Letter | Published:

Cryo-EM structure of the protein-conducting ERAD channel Hrd1 in complex with Hrd3

Nature volume 548, pages 352355 (17 August 2017) | Download Citation

Abstract

Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome1,2,3,4—a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain5,6. Hrd1 is the crucial membrane component for retro-translocation7,8, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction—from the cytosol into or across membranes9,10,11. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.

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Acknowledgements

We thank Z. Yu, R. Huang and C. Hong at the HHMI Janelia Cryo-EM Facility for help in microscope operation and data collection, Z. Li for technical support at our in-house EM facility, and S. Andrei Anghel, for help with experiments, and X. Wu, P. Carvalho and T. Walz for comments on the manuscript. This work was supported by the European Molecular Biology Organization (EMBO LTF #1437-2012) to S. S., by the European Research Council (ERC) under the Horizon 2020 research and innovation program (grant # 677770) to A.S., by the National Key Research and Development Program of China (grant # 2016YFB1000101) to D.L., and by NIGMS Award R01GM052586 to T.A.R. T.A.R. is a Howard Hughes Medical Institute Investigator.

Author information

Author notes

    • Stefan Schoebel

    Present address: University of Gothenburg, Department of Chemistry & Molecular Biology, Gothenburg 40530, Sweden.

    • Stefan Schoebel
    •  & Wei Mi

    These authors contributed equally to this work.

Affiliations

  1. Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Stefan Schoebel
    •  & Tom A. Rapoport
  2. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Wei Mi
    • , Melissa G. Chambers
    •  & Maofu Liao
  3. Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany

    • Alexander Stein
  4. Institute for Protein Design, University of Washington, Seattle, Washington, USA

    • Sergey Ovchinnikov
    • , Ryan Pavlovicz
    • , Frank DiMaio
    •  & David Baker
  5. National Lab for Parallel and Distributed Processing (PDL), School of Computer Science, National University of Defense Technology, Changsha, China

    • Huayou Su
    •  & Dongsheng Li

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Contributions

S.S. prepared protein and built the models, W.M. and M.L. collected and analysed EM data, A.S. designed the construct and performed sequence alignments, S.O. and R.P. and their advisors F.D. and D.B. built models based on evolutionary couplings and energy minimization, M.G.C. helped with EM data collection, H.S. and D.L. developed DSS in GeRelion, T.A.R. and M.L. supervised the project and T.A.R. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Tom A. Rapoport or Maofu Liao.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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    Supplementary Figure

    This file contains the uncropped scan of SDS-PAGE used in Extended Data Figure 1b.

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https://doi.org/10.1038/nature23314

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