Molecular Structure and Function of an Endoplasmic Reticulum-Mitochondrion Tether

内质网-线粒体系链的分子结构和功能

• 批准号: 10248518
• 负责人: Pascal Francois Egea
• 金额: $ 30.62万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248518
• 关键词: 3-Dimensional; Affect; Alzheimer' s Disease; Architecture; Binding; Biochemical; Biological Assay; Biological Models; Biophysics; Cell physiology; Cells; Cellular biology; Complex; Cryoelectron Microscopy; Crystallization; Diabetes Mellitus; Disease; Electron Microscopy; Endoplasmic Reticulum; Eukaryota; Eukaryotic Cell; Fluorescence; Fluorescence Microscopy; Genetic; Genetic Screening; Goals; Guanosine Triphosphate Phosphohydrolases; Hand; Human; Hybrids; Image; Impairment; In Vitro; Label; Lecithin; Ligands; Link; Lipid Binding; Lipids; Liposomes; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Membrane; Metabolic Diseases; Methods; Mitochondria; Mitochondrial Membrane Protein; Modeling; Molecular; Molecular Structure; Monitor; Names; Negative Staining; Neurodegenerative Disorders; Obesity; Organelles; Organism; Parkinson Disease; Phospholipids; Protein Binding Domain; Proteins; Proteomics; Public Health; Publishing; Recombinants; Regulation; Research; Resolution; Role; Route; Scaffolding Protein; Signal Transduction; Site; Source; Structural Models; Structure; System; Tail; Tubular formation; Ursidae Family; X ray diffraction analysis; X-Ray Crystallography; Yeasts; base; biophysical techniques; cellular imaging; design; human disease; imaging modality; improved; insight; light scattering; lipid metabolism; lipid transport; membrane assembly; metabolomics; mitochondrial membrane; mutant; nanodisk; particle; protein complex; proteoliposomes; reconstitution; reconstruction; stoichiometry; surfactant; vesicle transport

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.

抽象的 真核细胞的特点是其精致的区室化。膜结合细胞器的形成 高度动态且互连的网络。这种复杂性使得之间存在永久的串扰 细胞器是协调细胞功能的必需品。膜的紧密并置 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 由各种细胞器束缚蛋白复合物介导。小代谢物和信使，例如 磷脂 (PL) 和 Ca2+ 在这些膜接触位点 (MCS) 进行交换。了解 调节细胞器之间相互作用的分子机制将为这一问题提供新的见解 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），在模型真核生物酵母中鉴定的系绳，以及 在内质网-线粒体连接处发挥作用，也称为线粒体相关膜 (MAM)。尽管 许多团体研究 MCS，其中大多数使用基于遗传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。我们运用生物化学、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，但除了其亚基组成之外，没有任何其他成分 else 的架构和 MAM 组装模式已为人所知。其中三个亚基，ER- 锚定蛋白 Mmm1、可溶性亚基 Mdm12 和线粒体膜蛋白 Mdm34， 含有 SMP 结构域，这是一种脂质结合蛋白结构域，专门存在于位于酵母 MCS 的蛋白质中 对人类。使用质谱分析法，我们表明 Mdm12 优先结合磷脂酰胆碱，而 我们的 Mmm1/Mdm12 异源四聚体的 17 Å 分辨率负染色电镜结构揭示了可溶性 SMP 结构域不仅可以结合磷脂，还可以作为特定的蛋白质支架来组装系链。 这促使我们提出了第一个非常基本的结构模型，用于 ERMES 介导的 PL 交换 在MAM。缺乏体外重建的生化易处理系统阻碍了明确的努力 建立 ERMES 的职能。在这里，我们建议完成ERMES的重构来表征 其亚基化学计量并鉴定其真正的脂质配体。使用纯化的亚基，我们将重构 ERMES 的 SMP 核心在两种不同标记类型的脂蛋白体上，并评估束缚和 PL 使用基于荧光的生物物理方法进行体外交换。通过这个系统，我们还将剖析 尾锚定线粒体 GTPase Gem1（一个完整的 ERMES）对 ERMES 的调节机制 亚基被证明可以控制系链组装和脂质交换。最后，我们将确定结构 ERMES 通过“混合”方法结合单粒子高分辨率冷冻电镜分析来改善 我们当前重建的分辨率，以及最近获得的复合物晶体的 X 射线衍射分析。

项目成果

Moving Lipids, by the Numbers.

• DOI: 10.1177/25152564221103080
• 发表时间: 2022-01
• 期刊: Contact (Thousand Oaks (Ventura County, Calif.))
• 作者: Egea, Pascal F

Mechanisms of Non-Vesicular Exchange of Lipids at Membrane Contact Sites: Of Shuttles, Tunnels and, Funnels.

• DOI: 10.3389/fcell.2021.784367
• 发表时间: 2021
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5
• 作者: Egea PF

Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes.

傅立叶转化离子回旋共振质谱法作为表征多聚体膜蛋白复合物的平台。

• DOI: 10.1007/s13361-017-1799-4
• 发表时间: 2018-01
• 期刊: Journal of the American Society for Mass Spectrometry
• 影响因子: 3.2
• 作者: Lippens JL;Nshanian M;Spahr C;Egea PF;Loo JA;Campuzano IDG
• 通讯作者: Campuzano IDG

Highlighting membrane protein structure and function: A celebration of the Protein Data Bank.

• DOI: 10.1016/j.jbc.2021.100557
• 发表时间: 2021-01
• 期刊: The Journal of biological chemistry
• 作者: Li F;Egea PF;Vecchio AJ;Asial I;Gupta M;Paulino J;Bajaj R;Dickinson MS;Ferguson-Miller S;Monk BC;Stroud RM
• 通讯作者: Stroud RM