1. Real-Time Monitoring of APC /C-Mediated Substrate Degradation Using Xenopus laevis Egg Extracts
- Author
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Qiong Yang, Julia Kamenz, Renping Qiao, James E. Ferrell, and Molecular Systems Biology
- Subjects
Cyclin B ,Mitosis ,Cell Cycle Proteins ,Xenopus Proteins ,Cell cycle ,Anaphase-Promoting Complex-Cyclosome ,Article ,03 medical and health sciences ,Xenopus laevis ,0302 clinical medicine ,Ubiquitin ,Animals ,Anaphase promoting complex/cyclosome (APC/C) ,Ovum ,030304 developmental biology ,Feedback, Physiological ,0303 health sciences ,Cyclin-dependent kinase 1 ,Enzymatic activity ,biology ,Chemistry ,Optical Imaging ,Recombinant Proteins ,Cell biology ,Ubiquitin ligase ,Securin ,Luminescent Proteins ,Mitotic exit ,Frog egg extracts ,Proteolysis ,biology.protein ,Plate reader assay ,Female ,Anaphase-promoting complex ,Protein Kinases ,030217 neurology & neurosurgery - Abstract
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.
- Published
- 2021