Mechanism and Role of Membrane Fusion by the Atlastin GTPase

膜融合对于各种各样的生物过程是必不可少的。病毒与陷阱融合的研究 蛋白质催化剂揭示了一种共同的策略，即蛋白质固定在相对的膜上 经历有利的蛋白质折叠反应，将膜拉近并驱动脂质 核聚变所必需的重新安排。最近，一种新的融合范式出现了，发现 阿特拉斯汀(atlastin，ATL)是一种膜锚定的动力蛋白相关GTP酶，能触发合成脂质体的融合，是 内质网的分支形态所必需的。ATL有别于以前研究过的聚变催化剂 因为它是一种机械力化学酶，将GTP的水解与融合催化偶联。重要的是，虽然 取得了实质性进展，基本问题仍然悬而未决，在以下问题上仍缺乏共识 机制。在GTP存在下，ATL的N-末端胞液结构域发生反式二聚化和 一种交叉构象变化，假设将膜拉近到足够接近以驱动 核聚变。然而，在C末端胞浆内没有两亲性螺旋的情况下，不能观察到融合 ATL的尾部，暗示了一种顺序模型，其中交叉队形构成了 膜对接，尾巴随后起驱动脂质混合的作用。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 尾巴。因此，跨界是否主要用于调停对接，或者是否推动融合，需要 解决了。同样，GTP水解酶如何为融合反应循环提供能量也在争论中。主流机型 他们认为，GTP的水解力直接形成ATL交叉二聚体用于融合。然而， 我们最近的工作表明，GTP水解物是用来拆解而不是组装交叉的 二聚体，更可能用于在融合完成后回收融合机器。这一变化 这构成了一种范式转变，需要牢牢确立。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时间的融合中的二聚化 并确定交叉编队是否总是与融合重合，以及交叉编队是否 需要ATL尾部。在目标2中，我们将扩展我们对GTP水解反应周期的分析，从可溶的 根据膜的情况来确定GTP的水解性是否如我们新提出的那样 模型，只有在完成融合后才能发挥作用，以亚基回收为目的。总而言之， 拟议中的研究有望揭示GTP依赖的融合蛋白是如何 催化膜融合以及揭示不同融合催化剂之间的共同原理。另外， 因为人类ATL1的突变导致运动神经性疾病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.