西亚试剂：Detection of sequential polyubiquitylation on a millisecond

Detection of sequential polyubiquitylation on a millisecond timescale

Nathan W. Pierce1, Gary Kleiger1, Shu-ou Shan2,3 & Raymond J. Deshaies1,3

1 Howard Hughes Medical Institute, Division of Biology, MC 156-29,
2 Division of Chemistry and Chemical Engineering, MC 147-75, California Institute of Technology, 1200 East California Boulevard, Pasadena, California 91125, USA
3 These authors contributed equally to this work.
Correspondence to: Raymond J. Deshaies1,3 Correspondence and requests for materials should be addressed to R.J.D.

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 SCFCdc4 and SCF-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.

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