Abstract
The anaphase-promoting complex/cyclosome (APC/C) is a 1.2 MDa ubiquitin ligase complex with important functions in both proliferating and post-mitotic differentiated cells. In proliferating cells, APC/C controls cell cycle progression by targeting inhibitors of chromosome segregation and mitotic exit for degradation by the 26S proteasome. To understand how APC/C recruits and ubiquitylates its substrate proteins and how these processes are controlled, it is essential to analyze APC/C activity in vitro. In the past, such experiments have been limited by the fact that large quantities of purified APC/C were difficult to obtain and that mutated versions of the APC/C could not be easily generated. In this chapter we review recent advances in generating and purifying recombinant forms of the human APC/C and its co-activators, using methods that are scalable and compatible with mutagenesis. We also describe a method that allows the quantitative analysis of APC/C activity using fluorescently labeled substrate proteins.
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Jarvis, M.A., Brown, N.G., Watson, E.R., VanderLinden, R., Schulman, B.A., Peters, JM. (2016). Measuring APC/C-Dependent Ubiquitylation In Vitro. In: Coutts, A., Weston, L. (eds) Cell Cycle Oscillators. Methods in Molecular Biology, vol 1342. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2957-3_18
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DOI: https://doi.org/10.1007/978-1-4939-2957-3_18
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2956-6
Online ISBN: 978-1-4939-2957-3
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