Abstract
Identification of protein–protein interactions (PPIs) and protein kinase substrates is fundamental for understanding how proteins exert biological functions with their partners and targets. However, it is still technically challenging, especially for transient and weak interactions involved in most cellular processes. The proximity-tagging systems enable capturing snapshots of both stable and transient PPIs. In this chapter, we describe in detail the methodology of a novel proximity-based labeling approach, PUP-IT (pupylation-based interaction tagging), to identify PPIs using a protoplast transient expression system. We have successfully identified potential kinase substrates by targeted screening and tandem mass spectrometry analysis.
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Acknowledgments
We thank former and current members of the Sheen Laboratory for their efforts in improving the Arabidopsis mesophyll protoplasts transient expression system. We thank Jenifer Bush for plant management. This work was supported by the NIH grants GM060493 and GM129093 to J.S. and R.Q.Y., National Natural Science Foundation of China grants 32170270 and 31870227 to K.-H. L., and USDA 2020-67013-31615/accession no. 1022409 grant to S.C. (co-PI) and Ping He (PI).
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Ye, R., Lin, Z., Liu, KH., Sheen, J., Chen, S. (2023). Dynamic Proximity Tagging in Living Plant Cells with Pupylation-Based Interaction Tagging. In: Mukhtar, S. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 2690. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3327-4_14
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DOI: https://doi.org/10.1007/978-1-0716-3327-4_14
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