School of Biochemistry, University of Bristol, University Walk, Bristol, United Kingdom.
School of Chemistry, University of Bristol, University Walk, Bristol, United Kingdom.
Elife. 2022 Apr 29;11:e77586. doi: 10.7554/eLife.77586.
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.
蛋白质跨膜运输和进入膜是一个基本的生物学过程，其中绝大多数是由普遍存在的 Sec 机制完成的。在细菌中，当 SecY 复合物与胞质 ATP 酶 SecA（分泌）或翻译核糖体（插入）结合时，通常会发生这种情况。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，仍有一些重要的问题有待解决——特别是构成运输的各个步骤的性质，以及质膜跨膜质子动力势（PMF）的作用。在这里，我们应用最近开发的高分辨率蛋白质运输测定法来探索这些问题。我们发现，前蛋白运输主要受到精氨酸残基在膜中扩散的限制，特别是在大的疏水性序列的情况下。这种精氨酸的特殊效应是由其正电荷引起的，而赖氨酸可以被去质子化，并以中性形式穿过膜运输，从而减轻了这种效应。这些观察结果对蛋白质分泌的机制具有有趣的启示，表明 PMF 可以通过允许“质子棘轮”来帮助运输的简单机制，其中退出的赖氨酸残基的再质子化可以防止通道重新进入，从而使运输偏向于向外方向。
