Purification and activity assays for Ubc9, the ubiquitin-conjugating enzyme for the small ubiquitin-like modifier SUMO.
The small ubiquitin-like modifier (SUMO) can be conjugated to lysine residues directly by the ubiquitin-conjugating protein Ubc9. SUMO conjugation can be catalyzed in vitro using only E1, Ubc9 (E2), mature SUMO, and ATP because Ubc9 directly recognizes consensus SUMO modification sites found in many identified targets of SUMO conjugation. This article describes methods to prepare Ubc9 and provides details for assay conditions used to evaluate E2 thioester formation and E2-mediated SUMO conjugation under single turnover and multiple turnover conditions. It also briefly describes parameters used to evaluate E3-mediated SUMO conjugation. Conservation of the SUMO conjugation apparatus from yeast to human has enabled in vivo assessment of human Ubc9 function through yeast complementation assays.
Yunus
Ali A
AA
Structural Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Lima
Christopher D
CD
D015153:Blotting, Western; D006801:Humans; D025842:SUMO-1 Protein / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D025842:SUMO-1 Protein; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9