Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.
Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.
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活酵母细胞质膜和液泡膜中磷脂酰肌醇 4-磷酸的纳米级结构域形成。
DOI：
10.1016/j.ejcb.2018.03.007
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发表时间：
2018
期刊：
影响因子：
6.6
通讯作者：
Fujita A.
中科院分区：
文献类型：
--
作者：
Tomioku KN;Shigekuni M;Hayashi H;Yoshida A;Futagami T;Tamaki H;Tanabe K;Fujita A.
在芽殖酵母Saccharomyces cerevisiae中，PtdIns（4）P作为高尔基复合体、内体系统和质膜中的重要信号分子，通过与PtdIns（4）P结合蛋白的直接相互作用参与多种细胞功能的控制。为了分析PtdIns（4）P在酵母细胞中的纳米级分布，我们采用了电子显微镜技术，该技术在酵母膜的冷冻断裂复制品上特异性地标记PtdIns（4）P。这种方法最大限度地减少了人为扰动的可能性，因为膜中的分子在物理上是固定在原位的。我们观察到PtdIns（4）P定位于质膜、高尔基体、液泡和泡状结构膜的胞质小叶，而不是外质小叶。PtdIns（4）P标记在内质网膜、核膜和线粒体的外膜和内膜中均未观察到。免疫金标记的点型分析表明，PtdIns（4）P在质膜和液泡膜上形成直径小于100 nm的团簇。在质膜的胞质小叶中有三种隔室。在本研究中，我们发现PtdIns（4）P特异性地定位于平坦的未分化质膜隔室。在液泡膜中，PtdIns（4）P集中在膜内颗粒（IMP）缺乏的筏状结构域，这些结构域与脂滴紧密结合，但在固定相中不以IMP几何分布模式围绕富含IMP的非筏结构域。这是第一份在纳米水平上显示出芽酵母细胞质膜和液泡膜中PtdIns（4）P微区形成的报告，这将阐明PtdIns（4）P在每个膜中的功能。
In budding yeastSaccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilisedin situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.