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Direct observation of autoubiquitination for an integral membrane ubiquitin ligase in ERAD

Author

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  • Basila Moochickal Assainar

    (University of Michigan Medical School, 1150 W Medical Center Drive)

  • Kaushik Ragunathan

    (Brandeis University)

  • Ryan D. Baldridge

    (University of Michigan Medical School, 1150 W Medical Center Drive
    University of Michigan Medical School, 1150 W Medical Center Drive)

Abstract

The endoplasmic reticulum associated degradation (ERAD) pathway regulates protein quality control at the endoplasmic reticulum. ERAD of lumenal and membrane proteins requires a conserved E3 ubiquitin ligase, called Hrd1. We do not understand the molecular configurations of Hrd1 that enable autoubiquitination and the subsequent retrotranslocation of misfolded protein substrates from the ER to the cytosol. Here, we have established a generalizable, single-molecule platform that enables high-efficiency labeling, stoichiometry determination, and functional assays for any integral membrane protein. Using this approach, we directly count Hrd1 proteins reconstituted into individual proteoliposomes. We report that Hrd1 assembles in different oligomeric configurations with mostly monomers and dimers detected at limiting dilution. By correlating oligomeric states with ubiquitination in vitro, we conclude that Hrd1 monomers are inefficient in autoubiquitination while dimers efficiently assemble polyubiquitin chains. Therefore, our results reveal the minimal composition of a Hrd1 oligomer that is capable of autoubiquitination. Our methods are broadly applicable to studying other complex membrane protein functions using reconstituted bilayer systems.

Suggested Citation

  • Basila Moochickal Assainar & Kaushik Ragunathan & Ryan D. Baldridge, 2024. "Direct observation of autoubiquitination for an integral membrane ubiquitin ligase in ERAD," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45541-3
    DOI: 10.1038/s41467-024-45541-3
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    3. Hikaru Tsuchiya & Daocharad Burana & Fumiaki Ohtake & Naoko Arai & Ai Kaiho & Masayuki Komada & Keiji Tanaka & Yasushi Saeki, 2018. "Ub-ProT reveals global length and composition of protein ubiquitylation in cells," Nature Communications, Nature, vol. 9(1), pages 1-10, December.
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