title: E2/E3-mediated assembly of lysine 29-linked polyubiquitin chains.
abstract: Polyubiquitin (Ub) chains linked through Lys-48-Gly-76 isopeptide bonds represent the principal signal by which substrates of the Ub-dependent protein degradation pathway are targeted to the 26 S proteasome, but the mechanism(s) whereby these chains are assembled on substrate proteins is poorly understood. Nor have assembly mechanisms or definitive functions been assigned to polyubiquitin chains linked through several other lysine residues of ubiquitin. We show that rabbit reticulocyte lysate harbors enzymatic components that catalyze the assembly of unanchored Lys-29-linked polyubiquitin chains. This reaction can be reconstituted using the ubiquitin-conjugating enzyme (E2) known as UbcH5A, a 120-kDa protein(s) that behaves as a ubiquitin-protein ligase (E3), and ubiquitin-activating enzyme (E1). The same partially purified E3 preparation also catalyzes the assembly of unanchored chains linked through Lys-48. Kinetic studies revealed a K(m) of approximately 9 microM for the acceptor ubiquitin in the synthesis of diubiquitin; this value is similar to the concentration of free ubiquitin in most cells. Similar kinetic behavior was observed for conjugation to Lys-48 versus Lys-29 and for conjugation to tetraubiquitin versus monoubiquitin. The properties of these enzymes suggest that there may be distinct pathways for ubiquitin-ubiquitin ligation versus substrate-ubiquitin ligation in vivo.
authors.affiliation: Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214, USA.
authors.forename: L D
authors.initials: LD
authors.lastname: Mastrandrea
authors.forename: J
authors.initials: J
authors.lastname: You
authors.forename: E G
authors.initials: EG
authors.lastname: Niles
authors.forename: C M
authors.initials: CM
authors.lastname: Pickart
mesh_terms: D000818:Animals; D001704:Biopolymers / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011817:Rabbits; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D014452:Ubiquitins; D025821:Polyubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D008025:Ligases; D008239:Lysine