Measuring APC/C-Dependent Ubiquitylation In Vitro

• DOI: 10.1007/978-1-4939-2957-3_18
• Publication Date: 2015-08-08T07:28:41Z
• Authors: Marc A. Jarvis; Nicholas G. Brown; Edmond R. Watson; Ryan VanderLinden; Brenda A. Schulman; Jan-Michael Peters
• Keywords: 0303 health sciences; Cdc20 Proteins; Ubiquitin; Ubiquitination; Ubiquitin-Activating Enzymes; Cyclin B; Spodoptera; Anaphase-Promoting Complex-Cyclosome; Cdh1 Proteins; Recombinant Proteins; 03 medical and health sciences; Ubiquitin-Conjugating Enzymes; Sf9 Cells; Animals; Humans

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.