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A collection of cutting-edge techniques for studying ubiquitin-dependent protein degradation via the proteasome. The topics covered range broadly from basic biochemistry to cellular assays to discovery techniques using mass spectrometric analysis. These biochemical and cellular methods are necessary to explore the ubiquitin-proteasome system and ubiquitin-proteasome-dependent functions. State-of-the-art and user-friendly, Ubiquitin-Proteasome Protocols offers novice and experienced bench scientists alike a thorough compendium of readily reproducible techniques that will accelerate discovery, enhance productivity, and permit manipulation of the system for varied research purposes.
Contents Preface Contributors Part I Biochemical Methods to Study the Ubiquitin Proteasome System 1 Small-Molecule Inhibitors of Proteasome Activity Maria Gaczynska and Pawel A. Osmulski 2 Purification of E1 and E1-like Enzymes Arthur L. Haas 3 Assays for RING Family Ubiquitin Ligases Manabu Furukawa, Paul Andrews, and Yue Xiong 4 Ubiquitin Chain Synthesis Shahri Raasi and Cecile M. Pickart 5 Purification of Proteasomes, Proteasome Subcomplexes and Proteasome Associated Proteins from Budding Yeast David S. Leggett, Michael H. Glickman, and Daniel Finley 6 Recognition and Processing of Misfolded Proteins by PA700, the 19S Regulatory Complex of the 26S Proteasome Chang-Wei Liu, Elizabeth Strickland, George N. DeMartino, and Philip J. Thomas 7 Cell-free Assay for Ubiquitin-Independent Proteasomal Protein Degradation Chaim Kahana and Yuval Reiss 8 Assays of Proteasome-Dependent Cleavage Products Stefan Tenzer and Hansjorg Schild 9 Identification of Components of Protein Complexes Carol E. Parker, David R. Loiselle, Nedyalka N. Dicheva, Maria R. Warren, and Christoph H. Borchers 10 Mass Spectroscometric Determination of Protein Ubiqitination Carol E. Parker, Viorel Mocanu, Susanna F. Greer, and Christoph H. Borchers 11 Reconstitution of ER Associated Degradation Using Yeast Membranes and Cytosol Robert J. Lee, Ardythe A. McCracken, and Jeffrey L. Brodsky 12 Reticulocyte Lysate as a Model System to Study ER Membrane Protein Degradation Eric Carlson, Nathan Bays, Larry David, George Hilliard and William R. Skach 13 Deubiquitinating Enzyme Purifucation, Assay Inhibitors and Characterization Nathaniel S. Russell and Keith D. Wilkinson Part II Cellular Methods to Study Ubiquitin Proteasome Dependent Functions 14 Measuring Ubiquitin Conjugation in Cells Edward G. Mimnaugh and Leonard M. Neckers 15 Assays for Proteasome Assembly and Maturation R. Jurgen Dohmen, Markus K. London, Christoph Glanemann, and Paula C. Ramos 16 N-terminal Ubiquitination Aaron Ciechanover 17 Quantitating Defective Ribosome Products Shu-Bing Qian, Jack R. Bennink, and Jonathan W. Yewdell 18 Study of ER Associated Protein Quality Control and Degradation: Screen for ERAD Mutants after EMS Mutagenesis Antje Schaefer and Dieter H. Wolf 19 Study of ER Associated Protein Quality Control and Degradation: Genome Wide Screen for ERAD Components Antje Schaefer and Dieter H. Wolf 20 Use of the Cystic Fibrosis Transmembrane Conducatance Regulator as a Model Substrate to Study ER Protein Quality Control in Mammalian Cells J. Michael Younger, Chun Yang Fan, Liling Chen, Meredith F.N. Rosser, Andrea Fields, Hong-Yu Ren, Cam Patterson, and Douglas M. Cyr 21 Aggresome Formation Michael J. Corboy, Philip J. Thomas, and W. Christian Wigley 22 Detection of Sumoylated Proteins Kevin D. Sarge and Roland S. Hilgarth 23 Proteasome Inhibitors in Cancer Therapy Robert Z. Orlowski 24 Parkinson Disease: Assays for the Ubiquitin Ligase Activity of Neural Parkin Michael G. Schlossmacher and Hideki Shimura