Insights into ubiquitin chain architecture using Ub-clipping

Kirby N. Swatek, Joanne L. Usher, Anja F. Kueck, Christina Gladkova, Tycho E.T. Mevissen, Jonathan N. Pruneda, Tim Skern, David Komander

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1–3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10–20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.

Original languageEnglish (US)
Pages (from-to)533-537
Number of pages5
JournalNature
Volume572
Issue number7770
DOIs
StatePublished - Aug 22 2019

ASJC Scopus subject areas

  • General

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    Swatek, K. N., Usher, J. L., Kueck, A. F., Gladkova, C., Mevissen, T. E. T., Pruneda, J. N., Skern, T., & Komander, D. (2019). Insights into ubiquitin chain architecture using Ub-clipping. Nature, 572(7770), 533-537. https://doi.org/10.1038/s41586-019-1482-y