Detection of sequential polyubiquitylation on a millisecond timescale

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Final product distribution for SCF Cdc4 and CYCE.
Figure 2: Millisecond kinetics of a single-encounter reaction reveal sequential processivity.
Figure 3: Human Cdc34–SCF β-TrCP is sequentially processive.
Figure 4: Kinetic basis for Cdc34–SCF processivity.

References

  1. 1

    Thrower, J. S. et al. Recognition of the polyubiquitin proteolytic signal. EMBO J. 19, 94–102 (2000)

  2. 2

    Dye, B. T. & Schulman, B. Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu. Rev. Biophys. Biomol. Struct. 36, 131–150 (2007)

  3. 3

    Petroski, M. & Deshaies, R. Function and regulation of cullin–RING ubiquitin ligases. Nature Rev. Mol. Cell Biol. 6, 9–20 (2005)

  4. 4

    Saha, A. & Deshaies, R. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31 (2008)

  5. 5

    Petroski, M. & Deshaies, R. Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34. Cell 123, 1107–1120 (2005)

  6. 6

    Ravid, T. & Hochstrasser, M. Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue. Nature Cell Biol. 9, 422–427 (2007)

  7. 7

    Li, W. et al. A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate. Nature 446, 333–337 (2007)

  8. 8

    Hochstrasser, M. Lingering mysteries of ubiquitin-chain assembly. Cell 124, 27–34 (2006)

  9. 9

    Li, W. et al. Mechanistic insights into active site-associated polyubiquitination by the ubiquitin-conjugating enzyme Ube2g2. Proc. Natl Acad. Sci. USA 106, 3722–3727 (2009)

  10. 10

    Deshaies, R. J. & Joazeiro, C. A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009)

  11. 11

    Fersht, A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding Ch. 14 (Freeman, 1999)

  12. 12

    Nash, P. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521 (2001)

  13. 13

    Orlicky, S. et al. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase. Cell 112, 243–256 (2003)

  14. 14

    Strohmaier, H. et al. Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line. Nature 413, 316–322 (2001)

  15. 15

    Kleiger, G., Saha, A., Lewis, S., Kuhlman, B. & Deshaies, R. Rapid E2–E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell (in the press)

  16. 16

    Kati, W. M. et al. Mechanism and fidelity of HIV reverse transcriptase. J. Biol. Chem. 267, 25988–25997 (1992)

  17. 17

    Petroski, M. et al. Evaluation of a diffusion-driven mechanism for substrate ubiquitination by the SCF-Cdc34 ubiquitin ligase complex. Mol. Cell 24, 523–534 (2006)

  18. 18

    Feldman, R. M. et al. A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. Cell 91, 221–230 (1997)

  19. 19

    Kamura, T. et al. The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modification of cullins Cdc53 and Cul2. Genes Dev. 13, 2928–2933 (1999)

  20. 20

    Petroski, M. & Deshaies, R. In vitro reconstitution of SCF substrate ubiquitination with purified proteins. Methods Enzymol. 398, 143–158 (2005)

  21. 21

    Hao, B. et al. Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. Mol. Cell 26, 131–143 (2007)

  22. 22

    Wu, G. et al. Structure of a β-TrCP1-Skp1-β-catenin complex. Mol. Cell 11, 1445–1456 (2003)

Download references

Acknowledgements

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.

Author information

Correspondence to Raymond J. Deshaies.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-16 with Legends. (PDF 1442 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.