it is unknown how the PM retains sterols at a high concentration. Phospholipids are asymmetrically distributed in the PM, and phospholipid flippases play an important role in generating this phospholipid asymmetry. Here, we provide evidence that phospholipid flippases are essential for retaining ergosterol in the PM of yeast. A mutant in three flippases, Dnf1-Lem3, Dnf2-Lem3, and Dnf3-Crf1, and a membrane protein, Sfk1, showed a severe growth defect. We recently identified Sfk1 as a PM protein involved in phospholipid asymmetry. The PM of this mutant showed high permeability and low density, and many nutrient transporters failed to localize to the PM. Staining with the sterol probe filipin and the expression of a sterol biosensor revealed that ergosterol was not retained in the PM. Instead, ergosterol accumulated in an esterified form in lipid droplets. We propose that ergosterol is retained in the PM by the asymmetrical distribution of phospholipids and the action of Sfk1. Once phospholipid asymmetry is severely disrupted, sterols might be exposed on the cytoplasmic leaflet of the PM and actively transported to the endoplasmic reticulum by sterol transfer proteins.

中文翻译：

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磷脂翻转酶和Sfk1对麦角固醇在质膜中的保留至关重要

固醇是真核细胞质膜（PM）的重要脂质成分，但尚不清楚PM如何以高浓度保留甾醇。磷脂在PM中不对称分布，而磷脂脂蛋白酶在产生这种磷脂不对称性中起重要作用。在这里，我们提供的证据表明磷脂脂蛋白酶对于将麦角固醇保留在酵母的PM中至关重要。Dnf1-Lem3，Dnf2-Lem3和Dnf3-Crf1和三个膜蛋白Sfk1中的三个突变体的突变体显示出严重的生长缺陷。我们最近确定Sfk1是参与磷脂不对称的PM蛋白。该突变体的PM显示出高渗透性和低密度，并且许多营养转运蛋白未能定位于PM。用固醇探针菲律宾蛋白染色和固醇生物传感器的表达表明麦角固醇未保留在PM中。相反，麦角固醇以酯化形式积累在脂质小滴中。我们建议麦角固醇通过磷脂的不对称分布和Sfk1的作用保留在PM中。一旦磷脂的不对称性受到严重破坏，固醇可能会暴露于PM的细胞质小叶上，并通过固醇转移蛋白主动转运至内质网。