In vitro systems for NEDD8 conjugation by Ubc12.
Nedd8 is a ubiquitin-like molecule that is highly conserved in eukaryotes. Similar to ubiquitin, Nedd8 attaches to target proteins through an enzymatic cascade composed of Nedd8-specific E1 (activating)- and E2 (conjugating)-enzymes. The E1 for Nedd8 is a heterodimer of APP-BP1 and Uba3, while the E2 is Ubc12. The most well-characterized targets of Nedd8 are proteins of the Cullin family, a core component of SCF (Skp1/Cullin1/F-box proteins) and/or SCF-like ubiquitin ligase complexes. The Nedd8 modification of Cullin (Cul) family proteins is evolutionarily conserved, and genetic analyses in various organisms suggest a positive role of the NEDD8 for the function of Cul family proteins. Further biochemical analysis reveals that NEDD8 modification augments the ubiquitin ligase activity of Cullin-based complexes through the recruitment of ubiquitin-charged E2 to the complex. This chapter describes methods for the purification of NEDD8 conjugation enzymes and in vitro Nedd8 conjugation.
Chiba
Tomoki
T
Laboratory of Frontier Science, The Tokyo Metropolitan Institute of Medical Science, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D020672:I-kappa B Proteins / Q000032:analysis; D000075687:NEDD8 Protein; D000072000:NF-KappaB Inhibitor alpha; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D014452:Ubiquitins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D020672:I-kappa B Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C000606204:NFKBIA protein, human; D029701:Saccharomyces cerevisiae Proteins; C112595:UBC12 protein, S cerevisiae; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha