Detection of sequential polyubiquitylation on a millisecond timescale.
The pathway by which ubiquitin chains are generated on substrate through a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3 enzymes SCF(Cdc4) and SCF(beta-TrCP) work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.
Pierce
Nathan W
NW
Howard Hughes Medical Institute, Division of Biology, MC 156-29, Pasadena, California 91125, USA.
Kleiger
Gary
G
Shan
Shu-ou
SO
Deshaies
Raymond J
RJ
D001671:Biochemistry / Q000379:methods*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D013997:Time Factors; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
D044843:SKP Cullin F-Box Protein Ligases; D044764:Ubiquitin-Activating Enzymes