An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules.
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ICR Authors
Authors
Bakos, G
Yu, L
Gak, IA
Roumeliotis, TI
Liakopoulos, D
Choudhary, JS
Mansfeld, J
Yu, L
Gak, IA
Roumeliotis, TI
Liakopoulos, D
Choudhary, JS
Mansfeld, J
Document Type
Journal Article
Date
2018-11-14
Date Accepted
2018-10-10
Abstract
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.
Citation
Nature communications, 2018, 9 (1), pp. 4776 - ?
Source Title
Publisher
NATURE PUBLISHING GROUP
ISSN
2041-1723
eISSN
2041-1723
Collections
Research Team
Post-translational modifications and cell proliferation