Abstract
The timing and amplitude of plant signaling are frequently regulated through posttranslational modification of key signaling sectors, which facilitates rapid and flexible responses. Protein ubiquitination can serve as a degradation marker, influence subcellular localization, alter protein-protein interactions, and affect protein activity. Identification of polyubiquitinated proteins has been challenging due to their rapid degradation by the proteasome or removal of modifications by deubiquitination enzymes (DUBs). Tandem ubiquitin binding entities (TUBEs) are based on ubiquitin-associated domains and protect against both proteasomal degradation and DUBs. Here, we provide a protocol for purification of ubiquitinated plant proteins using TUBEs after transient expression in Nicotiana benthamiana. This protocol can also be applied to other plants to purify multiple ubiquitinated proteins or track ubiquitination of a target protein. This methodology provides an effective method for identification of ubiquitin ligase substrates and can be coupled with TUBEs targeting specific ubiquitination linkages.
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Acknowledgments
GC and DH were supported by a grant from the National Institutes of Health (NIH 1R35GM136402).
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Lee, D., Coaker, G. (2023). Purification and Detection of Ubiquitinated Plant Proteins Using Tandem Ubiquitin Binding Entities. 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_17
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DOI: https://doi.org/10.1007/978-1-0716-2784-6_17
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2783-9
Online ISBN: 978-1-0716-2784-6
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