Abstract
The ubiquitin-proteasome system (UPS) is the predominant protein degradation machinery in eukaryotic cells. It is highly conserved among eukaryotes and essential for their survival. Through regulated proteolysis the UPS plays a key role in a myriad of cellular functions, including developmental and stress signaling, cell differentiation, and cell death. Attachment of a ubiquitin chain to a substrate can trigger its recruitment to the proteasome for proteolysis. To efficiently degrade substrates, however, the proteasome employs HECT-type ubiquitin ligases that can further remodel ubiquitin chains of proteasome-captured substrates. It is thought that this remodeling process is necessary to maintain substrate affinity for the proteasome and to completely translocate the substrate into the 20S proteolytic barrel. Here, we describe a protocol for purifying proteasomes and their associated accessory proteins and provide a practical way to detect proteasome-associated E3 ligase activity. This assay is reliable and efficient for assessing the ability of proteasomes to form ubiquitin conjugates and is applicable to a wide range of eukaryotic species.
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Acknowledgments
This protocol was developed with funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 678511) and the Horizon Europe programme (grant agreement No. 101001137), the Biotechnology and Biological Sciences Research Council (BBSRC; grant no. BB/S016767/1), and a Royal Society University Research Fellowship (grant no. UF140600) to S.H.S, while Z.W. was funded by a PhD studentship from the Darwin Trust of Edinburgh.
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Wang, Z., Orosa-Puente, B., Spoel, S.H. (2023). Analysis of Proteasome-Associated Ubiquitin Ligase Activity. In: Lois, L.M., Trujillo, M. (eds) Plant Proteostasis. Methods in Molecular Biology, vol 2581. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2784-6_5
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DOI: https://doi.org/10.1007/978-1-0716-2784-6_5
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