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The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C-Cdk1 activating the APC/C and the APC/C inactivating Cdk1-constitutes the core of the biochemical cell cycle oscillator.Since its discovery three decades ago, the mechanisms of APC /C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC /C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC /C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC /C activity in Xenopus laevis egg extracts using APC /C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC /C mutants or investigate other mechanisms of APC /C regulation.
Alfieri,
Visualizing the complex functions and mechanisms of the anaphase promoting complex/cyclosome (APC/C).
2017, Pubmed
Alfieri,
Visualizing the complex functions and mechanisms of the anaphase promoting complex/cyclosome (APC/C).
2017,
Pubmed Banaszynski,
Analysis of histones and chromatin in Xenopus laevis egg and oocyte extracts.
2010,
Pubmed
,
Xenbase Blanco,
APC(ste9/srw1) promotes degradation of mitotic cyclins in G(1) and is inhibited by cdc2 phosphorylation.
2000,
Pubmed Carroll,
Enzymology of the anaphase-promoting complex.
2005,
Pubmed Chenevert,
The Spindle Assembly Checkpoint Functions during Early Development in Non-Chordate Embryos.
2020,
Pubmed
,
Xenbase Cooper,
Ama1p is a meiosis-specific regulator of the anaphase promoting complex/cyclosome in yeast.
2000,
Pubmed Davey,
Building a Regulatory Network with Short Linear Sequence Motifs: Lessons from the Degrons of the Anaphase-Promoting Complex.
2016,
Pubmed Delgado-Esteban,
APC/C-Cdh1 coordinates neurogenesis and cortical size during development.
2013,
Pubmed Dong,
Control of G1 in the developing Drosophila eye: rca1 regulates Cyclin A.
1997,
Pubmed Eguren,
The APC/C cofactor Cdh1 prevents replicative stress and p53-dependent cell death in neural progenitors.
2013,
Pubmed Floyd,
APC/C Cdh1 targets aurora kinase to control reorganization of the mitotic spindle at anaphase.
2008,
Pubmed Fujimitsu,
Cyclin-dependent kinase 1-dependent activation of APC/C ubiquitin ligase.
2016,
Pubmed
,
Xenbase Galli,
Cell Size Determines the Strength of the Spindle Assembly Checkpoint during Embryonic Development.
2016,
Pubmed Glotzer,
Cyclin is degraded by the ubiquitin pathway.
1991,
Pubmed
,
Xenbase Ha,
The Prozone Effect Accounts for the Paradoxical Function of the Cdk-Binding Protein Suc1/Cks.
2016,
Pubmed
,
Xenbase Irniger,
Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast.
1995,
Pubmed Izawa,
The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C.
2015,
Pubmed Jarvis,
Measuring APC/C-Dependent Ubiquitylation In Vitro.
2016,
Pubmed King,
A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.
1995,
Pubmed
,
Xenbase Kraft,
Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex.
2006,
Pubmed
,
Xenbase Kramer,
Mitotic regulation of the APC activator proteins CDC20 and CDH1.
2000,
Pubmed
,
Xenbase Lara-Gonzalez,
The spindle assembly checkpoint.
2012,
Pubmed Li,
The adaptor protein of the anaphase promoting complex Cdh1 is essential in maintaining replicative lifespan and in learning and memory.
2008,
Pubmed Minshull,
A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts.
1994,
Pubmed
,
Xenbase Murray,
Cell cycle extracts.
1991,
Pubmed Okaz,
Meiotic prophase requires proteolysis of M phase regulators mediated by the meiosis-specific APC/CAma1.
2012,
Pubmed Peters,
The anaphase promoting complex/cyclosome: a machine designed to destroy.
2006,
Pubmed Pines,
Cubism and the cell cycle: the many faces of the APC/C.
2011,
Pubmed Qiao,
Mechanism of APC/CCDC20 activation by mitotic phosphorylation.
2016,
Pubmed Reimann,
Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex.
2001,
Pubmed
,
Xenbase Sigrist,
Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles.
1997,
Pubmed
,
Xenbase Sudakin,
The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.
1995,
Pubmed Tischer,
The APC/C inhibitor XErp1/Emi2 is essential for Xenopus early embryonic divisions.
2012,
Pubmed
,
Xenbase Tugendreich,
CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition.
1995,
Pubmed Yamaguchi,
Structure of an APC3-APC16 complex: insights into assembly of the anaphase-promoting complex/cyclosome.
2015,
Pubmed Yamano,
In vitro assays for the anaphase-promoting complex/cyclosome (APC/C) in Xenopus egg extracts.
2009,
Pubmed
,
Xenbase Yang,
The Cdk1-APC/C cell cycle oscillator circuit functions as a time-delayed, ultrasensitive switch.
2013,
Pubmed
,
Xenbase Zachariae,
Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex.
1998,
Pubmed Zhang,
Molecular mechanism of APC/C activation by mitotic phosphorylation.
2016,
Pubmed
