title: Assembly of different length of polyubiquitins on the catalytic cysteine of E2 enzymes without E3 ligase; a novel application of non-reduced/reduced 2-dimensional electrophoresis.
abstract: In this study using non-reduced/reduced 2-dimensional electrophoresis (NR/R-2DE), we clearly demonstrated that E3-independent ubiquitination by Ube2K produced not only unanchored but also Ube2K-linked polyubiquitins through thioester and isopeptide bonds. E3-independent assembly of polyubiquitins on the catalytic cysteine of Ube2K strongly supports the possibility of 'en bloc transfer' for polyubiquitination. From the same analyses of E3-independent ubiquitination products by other E2s, we also found that different lengths of polyubiquitins were linked to different E2s through thioester bond; longer chains by Cdc34 like Ube2K, short chains by Ube2g2, and mono-ubiquitin by UbcH10. Our results suggest that E2s possess the different intrinsic catalytic activities for polyubiquitination.
authors.affiliation: School of Life Sciences, Gwangju Institute of Science & Technology (GIST), Gwangju, Republic of Korea.
authors.forename: Dong Yeon
authors.initials: DY
authors.lastname: Shin
authors.forename: Hyunju
authors.initials: H
authors.lastname: Lee
authors.forename: Eun Sil
authors.initials: ES
authors.lastname: Park
authors.forename: Yung Joon
authors.initials: YJ
authors.lastname: Yoo
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D003545:Cysteine; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004952:Esters; D025821:Polyubiquitin / Q000302:isolation & purification* / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D004952:Esters; D025821:Polyubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D003545:Cysteine