Rate-limiting transport of positively charged arginine residues through the Sec-machinery is integral to the mechanism of protein secretion.
Rate-limiting transport of positively charged arginine residues through the Sec-machinery is integral to the mechanism of protein secretion.
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DOI：
10.7554/elife.77586
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发表时间：
2022-04-29
期刊：
影响因子：
7.7
通讯作者：
Collinson, Ian
中科院分区：
文献类型：
--
作者：
Allen, William J.;Corey, Robin A.;Watkins, Daniel W.;Oliveira, A. Sofia F.;Hards, Kiel;Cook, Gregory M.;Collinson, Ian
蛋白质跨膜和跨膜转运是一个基本的生物学过程，其中绝大多数是由无处不在的Sec机制进行的。在细菌中，这通常是当SecY复合物接合胞质ATP酶SecA（分泌）或翻译核糖体（插入）时实现的。在理解蛋白质易位的机制方面已经取得了很大的进展。然而，重要的问题仍然存在-值得注意的是，构成运输的各个步骤的性质，以及质子动力（PMF）如何穿过质膜。在这里，我们应用最近开发的高分辨率蛋白质转运试验来探索这些问题。我们发现，前蛋白质运输主要是有限的精氨酸残基跨膜的扩散，特别是在庞大的疏水序列的背景下。精氨酸的这种由其正电荷引起的特异性作用被赖氨酸减轻，赖氨酸可以去质子化并以其中性形式跨膜转运。这些观察结果对蛋白质分泌的机制具有有趣的影响，表明PMF可以通过启用“质子棘轮”来帮助运输的简单机制，其中退出赖氨酸残基的再质子化防止通道重新进入，使运输偏向向外方向。
Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain – notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism through which the PMF can aid transport by enabling a 'proton ratchet', wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.