An E2-ubiquitin thioester-driven approach to identify substrates modified with ubiquitin and ubiquitin-like molecules

Gábor Bakos, Lu Yu, Igor A. Gak, Theodoros I. Roumeliotis, Dimitris Liakopoulos, Jyoti S. Choudhary and Jörg Mansfeld ()
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Gábor Bakos: Technische Universität Dresden
Lu Yu: The Institute of Cancer Research
Igor A. Gak: Technische Universität Dresden
Theodoros I. Roumeliotis: The Institute of Cancer Research
Dimitris Liakopoulos: Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR 5237
Jyoti S. Choudhary: The Institute of Cancer Research
Jörg Mansfeld: Technische Universität Dresden

Nature Communications, 2018, vol. 9, issue 1, 1-15

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-07251-5

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