Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex

• Published in: Methods (San Diego, Calif.) Vol. 38; no. 1; pp. 39 - 51
• Main Authors: Kraft, Claudine, Gmachl, Michael, Peters, Jan-Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2006
• Subjects: Anaphase promoting complex; Anaphase-Promoting Complex-Cyclosome; Animals; Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome; Assay; Cdc20 Proteins; Cell Cycle Proteins - biosynthesis; Cell Cycle Proteins - metabolism; Cell Extracts; Cyclosome; Electrophoresis, Polyacrylamide Gel; Female; HeLa Cells; Humans; Immunoprecipitation; Mitosis; Neoplasm Proteins - isolation & purification; Nuclear Proteins - isolation & purification; Protein Processing, Post-Translational; Proteins - analysis; Proteins - metabolism; Proteolysis; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - metabolism; Ubiquitin ligase; Ubiquitin-Protein Ligase Complexes - isolation & purification; Ubiquitin-Protein Ligase Complexes - metabolism; Ubiquitin-Protein Ligase Complexes - physiology; Ubiquitins - metabolism; Xenopus; Assay; Cyclosome; Ubiquitin ligase; Mitosis; Proteolysis; Anaphase promoting complex
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2005.07.005

The anaphase-promoting complex (APC) or cyclosome is a multi-subunit ubiquitin ligase that controls progression through mitosis and the G1-phase of the cell cycle. The APC ubiquitinates regulatory proteins such as securin and cyclin B and thereby targets them for destruction by the 26S proteasome. Activation of the APC depends on the activator proteins Cdc20 and Cdh1, which are thought to recruit substrates to the APC. In vitro, APC’s RING finger subunit Apc11 alone can also function as a ubiquitin ligase. Here, we review different methods that have been used to measure the ubiquitination activity of the APC in vitro and to analyze APC-mediated degradation reactions either in vitro or in vivo. We describe procedures to isolate the APC from human cells or from Xenopus eggs, to activate purified APC with recombinant Cdc20 or Cdh1 and to measure the ubiquitination activity of the resulting APC Cdc20 and APC Cdh1 complexes. We also describe procedures to analyze the ubiquitination activity associated with recombinant Apc11.