Abstract
The anaphase-promoting complex/cyclosome (APC/C), a large (20S) multisubunit E3 ligase, has an essential role to ubiquitylate numerous substrates at specific times during mitosis and G1 phase as well as in meiosis. The deregulation of the APC/C causes cell death or genomic instability, which is a hallmark of cancers. Although 13 years have passed since its discovery, the molecular mechanisms of the APC/C-dependent ubiquitylation and proteolysis are still poorly understood. The development of in vitro systems enables the identification of new substrates and investigation of the molecular mechanisms by which the APC/C recognizes its substrates. This chapter describes in vitro assays reconstituted in Xenopus egg extracts.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annu. Rev. Biochem. 67, 425–479.
Morgan, D. O. (2007). The Cell Cycle: Principles of Control. London: New Science Press.
Peters, J. M. (2006). The anaphase promoting complex/cyclosome: a machine designed to destroy. Nat. Rev. Mol. Cell Biol., 644–656.
Thornton, B. R., and Toczyski, D. P. (2006). Precise destruction: an emerging picture of the APC. Genes Dev. 20, 3069–3078.
Acquaviva, C., and Pines, J. (2006). The anaphase-promoting complex/cyclosome: APC/C. J. Cell Sci. 119, 2401–2404.
Musacchio, A., and Salmon, E. D. (2007). The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393.
Kops, G. J., Weaver, B. A., and Cleveland, D. W. (2005). On the road to cancer: aneuploidy and the mitotic checkpoint. Nat. Rev. Cancer 5, 773–785.
Yu, H. (2007). Cdc20: a WD40 activator for a cell cycle degradation machine. Mol. Cell 27, 3–16.
Araki, M., Yu, H., and Asano, M. (2005). A novel motif governs APC-dependent degradation of Drosophila ORC1 in vivo. Genes Dev. 19, 2458–2465.
Castro, A., Vigneron, S., Bernis, C., Labbe, J. C., and Lorca, T. (2003). Xkid is degraded in a D-box, KEN-box, and A-box-independent pathway. Mol. Cell Biol. 23, 4126–4138.
Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991). Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138.
Littlepage, L. E., and Ruderman, J. V. (2002). Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit. Genes Dev. 16, 2274–2285.
Pfleger, C. M., and Kirschner, M. W. (2000). The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev. 14, 655–665.
Hayes, M. J., Kimata, Y., Wattam, S. L., Lindon, C., Mao, G., Yamano, H., and Fry, A. M. (2006). Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C. Nat. Cell Biol. 8, 607–614.
Lohka, M. J., and Maller, J. L. (1985). Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J. Cell Biol. 101, 518–523.
Murray, A. W., Solomon, M. J., and Kirschner, M. W. (1989). The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature 339, 280–286.
Lorca, T., Devault, A., Colas, P., Van Loon, A., Fesquet, D., Lazaro, J. B., and Doree, M. (1992). Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts. FEBS Lett. 306, 90–93.
van der Velden, H. M., and Lohka, M. J. (1993). Mitotic arrest caused by the amino terminus of Xenopus cyclin B2. Mol. Cell Biol. 13, 1480–1488.
Stewart, E., Kobayashi, H., Harrison, D., and Hunt, T. (1994). Destruction of Xenopus cyclins A and B2, but not B1, requires binding to p34cdc2. EMBO J. 13, 584–594.
Lorca, T., Castro, A., Martinez, A. M., Vigneron, S., Morin, N., Sigrist, S., Lehner, C., Doree, M., and Labbe, J. C. (1998). Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts. EMBO J. 17, 3565–3575.
King, R. W., Glotzer, M., and Kirschner, M. W. (1996). Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates. Mol. Biol. Cell 7, 1343–1357.
Yamano, H., Gannon, J., Mahbubani, H., and Hunt, T. (2004). Cell cycle-regulated recognition of the destruction box of cyclin B by the APC/C in Xenopus egg extracts. Mol. Cell 13, 137–147.
Yamano, H., Tsurumi, C., Gannon, J., and Hunt, T. (1998). The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor. EMBO J. 17, 5670–5678.
Murray, A. W. (1991). Cell cycle extracts. Methods Cell Biol. 36, 581–605.
Masui, Y., and Markert, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129–145.
Geley, S., Kramer, E., Gieffers, C., Gannon, J., Peters, J. M., and Hunt, T. (2001). Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint. J. Cell Biol. 153, 137–148.
Yamano, H., Gannon, J., and Hunt, T. (1996). The role of proteolysis in cell cycle progression in Schizosaccharomyces pombe. EMBO J. 15, 5268–5279.
Kimata, Y., Trickey, M., Izawa, D., Gannon, J., Yamamoto, M., and Yamano, H. (2008). A mutual inhibition between APC/C and its substrate Mes1 required for meiotic progression in fission yeast. Dev. Cell, 14, 446–454.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Yamano, H., Trickey, M., Grimaldi, M., Kimata, Y. (2009). In Vitro Assays for the Anaphase-Promoting Complex/Cyclosome (APC/C) in Xenopus Egg Extracts. In: McAinsh, A. (eds) Mitosis. Methods in Molecular Biology, vol 545. Humana Press. https://doi.org/10.1007/978-1-60327-993-2_18
Download citation
DOI: https://doi.org/10.1007/978-1-60327-993-2_18
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-992-5
Online ISBN: 978-1-60327-993-2
eBook Packages: Springer Protocols