biostudies-literatureUnknown16(1)Wu XThe K29/K48-linked ubiquitination generated by the cooperative catalysis of E3 ligase Ufd4 and Ubr1 is an enhanced protein degradation signal, in which Ufd4 is responsible for introducing K29-linked ubiquitination to K48-linked ubiquitin chains to augment polyubiquitination. How HECT-E3 ligase Ufd4 mediates the ubiquitination event remains unclear. Here, we biochemically determine that Ufd4 preferentially catalyses K29-linked ubiquitination on K48-linked ubiquitin chains to generate K29/K48-branched ubiquitin chains and capture structural snapshots of Ub transfer cascades for Ufd4-mediated ubiquitination. The N-terminal ARM region and HECT domain C-lobe of Ufd4 are identified and characterized as key structural elements that together recruit K48-linked diUb and orient Lys29 of its proximalNature communications4313https://www.ebi.ac.uk/biostudies/studies/S-EPMC12062229biostudies-literatureStructural visualization of HECT-type E3 ligase Ufd4 accepting and transferring ubiquitin to form K29/K48-branched polyubiquitination.PMC12062229Liu LTong ZDeng ZLiang LJZheng QCai HWu XAi HMao JPan MfalseStructural visualization of HECT-type E3 ligase Ufd4 accepting and transferring ubiquitin to form K29/K48-branched polyubiquitination.The K29/K48-linked ubiquitination generated by the cooperative catalysis of E3 ligase Ufd4 and Ubr1 is an enhanced protein degradation signal, in which Ufd4 is responsible for introducing K29-linked ubiquitination to K48-linked ubiquitin chains to augment polyubiquitination. How HECT-E3 ligase Ufd4 mediates the ubiquitination event remains unclear. Here, we biochemically determine that Ufd4 preferentially catalyses K29-linked ubiquitination on K48-linked ubiquitin chains to generate K29/K48-branched ubiquitin chains and capture structural snapshots of Ub transfer cascades for Ufd4-mediated ubiquitination. The N-terminal ARM region and HECT domain C-lobe of Ufd4 are identified and characterized as key structural elements that together recruit K48-linked diUb and orient Lys29 of its proximal2025-01-01T00:00:00Z2025 May2026-06-02T12:09:09.324Z2026-04-18T03:11:07.396ZS-EPMC120622294034112110.1038/s41467-025-59569-6