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Ub-ProT reveals global length and composition of protein ubiquitylation in cells

Author

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  • Hikaru Tsuchiya

    (Tokyo Metropolitan Institute of Medical Science)

  • Daocharad Burana

    (Tokyo Metropolitan Institute of Medical Science
    Tokyo Institute of Technology)

  • Fumiaki Ohtake

    (Tokyo Metropolitan Institute of Medical Science)

  • Naoko Arai

    (Tokyo Metropolitan Institute of Medical Science)

  • Ai Kaiho

    (Tokyo Metropolitan Institute of Medical Science)

  • Masayuki Komada

    (Tokyo Institute of Technology)

  • Keiji Tanaka

    (Tokyo Metropolitan Institute of Medical Science)

  • Yasushi Saeki

    (Tokyo Metropolitan Institute of Medical Science)

Abstract

Protein ubiquitylation regulates diverse cellular processes via distinct ubiquitin chains that differ by linkage type and length. However, a comprehensive method for measuring these properties has not been developed. Here we describe a method for assessing the length of substrate-attached polyubiquitin chains, “ubiquitin chain protection from trypsinization (Ub-ProT).” Using Ub-ProT, we found that most ubiquitylated substrates in yeast-soluble lysate are attached to chains of up to seven ubiquitin molecules. Inactivation of the ubiquitin-selective chaperone Cdc48 caused a dramatic increase in chain lengths on substrate proteins, suggesting that Cdc48 complex terminates chain elongation by substrate extraction. In mammalian cells, we found that ligand-activated epidermal growth factor receptor (EGFR) is rapidly modified with K63-linked tetra- to hexa-ubiquitin chains following EGF treatment in human cells. Thus, the Ub-ProT method can contribute to our understanding of mechanisms regulating physiological ubiquitin chain lengths and composition.

Suggested Citation

  • 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.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-02869-x
    DOI: 10.1038/s41467-018-02869-x
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    Cited by:

    1. Ganga B. Vamisetti & Abhishek Saha & Yichao J. Huang & Rajeshwer Vanjari & Guy Mann & Julia Gutbrod & Nabieh Ayoub & Hiroaki Suga & Ashraf Brik, 2022. "Selective macrocyclic peptide modulators of Lys63-linked ubiquitin chains disrupt DNA damage repair," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    2. 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.
    3. Vishwas Mishra & Anna Crespo-Puig & Callum McCarthy & Tereza Masonou & Izabela Glegola-Madejska & Alice Dejoux & Gabriella Dow & Matthew J. G. Eldridge & Luciano H. Marinelli & Meihan Meng & Shijie Wa, 2023. "IL-1β turnover by the UBE2L3 ubiquitin conjugating enzyme and HECT E3 ligases limits inflammation," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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