Molecular Structure and Function of an Endoplasmic Reticulum-Mitochondrion Tether

摘要 真核细胞的特征在于其精细的区室化。膜结合细胞器形成 高度动态且相互关联的网络。这种复杂性使得 细胞器是协调细胞功能的必要条件。这些细胞膜紧密地并列在一起， 不同类型的细胞器对于细胞内物质和信息的受控交换至关重要， 是由各种细胞器-束缚蛋白复合物介导的。小的代谢物和信使，如 磷脂（PL）和Ca 2+在这些膜接触位点（MCS）交换。了解 调节细胞器之间相互作用的分子机制将为这一点提供新的见解 真核细胞生物学的基本方面。我们的研究重点是内质网- 线粒体相遇结构（ERMES），在模式真核生物酵母中鉴定的系链，以及 在ER-线粒体连接处起作用，也称为线粒体相关膜（MAMs）。而 许多研究小组对MCSs进行了研究，其中大多数采用基于基因筛选和细胞成像的方法 与蛋白质组学或代谢组学相结合的方法。我们将生物化学，生物物理和结构 在分子结构水平上表征ERMES的方法，以了解其精确的细胞功能 和作用机制。ERMES由五个亚基组成，但除了它的亚基组成外， 在MAMs上，其他人都知道它的架构和组装模式。其中三个亚单位，ER- 锚定蛋白Mmm 1、可溶性亚基Mdm 12和线粒体膜蛋白Mdm 34， 含有SMP结构域，一种仅在位于酵母MCS的蛋白质中发现的脂质结合蛋白结构域 对人类使用质谱，我们发现Mdm 12优先结合磷脂酰胆碱， 我们对Mmm 1/Mdm 12异源四聚体的17-nm分辨率负染电镜结构显示， SMP结构域不仅结合磷脂，而且还作为特定的蛋白质支架来组装系链。 这使我们提出了ERMES介导的PL交换的第一个和非常基本的结构模型 在MAMs。缺乏一个在体外重建的生化易处理的系统阻碍了确定 建立ERMES的职能。在这里，我们建议完成ERMES的重建，以表征 其亚基化学计量并鉴定其真正脂质配体。使用纯化的亚基，我们将重组 ERMES的SMP核心在两种不同标记类型的脂蛋白体上，并评估栓系和PL 在体外使用基于荧光的生物物理方法进行交换。有了这个系统，我们还将剖析 ERMES调控机制的尾锚定线粒体GTME 1，一个完整的ERMES 该亚基被证明控制系链组装和脂质交换。最后，我们将确定 ERMES通过“混合”方法结合单颗粒高分辨率冷冻EM分析，以提高 我们目前重建的分辨率，和最近获得的晶体的复杂的X射线衍射分析。

ABSTRACT Eukaryotic cells are characterized by their exquisite compartmentalization. Membrane-bound organelles form highly dynamic and interconnected networks. This complexity makes a permanent crosstalk between the organelles a necessity for the coordination of cellular functions. The tight juxtaposition of membranes from different types of organelles is essential to the controlled exchanges of matter and information within cells and is mediated by various organelle-tethering protein complexes. Small metabolites and messengers such as phospholipids (PLs) and Ca2+ are exchanged at these membrane contact sites (MCSs). Understanding the molecular mechanisms that regulate interactions between organelles will offer new insights into this fundamental aspect of eukaryotic cell biology. Our research focuses on the Endoplasmic Reticulum- Mitochondrion Encounter Structure (ERMES), a tether identified in the model eukaryote organism yeast, and functioning at ER-mitochondrial junctions also named Mitochondrion-Associated Membranes (MAMs). While many groups investigate MCSs, most of them use approaches based on genetic screens and cellular imaging methods combined with proteomics or metabolomics. We bring to bear biochemical, biophysical and structural methods to characterize ERMES at the level of molecular structure to understand its precise cellular functions and mechanism of action. ERMES is composed of five subunits, but besides its subunit composition nothing else is known about its architecture and mode of assembly at MAMs. Three of these subunits, the ER- anchored protein Mmm1, the soluble subunit Mdm12, and the mitochondrial membrane protein Mdm34, contain a SMP domain, a lipid-binding protein domain exclusively found in proteins located at MCSs from yeast to humans. Using mass-spectrometry, we showed that Mdm12 preferentially binds phosphatidylcholines while our 17-Å resolution negative-stain EM structure of the Mmm1/Mdm12 hetero-tetramer revealed that the soluble SMP domains not only bind phospholipids but also function as specific protein scaffolds to assemble the tether. This led us to propose a first and very rudimentary structural model for the ERMES-mediated exchange of PLs at MAMs. The lack of a biochemically tractable system reconstituted in vitro has hindered efforts to definitely establish the function(s) of ERMES. Here, we propose to complete the reconstitution of ERMES to characterize its subunit stoichiometry and identify its bona-fide lipid ligands. Using purified subunits we will reconstitute the SMP-core of ERMES on two distinctly labeled types of proteoliposomes and assess tethering and PL exchange using fluorescence-based biophysical methods in vitro. With this system, we will also dissect the mechanisms of ERMES regulation by the tail-anchored mitochondrial GTPase Gem1, an integral ERMES subunit that was shown to control tether assembly and lipid exchange. Last, we will determine the structure of ERMES by a `hybrid' approach combining single particle high-resolution cryo-EM analysis to improve the resolution of our current reconstructions, and X-ray diffraction analysis of recently obtained crystals of complex.