Insertion and folding pathways of single membrane proteins guided by translocases and insertases
Tetiana Serdiuk1, Anja Steudle2, Stefania A. Mari1, Selen Manioglu1, H. Ronald Kaback3,4,5, Andreas Kuhn2 and Daniel J. Müller1,*
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Science Advances 30 Jan 2019: Vol. 5, no. 1, eaau6824
Source/Fonte: Spark Notes
Abstract
Biogenesis in prokaryotes and eukaryotes requires the insertion of α-helical proteins into cellular membranes for which they use universally conserved cellular machineries. In bacterial inner membranes, insertion is facilitated by YidC insertase and SecYEG translocon working individually or cooperatively. How insertase and translocon fold a polypeptide into the native protein in the membrane is largely unknown. We apply single-molecule force spectroscopy assays to investigate the insertion and folding process of single lactose permease (LacY) precursors assisted by YidC and SecYEG. Both YidC and SecYEG initiate folding of the completely unfolded polypeptide by inserting a single structural segment. YidC then inserts the remaining segments in random order, whereas SecYEG inserts them sequentially. Each type of insertion process proceeds until LacY folding is complete. When YidC and SecYEG cooperate, the folding pathway of the membrane protein is dominated by the translocase. We propose that both of the fundamentally different pathways along which YidC and SecYEG insert and fold a polypeptide are essential components of membrane protein biogenesis.
Acknowledgments:
We thank R. Newton for discussing the manuscript, R. E. Dalbey for providing plasmid pT7-7 encoding YidC with a His10-tag at the C terminus, D. Balasubramaniam and J. Sugihara for providing some of the LacY and YidC samples used, D. Fotiadis for providing plasmid encoding the PreScission protease, the BioEM Lab of the University of Basel for providing TEM access and assistance, and N. Beerenwinkel for encouraging and constructive comments. Funding: This work was supported by the ETH-Zürich (to D.J.M.), the Swiss National Science Foundation (grant 205320_160199 to D.J.M.), and the National Center of Competence in Research “NCCR Molecular Systems Engineering” (to D.J.M.). Author contributions: T.S., D.J.M., A.K., and H.R.K. designed the experiments. A.K. and A.S. designed the SecY-YidC fusion construct. A.S. cloned, expressed, purified, and reconstituted the SecYEG and SecYEG-YidC fusion construct. T.S. performed the SMFS experiments. S.A.M. recorded AFM and TEM images. S.M. recorded Western blots. All authors analyzed experimental data and wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.
Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).
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