Mechanism and Role of Membrane Fusion by the Atlastin GTPase

Atlastin GTPase 膜融合的机制和作用

• 批准号: 10436798
• 负责人: Christina H Lee
• 金额: $ 30.58万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436798
• 关键词: Binding; Biochemical; Biological Assay; Biological Process; Biophysics; C-terminal; Catalysis; Cells; Chimeric Proteins; Consensus; Couples; Cytoplasmic Tail; Dimerization; Disease; Docking; Drosophila melanogaster; Dynamin; Enzymes; Etiology; Fluorescence Resonance Energy Transfer; Foundations; Future; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Spastic Paraplegia; Human; Hydrolysis; Kinetics; Length; Light; Lipids; Liposomes; Measurement; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Conformation; Monitor; Morphology; Motor; Mutation; N-terminal; Nucleotides; Organelles; Outcome; Phase; Point Mutation; Process; Protein Precursors; Proteins; Reaction; Recycling; Role; SNAP receptor; Scheme; Structure; Tail; Testing; Therapeutic; Variant; Viral; Virus Diseases; Work; analog; base; catalyst; dimer; driving force; insight; nervous system disorder; novel; prevent; protein folding; protein protein interaction; trafficking

Membrane fusion is essential for a wide variety of biological processes. Studies on viral and SNARE fusion protein catalysts have revealed a common strategy by which proteins anchored in opposing membranes undergo favorable protein-folding reactions that draw the membranes into close apposition and drive the lipid rearrangements necessary for fusion. More recently, a new fusion 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 distinct from previously studied fusion catalysts because it is a mechanochemical enzyme that couples hydrolysis of GTP to fusion catalysis. Importantly, while substantial progress has been made, basic questions remain unresolved and there is still little consensus on mechanism. In the presence of GTP, the N-terminal cytosolic domain of ATL undergoes trans dimerization and a crossover conformational change hypothesized to draw membranes sufficiently close together to drive fusion. However, no fusion is observed in the absence of an amphipathic helix within the C-terminal cytosolic tail of ATL, suggesting a sequential model in which crossover formation constitutes an upstream step for membrane docking, and the tail functions subsequently to drive lipid mixing. On the other hand, our recent work suggests that crossover dimerization provides the energy for fusion, but does not explain the role of the tail. Thus whether crossover serves primarily to mediate docking, or whether it drives fusion, needs to be resolved. Similarly, how GTP hydrolysis energizes the fusion reaction cycle is under debate. Prevailing models have held that the hydrolysis of GTP powers formation of the ATL crossover dimer directly for fusion. However, our recent work suggests that GTP hydrolysis serves to disassemble, rather than to assemble, the crossover dimer, and more likely serves to recycle the fusion machinery after the completion of fusion. This change constitutes a paradigm shift, and needs to be firmly established. In aim 1 we will ascertain the role of crossover dimerization in fusion using FRET probes to monitor the timing of crossover dimerization relative to lipid mixing and determine whether crossover formation invariably coincides with fusion, and whether crossover formation requires the ATL tail. In aim 2, we will extend our analysis of the GTP hydrolysis reaction cycle from the soluble phase to the context of membranes to ascertain whether the hydrolysis of GTP, as suggested by our new model, functions only after the completion of fusion for the purpose of subunit recycling. Altogether, the proposed studies promise to reveal broad mechanistic insights into how GTP-dependent fusion proteins catalyze membrane fusion as well as to uncover shared principles among disparate fusion catalysts. Also, 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.

膜融合对于多种生物过程至关重要。病毒与 SNARE 融合的研究 蛋白质催化剂揭示了蛋白质锚定在相对膜上的共同策略 经历有利的蛋白质折叠反应，使膜紧密贴合并驱动脂质 融合所需的重新排列。最近，随着发现出现了一种新的融合范式 atlastin (ATL) 是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 内质网分支形态所需的。 ATL 与之前研究的聚变催化剂不同 因为它是一种将 GTP 水解与融合催化耦合的机械化学酶。重要的是，虽然 取得了实质性进展，但基本问题仍未解决，各方仍缺乏共识 机制。在 GTP 存在的情况下，ATL 的 N 端胞质结构域发生反式二聚化并 假设交叉构象变化使膜足够靠近以驱动 融合。然而，在 C 端胞质内不存在两亲性螺旋的情况下，没有观察到融合。 ATL 的尾部，表明有一个顺序模型，其中交叉形成构成了 ATL 的上游步骤 膜对接，尾部随后发挥作用以驱动脂质混合。另一方面，我们最近 研究表明交叉二聚化为聚变提供了能量，但没有解释交叉二聚化的作用 尾巴。因此，交叉是否主要用于介导对接，或者是否驱动融合，需要确定 解决了。同样，GTP 水解如何为聚变反应循环提供能量也存在争议。流行型号 认为GTP的水解能直接形成ATL交叉二聚体以进行融合。然而， 我们最近的工作表明，GTP 水解作用是分解而不是组装交叉 二聚体，更有可能在聚变完成后回收聚变机器。这个改变 构成了范式转变，需要牢固确立。在目标 1 中，我们将确定交叉的作用 使用 FRET 探针监测融合中的二聚化相对于脂质混合的交叉二聚化的时间 并确定交叉形成是否总是与融合同时发生，以及交叉形成是否一致 需要 ATL 尾部。在目标 2 中，我们将从可溶性物质扩展 GTP 水解反应循环的分析。 相到膜的背景以确定 GTP 是否水解，正如我们的新研究所建议的 模型中，只有在完成融合后才发挥作用，以达到亚基回收的目的。总而言之， 拟议的研究有望揭示 GTP 依赖性融合蛋白如何产生广泛的机制见解 催化膜聚变并揭示不同聚变催化剂之间的共同原理。还， 因为人类 ATL1 的突变会导致运动神经系统疾病 HSP，其基础尚不清楚， 这些研究有可能揭示疾病的因果关系，并可能影响其治疗。