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

膜融合是生物学的基础。病毒和SNARE融合蛋白催化剂的研究揭示 锚定在相对膜上的蛋白质经历有利的蛋白质折叠的一种常见策略 将膜拉近并驱动融合所需的脂类重排的反应。 最近，一种新的融合范例出现了，发现阿特拉斯汀(ATL)是一种膜锚定的 动力蛋白相关的GTP酶可以触发合成囊泡的融合，是形成分支形态所必需的 急诊室。ATL不同于以前研究的融合蛋白，因为它将融合催化与 GTP的水解性。重要的是，虽然在ATL聚变机制方面取得了实质性进展，但 各方尚未达成共识。在GTP存在下，ATL的N-末端胞浆结构域经历 通过GTPase结构域的反式二聚和假设绘制的交叉构象变化 膜之间的距离足够紧密，以推动融合。然而，在没有核聚变的情况下，没有观察到融合。 ATL的C-末端胞浆尾部内的两亲性螺旋，暗示了一种序列模型，在该模型中， 形成构成膜对接的上游步骤，尾部随后起驱动脂质的作用 混合。另一方面，我们最近的工作表明，交叉二聚为聚变提供了能量 但并没有解释尾巴的作用。因此，交叉是主要用于调停对接，还是 它推动了核聚变，需要解决。同样，GTP水解酶如何为聚变反应循环提供能量仍然存在。 正在辩论中。流行的模型认为，GTP的水解力形成了ATL跨界 直接用于融合的二聚体。然而，我们最近的工作表明，GTP水解物用于分解 融合后的交叉二聚体用于亚基回收。在目标1中，我们将使用FRET探测器来区分 交叉二聚化是否可以从融合中分离出来，或者它是否发挥着核心作用，因此 离不开核聚变。在目标2中，我们将扩展我们对GTP水解反应周期的分析 以膜的上下文来确定GTP是否如我们所建议的那样被水解 最近的工作中，功能只有在融合完成后才能以亚基回收为目的。最后，上一篇 对ATL融合机制的研究依赖于使用果蝇ATL直系同源基因，这是唯一的ATL 融合活性的体外重建已经实现。这限制了我们对这一角色的理解 人类中存在的多个ATL同源基因ATL1-3的潜在调节。因此，第三个主要目标是 重建人类近亲的融合活性。总而言之，拟议的研究有望揭示 依赖GTP的融合蛋白如何催化膜融合的核心原理及其揭示的严重缺乏 对哺乳动物ATL1-3类鹦鹉的洞察。因为人类ATL1基因突变会导致运动神经病 基础尚不清楚的HSP障碍，这些研究有可能阐明疾病的因果关系 可能还会影响它的治疗方法。

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.