title: Purification and assay of the budding yeast anaphase-promoting complex.
abstract: The anaphase-promoting complex (APC) is a central regulator of the eukaryotic cell cycle and functions as an E3 ubiquitin protein ligase to catalyze the ubiquitination of a number of cell cycle regulatory proteins. The APC contains at least 13 subunits in addition to two activator subunits, Cdc20 and Cdh1, that associate with the APC in a cell cycle-dependent manner. This chapter describes methods for preparation and assay of the APC from Saccharomyces cerevisiae. Highly active APC is purified from cells expressing Cdc16 fused with a tandem affinity purification (TAP) tag. Enzymatically active APC is achieved upon addition of recombinant Cdc20 or Cdh1 together with E1, Ubc4, ATP, and ubiquitin. Activity assays toward several endogenous substrates, including Clb2 and Pds1, are described. In addition, methods for observation of APC-coactivator and APC-substrate complexes by native gel electrophoresis are described.
authors.affiliation: Section of Structural Biology, Institute for Cancer Research, London SW3 6JB, UK.
authors.forename: Lori A
authors.initials: LA
authors.lastname: Passmore
authors.forename: David
authors.initials: D
authors.lastname: Barford
authors.forename: J Wade
authors.initials: JW
authors.lastname: Harper
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D064194:Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome; D002147:Calmodulin; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D007074:Immunoglobulin G; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012156:Reticulocytes / Q000201:enzymology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics; D012685:Sepharose; D043743:Ubiquitin-Protein Ligase Complexes / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D064194:Apc6 Subunit, Anaphase-Promoting Complex-Cyclosome; C577397:CDC16 protein, S cerevisiae; D002147:Calmodulin; D018797:Cell Cycle Proteins; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012685:Sepharose; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome