Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum-plasma membrane contact sites
Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum-plasma membrane contact sites
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DOI：
10.1073/pnas.1503191112
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发表时间：
2015-04-21
影响因子：
11.1
通讯作者：
De Camilli, Pietro
中科院分区：
文献类型：
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作者：
Fernandez-Busnadiego, Ruben;Saheki, Yasunori;De Camilli, Pietro
内质网（ER）和质膜（PM）之间的密切联系在Ca2+稳态、信号传导和脂质代谢中起着重要作用。扩展的突触tagmins （E-Syts；酵母中的三白蛋白）是内质网锚定蛋白，介导内质网与PM的捆绑，并被认为介导两膜之间的脂质转移。E-Syt细胞质结构域包括一个突触蛋白样线粒体脂质结合蛋白（SMP）结构域，随后是E-Syt1中的5个C2结构域和E-Syt2/3中的3个C2结构域。在这里，我们使用低温电子断层扫描在分子分辨率上研究了e - syt介导的ER-PM接触的三维结构。在玻璃化的冷冻水合哺乳动物细胞中，过度表达单个E-Syts，其中e - syt依赖的接触是迄今为止主要的接触，ER-PM距离（19-22 nm）与E-Syts细胞质区域的氨基酸长度（即C2结构域的数量）相关。胞质Ca2+的升高缩短了e - syt1依赖接触位点的ER-PM距离。e - syt介导的接触在ER和PM之间显示出一个特征性的电子致密层。这些特征与暴露于基质相互作用分子1 （STIM1）和Ca2+通道Orai1介导的接触诱导条件下的细胞所观察到的明显不同，以及储存操作的Ca2+进入。在这些细胞中，内质网和PM之间的间隙由垂直于膜的丝状结构跨越。我们的研究结果定义了依赖e - syt的ER-PM接触的特定超微结构特征，并揭示了它们的结构可塑性，这可能影响ER和PM之间的串音以及E-Syts在两层脂质运输中的功能。
The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca2+ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt-mediated ER-PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt-dependent contacts were by far the predominant contacts, ER-PM distance (19-22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca2+ shortened the ER-PM distance at E-Syt1-dependent contacts sites. E-Syt-mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca2+ channel Orai1 as well as store operated Ca2+ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt-dependent ER-PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.