Purification of the COP9 signalosome from porcine spleen, human cell lines, and Arabidopsis thaliana plants.
COP9 signalosome (CSN) is an evolutionarily conserved multisubunit protein complex involved in diverse cellular and developmental processes in eukaryotes. CSN functions in the cell as proteases that deconjugate Nedd8/Rub1 from cullin family proteins and depolymerize ubiquitin chains. As such, CSN represents an important regulator of multiple cullin-based E3 ubiquitin ligases. CSN has also been shown to associate with protein kinase activities. This chapter describes purification of the CSN complex by classical chromatography from porcine spleen and by immunoaffinity purification procedures from cultured human cells and transgenic Arabidopsis plants expressing epitope-tagged CSN subunits. It also describes in vitro deneddylation assays using the HeLa cell extract or the Arabidopsis cell extract, which we have used to test and compare the activity of purified CSN complexes.
Menon
Suchithra
S
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8104, USA.
Rubio
Vicente
V
Wang
Xiping
X
Deng
Xing-Wang
XW
Wei
Ning
N
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000075686:COP9 Signalosome Complex; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000075687:NEDD8 Protein; D010447:Peptide Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols; D013154:Spleen / Q000201:enzymology*; D013552:Swine; D014452:Ubiquitins / Q000378:metabolism
D029681:Arabidopsis Proteins; D046912:Multiprotein Complexes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C513170:RUB1 protein, Arabidopsis; D014452:Ubiquitins; D011092:Polyethylene Glycols; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex