Abstract：
Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability became compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and caused a reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogues of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. Importantly, binding and membrane aggregation of natamycin was paralleled by a decrease of the dipole potential in the PM, and this effect was enhanced in the presence of myriocin. We conclude that ergosterol promotes binding and aggregation of natamycin in the yeast PM, which can be synergistically enhanced by inhibitors of sphingolipid synthesis.
摘要：
多烯大环内酯类是抗真菌物质，以甾醇依赖性方式与细胞相互作用。虽然被广泛使用，他们的行动方式知之甚少。这里,我们使用紫外线敏感（UV）显微镜显示抗真菌多烯那他霉素与酵母质膜（PM）结合，并导致碘化丙啶渗透到细胞中。就在膜通透性受损之前，我们观察到PM中游霉素的聚集与PM蛋白结构域无关.软X射线显微镜显示，游霉素的聚集与细胞变形和膜起泡平行。用麦角甾醇取代胆固醇可降低游霉素的结合力，并导致PM中游霉素的聚集减少。麦角甾醇合成的阻断需要通过ABC转运蛋白Aus1/Pdr11输入甾醇以确保纳他霉素结合。脱氢麦角甾醇（DHE）和胆甾烯（CTL）的定量成像，麦角固醇和胆固醇的两种类似物，分别,揭示了PM中基本均匀的横向固醇分布，排除纳他霉素与预组装的固醇结构域结合。使用肉豆蔻霉素消耗鞘脂增加了纳他霉素与酵母细胞的结合，可能是通过增加外部PM小叶中的麦角甾醇分数。重要的是,纳他霉素的结合和膜聚集与PM中偶极电位的降低平行，这种作用在多菌灵的存在下得到了增强。我们得出的结论是麦角甾醇促进酵母PM中那他霉素的结合和聚集，可以通过鞘脂合成抑制剂协同增强。
