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The Doc1 subunit is a processivity factor for the anaphase-promoting complex

Nature Cell Biology volume 4, pages 880–887 (2002)Cite this article

Abstract

Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APCdoc1Δ) indicates that Doc1 is required for processivity. The specific molecular defect in APCdoc1Δ is identified by a large increase in apparent KM for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APCdoc1Δ fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate–enzyme affinity.

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Figure 1: The APC is processive in vitro.
Figure 2: Substrate multi-ubiquitination requires multiple E2 enzymes.
Figure 3: Doc1 is required for APC activity but not assembly.
Figure 4: The Doc1 subunit is required for processive substrate ubiquitination.
Figure 5: Doc1 is not required for interaction of APC with E2 or Cdh1.
Figure 6: Doc1 enhances the APC–substrate interaction.
Figure 7: A model for cyclin ubiquitination by the APC.

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References

  1. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    CAS  PubMed  Google Scholar 

  2. Pickart, C. M. Mechanisms underlying ubiquitination. Annu. Rev. Biochem. 70, 503–533 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Scheffner, M., Nuber, U. & Huibregtse, J. M. . Protein ubiquitination involving an E1–E2–E3 enzyme ubiquitin thioester cascade. Nature 373, 81–83 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Peters, J. M. The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell 9, 931–943 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Harper, J. W., Burton, J. L. & Solomon, M. J. . The anaphase-promoting complex: it's not just for mitosis any more. Genes Dev. 16, 2179–2206 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Schwab, M. et al. Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC. EMBO J. 20, 5165–5175 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Burton, J. L. & Solomon, M. J. D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p. Genes Dev. 15, 2381–2395 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pfleger, C. M., Lee, E. & Kirschner, M. W. Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. Genes Dev. 15, 2396–2407 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hilioti, Z. et al. The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner. Curr. Biol. 11, 1347–1352 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Leverson, J. D. et al. The APC11 RING-H2 finger mediates E2-dependent ubiquitination. Mol. Biol. Cell 11, 2315–2325 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gmachl, M. et al. The RING-H2 finger protein APC11 and the E2 enzyme UBC4 are sufficient to ubiquitinate substrates of the anaphase-promoting complex. Proc. Natl Acad. Sci. USA 97, 8973–8978 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tang, Z. et al. Apc2 Cullin protein and Apc11 RING protein comprise the minimal ubiquitin ligase module of the anaphase-promoting complex. Mol. Biol. Cell 12, 3839–3851 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Grossberger, R. et al. Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex. J. Biol. Chem. 274, 14500–14507 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Hwang, L. H. & Murray, A. W. A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis. Mol. Biol. Cell 8, 1877–1887 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Au, S. W. et al. Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit. J. Mol. Biol. 316, 955–968 (2002).

    Article  CAS  PubMed  Google Scholar 

  16. Charles, J. F. et al. The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae. Curr. Biol. 8, 497–507 (1998).

    Article  CAS  PubMed  Google Scholar 

  17. Rigaut, G. et al. A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, 1030–1032 (1999).

    Article  CAS  Google Scholar 

  18. Pickart, C. M. Ubiquitin in chains. Trends Biochem. Sci. 25, 544–548 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Jaspersen, S. L., Charles, J. F. & Morgan, D. O. . Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14. Curr. Biol. 9, 227–236 (1999).

    Article  CAS  PubMed  Google Scholar 

  20. Hingorani, M. M. & O'Donnell, M. Sliding clamps: a (tail)ored fit. Curr. Biol. 10, R25–R29 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Gieffers, C. et al. Three-dimensional structure of the anaphase-promoting complex. Mol. Cell 7, 907–913 (2001).

    Article  CAS  PubMed  Google Scholar 

  22. Wendt, K. S. et al. Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex. Nature Struct. Biol. 8, 784–788 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure for protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  PubMed  Google Scholar 

  24. Gu, Y., Turck, C. W. & Morgan, D. O. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 366, 707–710 (1993).

    Article  CAS  PubMed  Google Scholar 

  25. Methods in Yeast Genetics — A Laboratory Course Manual (eds Rose, M., Winston, F. & Heiter, P.), (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1990).

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Acknowledgements

We thank A. Szidon, A. Rudner, S. Jaspersen and E. O'Shea for reagents. We also thank members of the Morgan lab for comments on the manuscript. This work was supported a grant from the National Institute of General Medical Sciences (GM53270) to D.O.M. and by a predoctoral fellowship from the National Science Foundation to C.W.C.

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  1. Departments of Physiology and Biochemistry & Biophysics, University of California, San Francisco, 94143-0444, CA, USA

    Christopher W. Carroll & David O. Morgan

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  1. Christopher W. Carroll
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Correspondence to David O. Morgan.

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Carroll, C., Morgan, D. The Doc1 subunit is a processivity factor for the anaphase-promoting complex. Nat Cell Biol 4, 880–887 (2002). https://doi.org/10.1038/ncb871

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