Mechanism and role of membrane fusion by the atlastin GTPase

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，其中锚定在相对膜中的复合物内的有利蛋白质折叠反应将脂质双层驱动在一起。然而，一个潜在的新的范例已经出现，发现atlastin（ATL），膜锚定的动力蛋白相关的GTdR，可以触发合成脂质体的融合，并需要为ER的分支形态。ATL是独特的，因为它是一种机械化学酶，直接将GTP水解与融合催化偶联。重要的是，尚未就如何运作达成一致意见。初始的膜束缚事件是在GTP水解的上游还是下游？核苷酸水解与束缚或融合耦合吗？这种类似于强力划动的交叉构象是用来束缚膜还是用来融合膜？Pi释放的作用是什么？本建议旨在回答这些机械问题。在目标1中，我们建立了一个膜拴系测定，基于动态光散射和冷冻电子显微镜，测量拴系除了融合。本试验用于确定初始拴系步骤是否需要GTP水解和/或交叉。初步结果表明，拴系严格依赖于水解的GTP，排除了一个模型，其中GTP结合的头部接触介导拴系上游的水解。目前正在测试网络共享事件是否也需要交叉。结果将有助于梳理出交叉对系留或融合步骤的贡献更大。在目标2中，我们将使用停流和猝灭流方法来更好地理解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ATL，是稳定地整合到脂质双层，结果应该给的融合反应的情况下，全长ATL蛋白的构象耦合的见解，从而有助于目前在该领域中的几个对比模型之间的竞争。最后，在目标3中，我们将描述 收集了导致运动神经疾病遗传性痉挛性截瘫HSP的ATL 1中已知的功能突变。将测定这些的体外融合活性以及细胞中的ER网络形成活性。进一步表征的突变体，融合本身的缺陷，可能会给融合机制的进一步见解，而突变体，可以融合膜，但还不能介导网络的形成，表征可能会揭示新的细胞调控机制ATL催化的融合。由于人类ATL 1突变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，并可能影响其治疗方法。

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.