Mechanism and role of membrane fusion by the atlastin GTPase

atlastin GTPase 膜融合的机制和作用

• 批准号: 9314318
• 负责人: Christina H Lee
• 金额: $ 26.06万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9314318
• 关键词: Agreement; Alleles; Binding; Biochemical; Biological Assay; Biophysics; Catalysis; Cells; Collection; Complex; Coupled; Couples; Coupling; Cryoelectron Microscopy; Cytoplasmic Tail; Data; Defect; Dimerization; Disease; Dynamin; Enzymes; Etiology; Event; GTP Binding; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Head; Health; Hereditary Spastic Paraplegia; Human; Hydrolysis; In Vitro; Individual; Kinetics; Length; Light; Lipid Bilayers; Lipids; Liposomes; Mammalian Cell; Measurement; Measures; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Conformation; Morphology; Motion; Motor; Mutation; Neurons; Nucleotides; Organelles; Outcome; Play; Point Mutation; Power stroke; Process; Protein Conformation; Protein Isoforms; Proteins; Reaction; Regulation; Role; Rotation; SNAP receptor; Structure; Synaptic Transmission; Testing; Therapeutic; Variant; Viral; Virus Diseases; Work; Yeasts; base; catalyst; dimer; in vitro Assay; insight; light scattering; mutant; nervous system disorder; protein folding; public health relevance; trafficking

DESCRIPTION (provided by applicant): Membrane fusion is an essential process. Studies on viral and SNARE fusion catalysts have revealed a common strategy to overcoming the energy barrier to fusion, wherein favorable protein-folding reactions within complexes anchored in opposing membranes drive lipid bilayers together. However, a potentially new paradigm has arisen with discovery that atlastin (ATL), a membrane-anchored dynamin-related GTPase, can trigger fusion of synthetic liposomes, and is required for the branched morphology of the ER. ATL is unique because it is a mechanochemical enzyme that directly couples hydrolysis of GTP to fusion catalysis. Importantly there is not yet agreement on how this works. Is the initial membrane-tethering event upstream or downstream of GTP hydrolysis? Is nucleotide hydrolysis coupled to tethering, or fusion? Does the power stroke-like crossover conformation serve to tether membranes or to fuse them? What is the role of Pi release? This proposal is directed at answering these mechanistic questions. In aim 1 we have established a membrane-tethering assay, based on dynamic light scattering and cryo-electron microscopy, to measure tethering apart from fusion. This assay is being used to determine whether the initial tethering step requires GTP hydrolysis and/or crossover. Preliminary results indicate that tethering depends strictly on hydrolysis of GTP, ruling out a model in which a GTP-bound head contact mediates tethering upstream of hydrolysis. Whether crossover is also required for the tethering event is currently being tested. The result will help tease apart whether crossover contributes more to the tethering or the fusion step. In aim 2 we will use stopped-flow and quench-flow approaches to better understand the coupling between GTP hydrolysis, Pi release, crossover dimer formation and lipid mixing. Because much of our analysis will focus on ATL that is stably integrated into the lipid bilayer, the outcomes should give insights into conformational coupling for the full-length ATL protein in the context of the fusion reaction, thereby helping to distinguih amongst several contrasting models currently in the field. Finally, in aim 3 we will characterize a collection of known functional mutations in ATL1 that cause the motor neurological disease hereditary spastic paraplegia HSP. These will be assayed for in vitro fusion activity as well as for ER network forming activity in cells. Further characterization of mutants, defective in fusion per se, may give further insights into the fusion mechanism; whereas, characterization of mutants that can fuse membranes but yet cannot mediate network formation, may reveal new cellular regulatory mechanisms for ATL-catalyzed fusion. Because mutations in human ATL1 cause the motor neurological disorder HSP whose basis is not understood, these studies have the potential to shed light on disease causality and possibly also impact its therapeutics.

描述（由申请人提供）：膜融合是一个重要的过程。对病毒和 SNARE 融合催化剂的研究揭示了克服融合能量障碍的共同策略，其中锚定在相对膜上的复合物内有利的蛋白质折叠反应将脂质双层驱动在一起。然而，随着发现 atlastin (ATL)（一种膜锚定动力相关 GTP 酶）可以触发合成脂质体的融合，并且是 ER 分支形态所必需的，一个潜在的新范例已经出现。 ATL 是独特的，因为它是一种机械化学酶，可直接将 GTP 水解与融合催化偶联。重要的是，目前尚未就其运作方式达成一致。初始膜束缚事件是在 GTP 水解的上游还是下游？核苷酸水解是否与束缚或融合有关？类似动力冲程的交叉构象是否用于束缚膜或融合它们？ Pi发布的作用是什么？该提案旨在回答这些机械问题。在目标 1 中，我们建立了一种基于动态光散射和冷冻电子显微镜的膜束缚测定，以测量融合以外的束缚。该测定用于确定初始束缚步骤是否需要 GTP 水解和/或交叉。初步结果表明，束缚严格依赖于 GTP 的水解，排除了 GTP 结合的头部接触介导水解上游束缚的模型。目前正在测试网络共享事件是否也需要交叉。结果将有助于弄清楚交叉是否对束缚或融合步骤贡献更大。在目标 2 中，我们将使用停流和猝灭流方法来更好地了解 GTP 水解、Pi 释放、交叉二聚体形成和脂质混合之间的耦合。由于我们的大部分分析将集中于稳定整合到脂质双层中的 ATL，因此结果应该能够深入了解融合反应背景下全长 ATL 蛋白的构象耦合，从而有助于区分当前该领域的几种对比模型。最后，在目标 3 中，我们将描述 ATL1 已知功能突变的集合，这些突变会导致运动神经系统疾病遗传性痉挛性截瘫 HSP。将对其进行体外融合活性以及细胞内 ER 网络形成活性的测定。对融合本身有缺陷的突变体的进一步表征，可能会进一步了解融合机制；然而，对能够融合膜但不能介导网络形成的突变体进行表征，可能会揭示 ATL 催化融合的新细胞调节机制。由于人类 ATL1 突变会导致运动神经系统疾病 HSP，其基础尚不清楚，因此这些研究有可能揭示疾病因果关系，并可能影响其治疗。