Abstract
A combination of coimmunoprecipitation (coIP) of tagged proteins followed by protein identification and quantitation using Liquid Chromatography Mass Spectrometry/Mass Spectrometry (LCMS/MS) has proven to be a reliable method to qualitatively characterize membrane-bound receptor complexes from plants. Success depends on a range of parameters, such as abundance and stability of the complex and functionality of the tagged receptors, efficiency of the protein complex isolation procedure, MS equipment, and analysis software in use. In this Chapter, we focus on the use of one of the green fluorescent protein-tagged receptors of the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, of which SERK3, also known as BRASSINOSTEROID INSENSITIVE1 (BRI1) ASSOCIATED KINASE1 (BAK1), is a coreceptor of BRI1. Like BRI1 itself, SERK3 is a leucine-rich repeat receptor kinase (LRR RK) with a single-pass transmembrane domain. The latest updated laboratory protocol is presented as well as examples of data analysis and typical results obtained. Potential drawbacks of the procedure employed for plant membrane proteins will be pointed out.
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References
Gou X, He K, Yang H et al (2010) Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana. BMC Genomics 11:19
Ntoukakis V, Schwessinger B, Segonzac C et al (2011) Cautionary notes on the use of C-terminal BAK1 fusion proteins for functional studies. Plant Cell 23:3871–3878
van Esse GW, Westphal AH, Surendran RP et al (2011) Quantification of the Brassinosteroid Insensitive1 receptor in planta. Plant Physiol 156:1691–1700
Karlova R, Boeren S, Russinova E et al (2006) The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 protein complex includes BRASSINOSTEROID-INSENSITIVE1. Plant Cell 18:626–638
Smaczniak C, Li N, Boeren S et al (2012) Proteomics-based identification of low-abundance signaling and regulatory protein complexes in native plant tissues. Nat Protoc 7:2144–2158
Wendrich JR, Boeren S, Möller BK et al (2017) In vivo identification of plant protein complexes using IP-MS/MS. In: Kleine-Vehn J, Sauer M (eds) Plant hormones, Methods in molecular biology, 3rd edn. Humana Press, Totowa, NJ 1497:147–158
Fàbregas N, Li N, Boeren S et al (2013) The BRASSINOSTEROID INSENSITIVE1-LIKE3 signalosome complex regulates Arabidopsis root development. Plant Cell 25:3377–3388
Bücherl C, van Esse GW, Kruis A et al (2013) Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling. Plant Physiol 162:1911–1925
Acknowledgements
We thank Jos Wendrich (Biochemistry, Wageningen University, Netherlands) and Bert de Rybel (VIB-Ghent University, Belgium) for providing the updated immunoprecipitation protocols currently in use.
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van Dongen, W., van Heerde, L., Boeren, S., de Vries, S.C. (2017). Identification of Brassinosteroid Signaling Complexes by Coimmunoprecipitation and Mass Spectrometry. In: Russinova, E., Caño-Delgado, A. (eds) Brassinosteroids. Methods in Molecular Biology, vol 1564. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6813-8_12
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DOI: https://doi.org/10.1007/978-1-4939-6813-8_12
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6813-8
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