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 and David Komander ()
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Kirby N. Swatek: Medical Research Council Laboratory of Molecular Biology
Joanne L. Usher: Medical Research Council Laboratory of Molecular Biology
Anja F. Kueck: Medical Research Council Laboratory of Molecular Biology
Christina Gladkova: Medical Research Council Laboratory of Molecular Biology
Tycho E. T. Mevissen: Medical Research Council Laboratory of Molecular Biology
Jonathan N. Pruneda: Medical Research Council Laboratory of Molecular Biology
Tim Skern: Medical University of Vienna
David Komander: Medical Research Council Laboratory of Molecular Biology

Nature, 2019, vol. 572, issue 7770, 533-537

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41586-019-1482-y

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