The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion.
The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion.
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DOI：
10.1083/jcb.201609071
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发表时间：
2017-05-01
期刊：
影响因子：
--
通讯作者：
Lee TH
中科院分区：
文献类型：
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作者：
Winsor J;Hackney DD;Lee TH
GTPase atlastin通过GTPase头间的反式二聚化介导异型膜ER融合。Winsor等人使用诱变方法表明，在阿特拉斯蛋白头之间接触时，蛋白质同时表现出GTP水解催化的头对头二聚化和交叉构象转移，这些变化为融合提供了能量。内质网膜的同型融合由atlasatin GTPase催化。其机制包括GTP酶头之间的反式二聚化和有利的交叉构象转移，由GTP水解催化，将二聚体从“预融合”状态转化为“后融合”状态。然而，交叉形成是否真的为核聚变提供能量仍然不清楚，围绕它的事件顺序也不清楚。在这里，我们对阿特拉斯汀进行了突变，以选择性地破坏交叉构象的稳定性，并使用基于荧光的动力学分析来分析变异。所有变异同时进行二聚化和交叉，并以野生型的速率进行。然而，某些变体在交叉二聚体构象中是不稳定的，并且交叉二聚体的稳定性与脂质混合活性密切相关。然而，在所有的突变变体中，捆绑似乎都没有受损。结果表明，GTP水解同时催化系聚和脂质混合，但脂质混合所需的能量超过系聚所需的能量，通过交叉形成释放的全部能量是聚变所必需的。
The GTPase atlastin mediates homotypic membrane ER fusion through trans-dimerization between GTPase heads. Winsor et al. use a mutagenesis approach to show that, upon contact between atlastin heads, the proteins concurrently display GTP hydrolysis-catalyzed head-to-head dimerization and a crossover conformational shift, and these changes energize fusion. The homotypic fusion of endoplasmic reticulum membranes is catalyzed by the atlastin GTPase. The mechanism involves trans-dimerization between GTPase heads and a favorable crossover conformational shift, catalyzed by GTP hydrolysis, that converts the dimer from a “prefusion” to “postfusion” state. However, whether crossover formation actually energizes fusion remains unclear, as do the sequence of events surrounding it. Here, we made mutations in atlastin to selectively destabilize the crossover conformation and used fluorescence-based kinetic assays to analyze the variants. All variants underwent dimerization and crossover concurrently, and at wild-type rates. However, certain variants were unstable once in the crossover dimer conformation, and crossover dimer stability closely paralleled lipid-mixing activity. Tethering, however, appeared to be unimpaired in all mutant variants. The results suggest that tethering and lipid mixing are catalyzed concurrently by GTP hydrolysis but that the energy requirement for lipid mixing exceeds that for tethering, and the full energy released through crossover formation is necessary for fusion.