We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules.
- Authors
Bakos, Gábor; Yu, Lu; Gak, Igor A.; Roumeliotis, Theodoros I.; Liakopoulos, Dimitris; Choudhary, Jyoti S.; Mansfeld, Jörg
- 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. Ubiquitination and ubiquitin-like modifications of proteins regulate multiple cellular processes but identifying substrates of specific E2 and E3 enzymes remains challenging. Here, the authors conjugate E2 enzymes with enrichable ubiquitin derivatives to identify substrates of specific E2/E3 pairs by mass spectrometry.
- Publication
Nature Communications, 2018, Vol 9, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-018-07251-5