Abstract
Posttranslational modifications by the ubiquitin-like family (UbL) of proteins determine the biological fate of a substrate, including new interaction partners. In the case of the small ubiquitin-like modifier (SUMO), this is achieved in part through its non-covalent interaction with SUMO-interacting motifs (SIMs) found in some proteins. Investigating such partner-complex formation is particularly challenging due to the fast dynamics and reversibility of SUMO modifications and the low affinity of SUMO-SIM interactions. Here, we present a detailed protocol of SUMO-ID, a technology that merges promiscuous proximity biotinylation by TurboID enzyme and protein-fragment complementation strategy to specifically biotinylate SUMO-dependent interactors of particular substrates. When coupled to streptavidin-affinity purification and mass spectrometry, SUMO-ID efficiently identifies SUMO-dependent interactors of a given protein. The methodology describes all the steps from SUMO-ID cell line generation to LC-MS sample preparation to study SUMO-dependent interactors of a particular protein. The protocol is generic and therefore adaptable to study other UbL-dependent interactors, such as ubiquitin.
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Acknowledgments
We acknowledge funding by the grants 765445-EU (UbiCODE Program), PID2020-114178GB-I00 (MINECO/FEDER, EU), SAF2017-90900-REDT (UBIRed Program). We acknowledge the COST Action CA20113 “PROTEOCURE” supported by COST (European Cooperation in Science and Technology). Additional support was provided by the Department of Industry, Tourism, and Trade of the Basque Country Government (Elkartek Research Programs) and by the Innovation Technology Department of the Bizkaia County.
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Barroso-Gomila, O., Mayor, U., Barrio, R., Sutherland, J.D. (2023). SUMO-ID: A Strategy for the Identification of SUMO-Dependent Proximal Interactors. In: Rodriguez, M.S., Barrio, R. (eds) The Ubiquitin Code. Methods in Molecular Biology, vol 2602. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2859-1_13
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DOI: https://doi.org/10.1007/978-1-0716-2859-1_13
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