Protease protection assays show polypeptide movement into the SecY channel by power strokes of the SecA ATPase
Protease protection assays show polypeptide movement into the SecY channel by power strokes of the SecA ATPase
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DOI：
10.15252/embr.202050905
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发表时间：
2020-09-24
期刊：
影响因子：
7.7
通讯作者：
Rapoport, Tom A.
中科院分区：
文献类型：
--
作者：
Catipovic, Marco A.;Rapoport, Tom A.
细菌分泌蛋白通过SecA ATP酶通过质膜中的蛋白传导SecY通道在细胞分裂后转运。在ATP水解周期中，SecA的双螺旋指和钳结构域经历了大的构象变化，但这些变化如何导致多肽运动尚不清楚。在这里，我们使用一个重建的纯化系统和蛋白酶保护试验表明，ATP结合SecA的结果在一段易位底物被推入通道。这种运动是通过指尖上保守残基的突变来阻止的。SecA夹的突变导致底物在ATP结合状态下的回滑。总之，这些数据支持易位的动力冲程模型，其中，在ATP结合时，双螺旋指状物将底物推入通道，在那里它被夹子固定，直到发生核苷酸水解。
Bacterial secretory proteins are translocated post-translationally by the SecA ATPase through the protein-conducting SecY channel in the plasma membrane. During the ATP hydrolysis cycle, SecA undergoes large conformational changes of its two-helix finger and clamp domains, but how these changes result in polypeptide movement is unclear. Here, we use a reconstituted purified system and protease protection assays to show that ATP binding to SecA results in a segment of the translocation substrate being pushed into the channel. This motion is prevented by mutation of conserved residues at the finger's tip. Mutation of SecA's clamp causes backsliding of the substrate in the ATP-bound state. Together, these data support a power stroke model of translocation in which, upon ATP binding, the two-helix finger pushes the substrate into the channel, where it is held by the clamp until nucleotide hydrolysis has occurred.