Format

Send to

Choose Destination
Nature. 2019 Aug;572(7770):533-537. doi: 10.1038/s41586-019-1482-y. Epub 2019 Aug 15.

Insights into ubiquitin chain architecture using Ub-clipping.

Author information

1
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
2
Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried, Germany.
3
Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA.
4
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
5
Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA.
6
Department of Medical Biochemistry, Max Perutz Labs, Vienna Biocenter, Medical University of Vienna, Vienna, Austria.
7
Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. dk@wehi.edu.au.
8
Ubiquitin Signalling Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. dk@wehi.edu.au.
9
Department of Medical Biology, The University of Melbourne, Melbourne, Victoria, Australia. dk@wehi.edu.au.

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.

PMID:
31413367
DOI:
10.1038/s41586-019-1482-y

Supplemental Content

Full text links

Icon for Nature Publishing Group
Loading ...
Support Center