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A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate

Abstract

In eukaryotic cells, many short-lived proteins are conjugated with Lys 48-linked ubiquitin chains and degraded by the proteasome1. Ubiquitination requires an activating enzyme (E1), a conjugating enzyme (E2) and a ligase (E3)2. Most ubiquitin ligases use either a HECT (homologous to E6-associated protein C terminus) or a RING (really interesting new gene) domain to catalyse polyubiquitination3, but the mechanism of E3 catalysis is poorly defined4. Here we dissect this process using mouse Ube2g2 (E2; identical at the amino acid level to human Ube2g2) and human gp78 (E3), an endoplasmic reticulum (ER)-associated conjugating system essential for the degradation of misfolded ER proteins5,6. We demonstrate by expressing recombinant proteins in Escherichia coli that Ube2g2/gp78-mediated polyubiquitination involves preassembly of Lys 48-linked ubiquitin chains at the catalytic cysteine of Ube2g2. The growth of Ube2g2-anchored ubiquitin chains seems to be mediated by an aminolysis-based transfer reaction between two Ube2g2 molecules that each carries a ubiquitin moiety in its active site. Intriguingly, polyubiquitination of a substrate can be achieved by transferring preassembled ubiquitin chains from Ube2g2 to a lysine residue in a substrate.

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Figure 1: Formation of Ube2g2-linked ubiquitin chains.
Figure 2: Formation of Ube2g2-linked di-ubiquitin by an aminolysis-based transfer reaction.
Figure 3: Transfer of ubiquitin chains from Ube2g2 to HERPc.
Figure 4: Ubiquitination of HERPc by chemically synthesized ubiquitin oligomers.

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Acknowledgements

We thank T. Rapoport, M. Gellert and M. Krause for critical reading of the manuscript; A. Weissman, Y. Zheng and M. Seeger for plasmids; P. Carvalho for the ΔUbc7 ΔIre1 strain; M. Hochstrasser for communicating results before publication; and the Taplin Biological Mass Spectrometry Facility at Harvard Medical School for mass spectrometry analysis. This work was supported by funding from the NIDDK intramural research program at the National Institutes of Health.

Author Contributions W.L. performed most of the experiments. D.T. and Y.Y. performed some of the experiments. D.T. and A.T.B. provided intellectual input. W.L. and Y.Y. designed the experiments. Y.Y. wrote the paper.

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Correspondence to Yihong Ye.

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Li, W., Tu, D., Brunger, A. et al. A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate. Nature 446, 333–337 (2007). https://doi.org/10.1038/nature05542

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