Membrane fusion by Drosophila atlastin does not require GTP hydrolysis.
Membrane fusion by Drosophila atlastin does not require GTP hydrolysis.
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DOI：
10.1091/mbc.e22-05-0164
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发表时间：
2022-12-01
影响因子：
3.3
通讯作者：
Lee, Tina H.
中科院分区：
文献类型：
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作者：
Crosby, Daniel;Lee, Tina H.
atlasatin (ATL) gtp酶通过反式二聚化和功率卒中样交叉构象重排来驱动内质网膜融合。融合依赖于GTP，但核苷酸水解的作用仍然存在争议。例如，不可水解的GTP类似物完全阻止了融合，这表明在ATL二聚化和交叉过程中需要GTP水解，但这没有回答ATL二聚体在融合后如何分解的问题。我们最近使用野生型果蝇ATL （DATL）的截断细胞质结构域和一种新的水解缺陷D127N变体进行单次翻转实验，发现二聚化和交叉始终先于GTP水解，而水解与二聚体的分解更紧密地吻合。此外，虽然非水解类似物可以结合DATL G结构域，但它们不能完全再现gtp结合状态。这预示着核苷酸的水解对于核聚变是必不可少的。在这里，我们报道了全长DATL的D127N变体驱动外叶和内叶膜融合，几乎没有检测到GTP的水解。然而，反式二聚体不能分解，随后的几轮融合也不能发生。我们的研究结果证实，ATL介导的融合是在gtp结合状态下驱动的，核苷酸水解有助于重置融合机制以进行循环。
Atlastin (ATL) GTPases undergo trans dimerization and a power strokelike crossover conformational rearrangement to drive endoplasmic reticulum membrane fusion. Fusion depends on GTP, but the role of nucleotide hydrolysis has remained controversial. For instance, nonhydrolyzable GTP analogs block fusion altogether, suggesting a requirement for GTP hydrolysis in ATL dimerization and crossover, but this leaves unanswered the question of how the ATL dimer is disassembled after fusion. We recently used the truncated cytoplasmic domain of wild-type Drosophila ATL (DATL) and a novel hydrolysis-deficient D127N variant in single turnover assays to reveal that dimerization and crossover consistently precede GTP hydrolysis, with hydrolysis coinciding more closely with dimer disassembly. Moreover, while nonhydrolyzable analogs can bind the DATL G domain, they fail to fully recapitulate the GTP-bound state. This predicted that nucleotide hydrolysis would be dispensable for fusion. Here we report that the D127N variant of full-length DATL drives both outer and inner leaflet membrane fusion with little to no detectable hydrolysis of GTP. However, the trans dimer fails to disassemble and subsequent rounds of fusion fail to occur. Our findings confirm that ATL mediated fusion is driven in the GTP-bound state, with nucleotide hydrolysis serving to reset the fusion machinery for recycling.
影响因子：
16.8
作者：
通讯作者：
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