Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover
Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover
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对 SecA 驱动的蛋白质转运的精确测量揭示了与 ATP 周转的间接耦合
DOI：
10.1101/2020.05.08.084160
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Allen W
中科院分区：
文献类型：
--
作者：
Allen W
普遍保守的Sec系统是细胞用于跨膜转运蛋白质的主要方法。直到最近，测量活性-了解生物系统如何工作的先决条件-一直限于时间分辨率差的不连续蛋白质转运测定，或用作干扰该过程的报告员大，非天然标签。基于分裂超亮荧光素酶（NanoLuc）的测定的开发改变了这一点。在这里，我们利用这项技术来解开构成细菌翻译后转运的步骤。在部署的条件下，模型前蛋白质底物proSpy的转运以每分钟200个氨基酸发生，数据最佳拟合为一系列大的、约30个氨基酸的步骤，每个步骤与许多（100个）ATP水解事件偶联。在此之前，没有证据表明存在明显的限速启动事件。动力学建模表明，SecA驱动的运输活动是由底物（多肽）浓度梯度促进的-与经典的膜转运蛋白保持一致。此外，我们描述的功能是一致的非确定性电机机制，如布朗棘轮。
The universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity – a prerequisite for understanding how biological systems works – has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, ∼30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient – in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.
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影响因子：
7.7
作者：
W. Allen;R. Corey;Peter Oatley;R. Sessions;S. Baldwin;S. Radford;R. Tuma;I. Collinson
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Alexandra Tsirigotaki;K. Chatzi;M. Koukaki;Jozefien De Geyter;Athina G. Portaliou;Georgia Orfanoudaki;M. Sardis;M. B. Trelle;T. Jørgensen;S. Karamanou;A. Economou
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作者：
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通讯作者：
WICKNER, W
影响因子：
3.4
作者：
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通讯作者：
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