Formation of a stable adduct between ubiquitin and the Arabidopsis ubiquitin-conjugating enzyme, AtUBC1+.
Ubiquitin conjugating enzymes (E2s) are an integral part of a multienzyme pathway that ligates ubiquitin to intracellular target proteins. This ligation has been implicated in a number of fundamental processes including protein degradation, cell cycle progression, DNA repair, and organelle biogenesis. To function, E2s form a labile thiol-ester intermediate between a specific cysteine within the E2 and the carboxyl terminus of ubiquitin; this high energy intermediate then serves as the donor for ubiquitin ligation. To aid in the characterization of E2s, we have created a stable ubiquitin-E2 intermediate using a mutant form of the 16-kDa E2 encoded by the Arabidopsis thaliana AtUBC1 gene in which the active-site cysteine at residue 88 was replaced with serine. The mutant protein synthesized in Escherichia coli formed an adduct with ubiquitin in vitro, but in this case the E2 and ubiquitin were linked via a more stable ester bond. The ester-linked ubiquitin could not be transferred subsequently to substrate proteins in an E3 alpha-dependent conjugation reaction. The ester adduct was sufficiently stable to survive purification by anion exchange high performance liquid chromatography. As a result, this adduct may prove useful for the structural analysis of ubiquitin-E2 intermediates and in the study of E2s interacting with other ubiquitin pathway enzymes.
Sullivan
M L
ML
Department of Horticulture, University of Wisconsin, Madison 53706.
Vierstra
R D
RD
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D002417:Cattle; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*
D029681:Arabidopsis Proteins; D004952:Esters; D014452:Ubiquitins; D004247:DNA; C489357:UBC1 protein, Arabidopsis; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases