Large-scale purification of the vertebrate anaphase-promoting complex/cyclosome.
The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle. The APC/C is a 1.5-MDa complex composed of at least 12 different core subunits. At different stages of mitosis and G1, the APC/C associates with a variety of regulatory proteins, such as the activator proteins Cdc20 and Cdh1 and the mitotic checkpoint complex (MCC), which regulate APC/C activity in a substrate-specific manner. Although APC/C and its regulators have been under intense investigation, it is still poorly understood how substrates are recognized and ubiquitinated by the APC/C, why so many subunits are required for these processes, and how regulators of the APC/C control its ubiquitin ligase activity in a substrate-specific manner. This chapter describes a simple and rapid procedure that allows the isolation of APC/C from vertebrate cells and tissues with reasonable purity and at high concentrations, yielding up to 0.5 mg of APC/C. This procedure should facilitate biochemical, biophysical, and structural analyses of the APC/C that will be needed for a better mechanistic understanding of its function and regulation.
Herzog
Franz
F
Research Institute of Molecular Pathology (IMP), 1030 Vienna, Austria.
Peters
Jan-Michael
JM
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D008863:Microspheres; D008969:Molecular Sequence Data; D010063:Ovum; D025801:Ubiquitin / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D014982:Xenopus laevis
D000906:Antibodies; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome