title: Measuring APC/C-Dependent Ubiquitylation In Vitro.
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.
authors.affiliation: Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Dr. Bohrgasse 7, 1030, Vienna, Austria.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Jarvis
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Brown
authors.forename: Edmond R
authors.initials: ER
authors.lastname: Watson
authors.forename: Ryan
authors.initials: R
authors.lastname: VanderLinden
authors.forename: Brenda A
authors.initials: BA
authors.lastname: Schulman
authors.forename: Jan-Michael
authors.initials: JM
authors.lastname: Peters
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D064199:Cdc20 Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D064200:Cdh1 Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019926:Cyclin B / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D025801:Ubiquitin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D019926:Cyclin B; D011994:Recombinant Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D064173:Anaphase-Promoting Complex-Cyclosome; D044764:Ubiquitin-Activating Enzymes