Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases)
Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases)
复制标题
酵母液泡膜脂质组从对数（一相）到稳定阶段（两相）的重塑
DOI：
10.1016/j.bpj.2023.01.009
复制
发表时间：
2023
影响因子：
3.4
通讯作者：
Keller, Sarah L.
中科院分区：
文献类型：
--
作者：
Reinhard, John;Leveille, Chantelle L.;Cornell, Caitlin E.;Merz, Alexey J.;Klose, Christian;Ernst, Robert;Keller, Sarah L.
在营养限制条件下，酿酒酵母芽生酵母从快速生长阶段(对数期)向静止阶段(稳定期)转变。这种转变伴随着液泡膜上的液-液相分离，液泡是一种内体细胞器。最近的工作表明，在液泡膜上形成的微米级结构域使酵母能够在应激状态下存活下来。一个悬而未决的问题是，哪些分子变化可能会导致这种膜相分离。在这里，我们对对数期和稳定期的液泡膜进行了类脂组学研究。在稳定期，当脂滴与液泡密切接触时，分离纯液泡膜是具有挑战性的。免疫分离法已被证明能成功地纯化具有高度细胞器特异性的对数阶段的液泡膜，但免疫分离稳定期的液泡膜是不可能的。在这里，我们开发了Mam3作为空泡免疫分离的诱饵蛋白，并展示了非空泡膜的低污染。我们发现，固定阶段的液泡膜含有高得惊人的磷脂酰胆碱脂(∼40%)，大约是对数阶段膜的两倍。此外，在静止阶段，由于更长和更饱和的酰基链，这些脂类具有更高的熔融温度。另一个令人惊讶的是，没有观察到甾醇含量的显著变化。这些脂体的变化主要反映在整个细胞水平，符合占主导地位的观点，即膜中的相分离至少需要存在三种类型的分子：高熔点的脂类、低熔点的脂类和甾醇。
Upon nutrient limitation, budding yeast ofSaccharomyces cerevisiaeshift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (∼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.