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CRL (Cullin-Ring ubiquitin ligases) are the major class of plant E3 ubiquitin ligases. Immunoprecipitation-based methods are useful techniques for revealing interactions among Cullin-Ring Ligase (CRL) subunits or between CRLs and other proteins, as well as for detecting poly-ubiquitin modifications of the CRLs themselves. Here, we describe two immunoprecipitation (IP) procedures suitable for CRLs in Arabidopsis: (1) a procedure for IP analysis of CRL subunits and their interactors and a second procedure for in vivo ubiquitination analysis of the CRLs. Both protocols can be divided into two major steps: (1) preparation of cell extracts without disruption of protein interactions and (2) affinity purification of the protein complexes and subsequent detection. We provide a thorough description of all the steps, as well as advice on how to choose proper buffers for these analyses. We also suggest a series of negative controls that can be used to verify the specificity of the procedure.

Titel:	Immunoprecipitation of Cullin-Ring Ligases (CRLs) in Arabidopsis thaliana Seedlings.
Autor/in / Beteiligte Person:	Casagrande, F ; Serino, G
Zeitschrift:	Methods in molecular biology (Clifton, N.J.), Jg. 2581 (2023), S. 31-42
Veröffentlichung:	Totowa, NJ : Humana Press ; <i>Original Publication</i>: Clifton, N.J. : Humana Press,, 2023
Medientyp:	academicJournal
ISSN:	1940-6029 (electronic)
DOI:	10.1007/978-1-0716-2784-6_3
Schlagwort:	Seedlings metabolism Cullin Proteins metabolism Ubiquitin metabolism Immunoprecipitation Arabidopsis metabolism
Sonstiges:	Nachgewiesen in: MEDLINE Sprachen: English Publication Type: Journal Article Language: English [Methods Mol Biol] 2023; Vol. 2581, pp. 31-42. MeSH Terms: Arabidopsis* / metabolism ; Seedlings / metabolism ; Cullin Proteins / metabolism ; Ubiquitin / metabolism ; Immunoprecipitation References: Baek K, Scott DC, Schulman BA (2021) NEDD8 and ubiquitin ligation by cullin RING E3 ligases. Curr Op Struct Biol 67:101–109. (PMID: 10.1016/j.sbi.2020.10.007) ; Bai C, Sen P, Hofmann K et al (1996) SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86: 263-74. 3. Feldman RMR, Correll C, Kaplan K et al. (1997) a complex of Cdc4p, Skp1p, and Cdc53p/Cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. Cell 91:221–230. ; Choi C, Gray W, Mooney S et al (2014) Composition, roles, and regulation of Cullin based ubiquitin E3 ligases. The Arabidopsis Book 12:e0175. (PMID: 10.1199/tab.0175255058534262284) ; Zheng N, Schulman B, Song L et al (2002) Structure of the Cul1–Rbx1–Skp1–F boxSkp2 SCF ubiquitin ligase complex. Nature 416:703–709. (PMID: 10.1038/416703a11961546) ; Wang K, Deshaies RJ, Liu X (2020) Assembly and regulation of CRL 3 ubiquitin ligases. Adv Exp Med Biol 1217:33–46. (PMID: 10.1007/978-981-15-1025-0_331898220) ; Schwechheimer C, Serino G, Callis J et al (2001) Interaction of the COP9 signalosome with the E3 ubiquitin ligase SCF TIR1 in mediating auxin-response. Science 292:1379–1382. (PMID: 10.1126/science.105977611337587) ; de Bie P, Ciechanover A (2011) Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms. Cell Death Differ 18:1393–1402. (PMID: 10.1038/cdd.2011.16213728473178436) ; Serino G, Deng XW (2007) Protein coimmunoprecipitation in Arabidopsis. CSH protocols. Cold Spring Harbor, Laboratory Press, p pdb.prot4683. ; Smith BJ (1994) SDS polyacrylamide gel electrophoresis of proteins. In: Walker JM (ed) Basic protein and peptide protocols. Methods in molecular biology, vol 32. Humana Press, pp 23–34. (PMID: 10.1385/0-89603-268-X:23) ; Walker JM (1994) Gradient SDS polyacrylamide gel electrophoresis of proteins. In: Walker JM (ed) Basic protein and peptide protocols. Methods in molecular biology, vol 32. Humana Press, pp 35–38. (PMID: 10.1385/0-89603-268-X:35) ; Franciosini A, Lombardi B, Iafrate S et al (2013) The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation. Mol Plant 6:1616–1629. (PMID: 10.1093/mp/sst04523475998) Contributed Indexing: Keywords: Co-immunoprecipitation; Cullin–Ring ubiquitin ligase; Immunoblot; Immunoprecipitation; NEDD8; Neddylation; Ubiquitin Substance Nomenclature: 0 (Cullin Proteins) ; 0 (Ubiquitin) Entry Date(s): Date Created: 20221122 Date Completed: 20221124 Latest Revision: 20221128 Update Code: 20231215

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Immunoprecipitation of Cullin-Ring Ligases (CRLs) in Arabidopsis thaliana Seedlings.