SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2, and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 6500 SUMO2/3 target proteins. The limited number of SUMO E3s provides the unique opportunity to systematically study E3 substrate wiring. We developed SUMO-activated target traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL (ZNF451-3), and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool to browse the dataset in an interactive manner. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

Automated summary

Ubiquitin and ubiquitin-like conjugation cascades involve E1, E2, and E3 enzymes, with E3s providing substrate specificity. Through mass spectrometry-based approaches, more than 6500 SUMO2/3 target proteins have been identified. Using SUMO-activated target traps (SATTs), the substrates for eight different SUMO E3s were systematically identified, revealing E3 substrate preference. Quantitative proteomics and the Polar SATTs web-based tool were developed to measure substrate specificity and browse the dataset interactively. Overall, the study uncovers the E3-to-target wiring of 1388 SUMO substrates for different SUMO E3 ligases.