Tracking the Stepwise Movement of a Membrane-inserting Protein In Vivo
Tracking the Stepwise Movement of a Membrane-inserting Protein In Vivo
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DOI：
10.1016/j.jmb.2019.10.010
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发表时间：
2020-01-17
影响因子：
5.6
通讯作者：
Dalbey, Ross E.
中科院分区：
文献类型：
--
作者：
He, Haoze;Kuhn, Andreas;Dalbey, Ross E.
正确的膜插入对于所有细胞中膜蛋白的结构和功能至关重要。YidC插入酶在这一过程中起着至关重要的作用，但YidC介导的插入的分子机制仍然未知。在这里，我们通过获得一系列的翻译停滞中间体跟踪Pf 3涂层通过YidC的逐步运动，并通过巯基交联研究它们。我们表明，Pf 3插入作为一个螺旋发夹，即，预期的跨膜区段沿着由TM 3和TM 5组成的YidC油脂载玻片移动，而N-末端尾部瞬时折回到位于膜的内小叶中的YidC的亲水性凹槽中，直到在涉及电化学膜电位的后续步骤中移位到周质。除了提供关于YidC如何插入单跨膜蛋白的虚拟见解外，我们的研究还展示了一种有价值的体内跟踪方法，可用于研究更复杂的底物或其他易位酶。(C)2019作者（S）出版社：Elsevier Ltd
Proper membrane insertion is crucial for the structure and function of membrane proteins in all cells. The YidC insertase plays an essential role in this process, but the molecular mechanism of YidC-mediated insertion remains unknown. Here we track the stepwise movement of Pf3 coat through YidC by obtaining a series of translational arrested intermediates, and investigate them by thiol cross-linking. We show that Pf3 is inserted as a helical hairpin, i.e., the prospective transmembrane segment moves along the YidC greasy slide comprised of TM3 and TM5, whereas the N-terminal tail transiently folds back into the hydrophilic groove of YidC located in the inner leaflet of the membrane until it is translocated to the periplasm in a subsequent step involving the electrochemical membrane potential. In addition to providing virtual insights about how YidC inserts single-spanning membrane proteins, our study also demonstrates a valuable in vivo tracking method that can be applied to study more complicated substrates or other translocases. (C) 2019 The Author(s). Published by Elsevier Ltd.