Mechanism and Role of Membrane Fusion by the Atlastin GTPase - Equipment Supplement

Atlastin GTPase 膜融合的机制和作用 - 设备补充

• 批准号: 10581823
• 负责人: Christina H Lee
• 金额: $ 4.24万
• 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/10581823
• 关键词: Biochemical; Biology; Biophysics; C-terminal; Catalysis; Cells; Chimeric Proteins; Consensus; Couples; Dimerization; Disease; Docking; Drosophila genus; Dynamin; Enzymes; Equipment; Etiology; Fluorescence Resonance Energy Transfer; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Spastic Paraplegia; Human; Human Activities; Hydrolysis; In Vitro; Light; Lipids; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Conformation; Morphology; Motor; Mutation; N-terminal; Nucleotides; Orthologous Gene; Outcome; Phase; Play; Process; Proteins; Reaction; Recycling; Regulation; Role; SNAP receptor; Tail; Therapeutic; Vesicle; Viral; Work; base; catalyst; dimer; insight; nervous system disorder; paralogous gene; protein folding; reconstitution

Membrane fusion is fundamental to biology. 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 vesicles and is required for the branched morphology of the ER. ATL is distinct from previously studied fusion proteins because it couples fusion catalysis to the hydrolysis of GTP. Importantly, while substantial progress has been made on the ATL fusion mechanism, a consensus has yet to be reached. In the presence of GTP, the N-terminal cytosolic domain of ATL undergoes trans dimerization through the GTPase domain 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 remains 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 the crossover dimer after fusion for the purpose of subunit recycling. In aim 1, we will use FRET probes to distinguish whether crossover dimerization can be uncoupled from fusion, or whether it plays a central role and therefore is inseparable from fusion. 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 recent work, functions only after the completion of fusion for the purpose of subunit recycling. Finally, previous studies on the ATL fusion mechanism have relied on use of the Drosophila ATL ortholog, the only ATL for which in vitro reconstitution of fusion activity has been achieved. This has limited our understanding of the role and potential regulation of the multiple ATL paralogs ATL1-3 present in humans. Thus, a third major goal is to reconstitute the fusion activity of the human paralogs. Altogether, the proposed studies promise to reveal the core principles of how GTP-dependent fusion proteins catalyze membrane fusion and to reveal sorely lacking insights into the mammalian ATL1-3 paralogs. 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.

膜融合是生物学的基础。对病毒和SNARE融合蛋白催化剂的研究表明， 锚定在相对膜上的蛋白质进行有利蛋白质折叠的常见策略 这些反应将细胞膜拉到紧密的位置，并驱动融合所需的脂质重排。 最近，一种新的融合范例已经出现，发现atlastin（ATL）是一种膜锚定的融合蛋白。 发动蛋白相关的GT3可以触发合成囊泡的融合，并且是细胞分支形态所必需的。 急诊室ATL与先前研究的融合蛋白不同，因为它将融合催化偶联到融合蛋白上。 GTP水解。重要的是，虽然在ATL融合机制方面取得了实质性进展，但 目前尚未达成共识。在GTP存在下，ATL的N-末端胞质结构域经历 通过GTTT结构域的反式二聚化和交叉构象变化假设绘制 细胞膜靠得足够近来推动融合。然而，在不存在一个或多个纳米颗粒的情况下，没有观察到融合。 ATL的C-末端胞质尾部内的两亲性螺旋，这表明了一个顺序模型，其中交叉 形成构成膜对接的上游步骤，并且尾部随后起作用以驱动脂质 混合另一方面，我们最近的工作表明，交叉二聚化为融合提供了能量 但没有解释尾巴的作用。因此，无论是交叉主要用于介导对接，还是 它推动了核聚变，需要解决。类似地，GTP水解如何为融合反应循环提供能量， 正在讨论中。流行的模型认为GTP的水解促进ATL交叉的形成 二聚体直接用于融合。然而，我们最近的工作表明，GTP水解有助于分解 融合后的交叉二聚体用于亚基再循环。在目标1中，我们将使用FRET探针来区分 交叉二聚化是否可以从融合中解偶联，或者它是否起着核心作用， 与融合密不可分。在目标2中，我们将从GTP水解反应循环扩展我们的分析。 可溶相的情况下，膜，以确定是否水解的GTP，建议我们 最近的工作，功能只有在融合完成后的亚基回收的目的。最后，前 ATL融合机制的研究依赖于果蝇ATL直系同源物的使用，这是唯一的ATL， 已经实现了融合活性的体外重建。这限制了我们对角色的理解， 人类中存在的多种ATL旁系同源物ATL 1 -3的潜在调节。因此，第三个主要目标是 重建人旁系同源物的融合活性。总之，拟议中的研究有望揭示 GTP依赖性融合蛋白如何催化膜融合的核心原理， 对哺乳动物ATL 1 -3旁系同源物的深入了解。因为人类ATL 1的突变会导致运动神经系统的 这些研究有可能阐明疾病的因果关系， 也可能影响其治疗方法。