The Ubiquitin Proteasome System: Methods and Protocols

Preface...

Table of Contents...

Contributors...

Part I Methods to Uncover the Mechanisms of Action for Enzymes that Assemble or Disassemble Poly-Ubiquitin Chains

1.Characterization of RING-Between-RING E3 Ubiquitin Transfer Mechanisms Katherine H. Reiter and Rachel E. Klevit

2.Single Turnover RING/U-Box E3-Mediated Lysine Discharge Assays

Lori Buetow, Mads Gabrielsen, and Danny T. Huang

3.Methods for NAD-Dependent Ubiquitination Catalyzed by Legionella pneumophila Effector Proteins 

Jiazhang Qiu and Zhao-Qing Luo

4.Using In Vitro Ubiquitylation Assays to Estimate the Affinities of Ubiquitin-Conjugating Enzymes for their Ubiquitin Ligase Partners

Spencer Hill, Connor Hill, and Gary Kleiger

5.Competition Assay for Measuring Deubiquitinating Enzyme Substrate Affinity

Michael T. Morgan and Cynthia Wolberger

Part II Tools to Study Ubiquitylation 

6.Enzymatic Assembly of Ubiquitin Chains

Martin A. Michel, David Komander, and Paul R. Elliott

7.Ubiquitin-Activated Interaction Traps (UBAITs): Tools for Capturing Protein-Protein Interactions

Hazel F. O'Connor, Caleb D. Swaim, Larissa A. Canadeo, and Jon M. Huibregtse

8.Generating Intracellular Modulators of E3 Ligases and Deubiquitinases from Phage-Displayed Ubiquitin Variant Libraries

Wei Zhang and Sachdev S. Sidhu

9.Integrated Proteogenomic Approach for Identifying Degradation Motifs in Eukaryotic Cells

Yifat Geffen, Alon Appleboim, Richard G. Gardner, and Tommer Ravid

10.A Method to Monitor Protein Turnover by Flow Cytometry and to Screen for Factors that Control Degradation by Fluorescence-Activated Cell Sorting

Sophie A. Comyn and Thibault Mayor

11.E. coli Based Selection and Expression Systems for Discovery, Characterization, and Purification of Ubiquitylated Proteins

Olga Levin-Kravets, Tal Keren-Kaplan, Ilan Attali, Itai Sharon, Neta Tanner, Dar Shapira, Ritu Rathi, Avinash Persaud, Noa Shohat, Anna Shusterman, and Gali Prag

Part III Structural Approaches as Applied to Enzymes that Participate in the Ubiquitin Proteasome System 

12.Strategies to Trap Enzyme-Substrate Complexes that Mimic Michaelis Intermediates during E3-Mediated Ubiquitin-Like Protein Ligation

Frederick C. Streich Jr. and Christopher D. Lima

13.Small-Angle X-Ray Scattering for the Study of Proteins in the Ubiquitin Pathway

Jean-François Trempe and Kalle Gehring

14.Methods for Preparing Cryo-EM Grids of Large Macromolecular Complexes

Leifu Chang and David Barford

Part IV Methods to Study 26S Proteasome Function

15.Recombinant Expression, Unnatural Amino-Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes

Jared A. M. Bard and Andreas Martin

16.Native Gel Approaches in Studying Proteasome Assembly and Chaperones

Jeroen Roelofs, Anjana Suppahia, Kenrick A. Waite, and Soyeon Park

17.Measuring the Overall Rate of Protein Breakdown in Cells and the Contributions of the Ubiquitin-Proteasome and Autophagy-Lysosomal Pathways

Zhe Sha, Jinghui Zhao, and Alfred L. Goldberg

18.Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis

Chueh-Ling Kuo, Galen A. Collins, and Alfred L. Goldberg

19.Measurement of the Multiple Activities of 26S Proteasomes

Hyoung Tae Kim, Galen A. Collins, and Alfred L. Goldberg

20.Exploring the Regulation of Proteasome Function by Subunit Phosphorylation

Jordan J.S. VerPlank and Alfred L. Goldberg 

21.Scalable In Vitro Proteasome Activity Assay

Amit Kumar Singh Gautam, Kirby Martinez-Fonts, and Andreas Matouschek

Part V Proteomic Methods to Study the Ubiquitin Proteasome System

22.Exploring the Rampant Expansion of Ubiquitin Proteomics

Amalia Rose and Thibault Mayor

23.Ubiquitin diGLY Proteomics as an Approach to Identify and Quantify the Ubiquitin-Modified Proteome

24.Interpreting the Language of Polyubiquitin with Linkage-Specific Antibodies and Mass Spectrometry

Marissa L. Matsumoto, Erick R. Castellanos, Yi Jimmy Zeng, and Donald S. Kirkpatrick

25.Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking Assisted Affinity Purification and Mass Spectrometry

Xiaorong Wang and Lan Huang