An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules.
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Date
2018-11-14Author
Bakos, G
Yu, L
Gak, IA
Roumeliotis, TI
Liakopoulos, D
Choudhary, JS
Mansfeld, J
Type
Journal Article
Metadata
Show full item recordAbstract
Covalent modifications of proteins with ubiquitin and ubiquitin-like molecules are instrumental to many biological processes. However, identifying the E3 ligase responsible for these modifications remains a major bottleneck in ubiquitin research. Here, we present an E2-thioester-driven identification (E2~dID) method for the targeted identification of substrates of specific E2 and E3 enzyme pairs. E2~dID exploits the central position of E2-conjugating enzymes in the ubiquitination cascade and provides in vitro generated biotinylated E2~ubiquitin thioester conjugates as the sole source for ubiquitination in extracts. This enables purification and mass spectrometry-based identification of modified proteins under stringent conditions independently of the biological source of the extract. We demonstrate the sensitivity and specificity of E2-dID by identifying and validating substrates of APC/C in human cells. Finally, we perform E2~dID with SUMO in S. cerevisiae, showing that this approach can be easily adapted to other ubiquitin-like modifiers and experimental models.
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Subject
Cell Line
Hela Cells
Humans
Saccharomyces cerevisiae
Ubiquitin-Activating Enzymes
Ubiquitin-Conjugating Enzymes
Ubiquitin-Protein Ligases
Saccharomyces cerevisiae Proteins
Ubiquitins
SUMO-1 Protein
Ubiquitin
Anaphase-Promoting Complex-Cyclosome
Research team
Post-translational modifications and cell proliferation
Language
eng
Date accepted
2018-10-10
License start date
2018-11-14
Citation
Nature communications, 2018, 9 (1), pp. 4776 - ?
Publisher
NATURE PUBLISHING GROUP
Except where otherwise noted, this item's license is described
as
https://creativecommons.org/licenses/by/4.0
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