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YidC assists the stepwise and stochastic folding of membrane proteins

Nature Chemical Biology volume 12, pages 911–917 (2016)Cite this article

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Abstract

How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone and insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and the membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, indicating that LacY can fold along variable pathways toward the native structure. Since YidC is essential in membrane protein biogenesis and LacY is a model for the major facilitator superfamily, our observations have general relevance.

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Figure 1: Unfolding steps and intermediates of native wild-type LacY.
Figure 2: Refolding single lactose permeases.
Figure 3: YidC suppresses misfolding and assists folding of LacY.
Figure 4: LacY shows variable folding intermediates toward full refolding.
Figure 5: Schematic model of the folding free-energy landscape of LacY sculpted by YidC.

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Acknowledgements

We thank R.E. Dalbey (The Ohio State University) for providing plasmid pT7-7 encoding YidC with a His10 tag at the C terminus; R. Newton, J. Thoma, S. Hiller and R.E. Dalbey for encouraging and constructive comments; and S. Weiser for assistance. This work was supported by the Eidgenössische Technische Hochschule Zürich (to D.J.M.), the Swiss National Science Foundation (grant 205320_160199 to D.J.M.), the Swiss National Center of Competence in Research “NCCR Molecular Systems Engineering” (to D.J.M.), the European Union Marie Curie Actions program through the ACRITAS Initial Training Network (FP7-PEOPLE-2012-ITN, project 317348 to D.J.M.), U.S. National Institutes of Health grants DK51131, DK069463 and GM073210 (to H.R.K.), and U.S. National Science Foundation grant MCB-1129551 (to H.R.K.).

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Authors and Affiliations

  1. Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, Basel, Switzerland

    Tetiana Serdiuk, Stefania A Mari & Daniel J Müller

  2. Department of Physiology, University of California–Los Angeles, Los Angeles, California, USA

    Dhandayuthapani Balasubramaniam, Junichi Sugihara & H Ronald Kaback

  3. Department of Microbiology, Immunology & Molecular Genetics, University of California–Los Angeles, Los Angeles, California, USA

    H Ronald Kaback

  4. Molecular Biology Institute, University of California–Los Angeles, Los Angeles, California, USA

    H Ronald Kaback

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  1. Tetiana Serdiuk
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Contributions

T.S., D.J.M. and H.R.K. designed the experiments. J.S. and D.B. cloned, expressed, purified and reconstituted LacY and YidC. T.S. performed the SMFS experiments. S.A.M. recorded AFM images. All authors analyzed experimental data and wrote the paper.

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Correspondence to H Ronald Kaback or Daniel J Müller.

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Serdiuk, T., Balasubramaniam, D., Sugihara, J. et al. YidC assists the stepwise and stochastic folding of membrane proteins. Nat Chem Biol 12, 911–917 (2016). https://doi.org/10.1038/nchembio.2169

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