Mechanism and Role of Membrane Fusion by the Atlastin GTPase

膜融合对于多种生物过程是必不可少的。病毒与SNARE融合的研究 蛋白质催化剂揭示了一种共同的策略，通过这种策略，蛋白质锚定在相对的膜上， 经历有利的蛋白质折叠反应，将膜拉到紧密的并置，并驱动脂质 融合所需的重排。最近，一个新的融合范例已经出现， atlastin（ATL）是一种膜锚定的动力蛋白相关的GT3，可以触发合成脂质体的融合， 这是ER的分支形态所需的。ATL不同于以前研究的聚变催化剂 因为它是一种将GTP水解与融合催化偶联的机械化学酶。重要的是，虽然 虽然取得了实质性进展，但基本问题仍未得到解决， 机制在GTP存在下，ATL的N-末端胞质结构域经历反式二聚化， 假设交叉构象变化将膜拉得足够近， 核聚变然而，在C-末端胞质内没有两亲性螺旋的情况下，没有观察到融合。 ATL的尾部，提出了一个顺序模型，其中交叉形成构成了上游步骤， 膜对接，并且尾部随后起作用以驱动脂质混合。另一方面，我们最近 工作表明，交叉二聚化提供了融合的能量，但没有解释的作用， 尾巴因此，交叉是否主要用于调解对接，或者它是否驱动融合，需要 解决了类似地，GTP水解如何为融合反应循环提供能量也在争论之中。流行车型 认为GTP的水解促进ATL交叉二聚体的形成，直接用于融合。然而，在这方面， 我们最近的工作表明，GTP水解作用是分解而不是组装交叉蛋白， 二聚体，并且更可能在融合完成后用于再循环融合机器。这种变化 这是一种范式转变，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一致，以及交叉形成是否 需要ATL的尾巴在目标2中，我们将从可溶性的GTP水解反应循环扩展我们的分析。 相的情况下，膜，以确定是否水解的GTP，建议我们的新 模型，功能只有在融合完成后的亚基回收的目的。一切器皿的银子 拟议的研究有望揭示广泛的机制见解如何GTP依赖性融合蛋白 催化膜融合以及揭示不同融合催化剂之间的共同原理。还有， 因为人ATL 1的突变引起运动神经障碍HSP（其基础尚不清楚）， 这些研究有可能揭示疾病的因果关系，也可能影响其治疗方法。

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.