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 SCFβ-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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We thank J. Vielmetter for providing SCFCdc4, β-TrCP-Skp1 and human E1; S. Hess, R. L. J. Graham and the Proteome Exploration Laboratory for providing assistance with mass spectrometry of CYCE and Cdc34 thioester. We thank S. Schwarz, B. Schulman and G. Wu for gifts of reagents. We thank D. Sprinzak and all the members of the Deshaies and Shan laboratories for support and discussions. N.W.P. was supported by the Gordon Ross Fellowship and a National Institutes of Health Training Grant. R.J.D. is an Investigator of the Howard Hughes Medical Institute. This work was supported in part by National Institutes of Health GM065997.
Author Contributions N.W.P. performed all computational modelling and experiments, except G.K. performed the mass spectrometry experiments in Fig. 1g. N.W.P., R.J.D. and S.-o.S. conceived the experiments. N.W.P. and R.J.D. wrote the manuscript with editorial input from the other authors.
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Pierce, N., Kleiger, G., Shan, S. et al. Detection of sequential polyubiquitylation on a millisecond timescale . Nature 462, 615–619 (2009). https://doi.org/10.1038/nature08595
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