Mechanism and role of membrane fusion by the atlastin GTPase

描述(申请人提供)：膜融合是一个必不可少的过程。对病毒和SNARE融合催化剂的研究揭示了一种克服融合能量障碍的共同策略，即锚定在相对膜上的复合体中有利的蛋白质折叠反应将脂质双层驱动在一起。然而，随着发现阿特拉斯汀(ATL)，一种膜锚定的动力蛋白相关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必需的，一个潜在的新范式已经出现。ATL是独一无二的，因为它是一种机械力化学酶，直接将GTP的水解与融合催化偶联。重要的是，目前还没有就这一点达成一致。最初的膜系留事件是在GTP水解酶的上游还是下游？核苷酸水解是否与系留或融合有关？这种动力冲程状的交叉构象是用来系膜还是用来融合它们？PI释放的作用是什么？这项提议旨在回答这些机械性的问题。在目标1中，我们建立了一种基于动态光散射和低温电子显微镜的膜系留实验，用于测量系留分离融合。这种分析方法被用来确定最初的系链步骤是否需要GTP水解和/或交叉。初步结果表明，系系严格依赖于GTP的水解，排除了一种模型，在该模型中，GTP结合的头部接触介导了系系的上游水解。系留项目是否也需要交叉目前正在测试中。结果将有助于梳理出交叉对系绳和融合步骤的贡献更大。在目标2中，我们将使用停流和急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在稳定整合到脂质双层中的ATL，因此结果应该有助于在融合反应的背景下深入了解全长ATL蛋白的构象耦合，从而帮助区分该领域当前几个不同的模型。最后，在目标3中，我们将描述一个 收集已知的导致运动神经性疾病遗传性痉挛性截瘫HSP的ATL1功能突变。这些将被检测体外融合活性以及细胞内内质网形成活性。对融合本身存在缺陷的突变体的进一步表征可能会进一步深入了解融合机制；而对能够融合膜但不能介导网络形成的突变体的表征，可能会揭示ATL催化融合的新的细胞调控机制。由于人类ATL1的突变导致运动神经疾病HSP，其基础尚不清楚，这些研究有可能阐明疾病的因果关系，并可能影响其治疗。

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.