SUMO Protocols

1. Front Matter

   Pages i-x

   PDF

2. Introduction

   1. The SUMO System: An Overview

      • Helle D. Ulrich

      Pages 3-16

3. Identification of Sumo Targets

   1. Identification of SUMO Target Proteins by Quantitative Proteomics

      • Jens S. Andersen, Ivan Matic, Alfred C O. Vertegaal

      Pages 19-31

   2. Identification of SUMO-Conjugated Proteins and their SUMO Attachment Sites Using Proteomic Mass Spectrometry

      • James A. Wohlschlegel

      Pages 33-49

   3. Identification of SUMO Targets Through In Vitro Expression Cloning

      • Christian B. Gocke, Hongtao Yu

      Pages 51-61

   4. Enhanced Detection of In Vivo SUMO Conjugation by Ubc9 Fusion-Dependent Sumoylation (UFDS)

      • Rainer Niedenthal

      Pages 63-79

   5. In Vivo Detection and Characterization of Sumoylation Targets in Saccharomyces cerevisiae

      • Helle D. Ulrich, Adelina A. Davies

      Pages 81-103

4. Identification Of Sumo Interactors

   1. Identification of SUMO-Interacting Proteins by Yeast Two-Hybrid Analysis

      • Mary B. Kroetz, Mark Hochstrasser

      Pages 107-120

   2. Identification of SUMO-Binding Motifs by NMR

      • Candace S. Seu, Yuan Chen

      Pages 121-138

5. Functional Analysis Of Sumoylation In Vivo

   1. Regulation of Transcription Factor Activity by SUMO Modification

      • Jian Ouyang, Alvaro Valin, Grace Gill

      Pages 141-152

   2. Characterization of the Effects and Functions of Sumoylation Through Rapamycin-Mediated Heterodimerization

      • Shanshan Zhu, Michael J. Matunis

      Pages 153-164

6. Biochemical Studies Of Sumo Conjugation

   1. Purification of SUMO Conjugating Enzymes and Kinetic Analysis of Substrate Conjugation

      • Ali A. Yunus, Christopher D. Lima

      Pages 167-186

   2. Performing In Vitro Sumoylation Reactions Using Recombinant Enzymes

      • Andreas Werner, Marie-Christine Moutty, Ulrike Möller, Frauke Melchior

      Pages 187-199

   3. Preparation of Sumoylated Substrates for Biochemical Analysis

      • Puck Knipscheer, Helene Klug, Titia K. Sixma, Andrea Pichler

      Pages 201-210

   4. Strategies for the Expression of SUMO-Modified Target Proteins in Escherichia coli

      • Hisato Saitoh, Junsuke Uwada, Kawasaki Azusa

      Pages 211-221

7. Biochemical Studies of Sumo Deconjugation

   1. Preparation of SUMO Proteases and Kinetic Analysis Using Endogenous Substrates

      • David Reverter, Christopher D. Lima

      Pages 225-239

   2. An In Vitro FRET-Based Assay for the Analysis of SUMO Conjugation and Isopeptidase Cleavage

      • Nicolas Stankovic-Valentin, Lukasz Kozaczkiewicz, Katja Curth, Frauke Melchior

      Pages 241-251

   3. FRET-Based In Vitro Assays for the Analysis of SUMO Protease Activities

      • Michael H. Tatham, Ronald T. Hay

      Pages 253-268

   4. Detection and Characterization of SUMO Protease Activity Using a Sensitive Enzyme-Based Reporter Assay

      • Craig A. Leach, Xufan Tian, Michael R. Mattern, Benjamin Nicholson

      Pages 269-281

8. Manipulation And Alternative Uses Of The Sumo System

   1. Inhibition of the SUMO Pathway by Gam1

      • Mariaelena Pozzebon, Chiara V. Segré, Susanna Chiocca

      Pages 285-301

Post-translational protein modifications by members of the ubiquitin family are widely recognized as important regulatory control systems for a variety of biological pathways. Their influence on eukaryotic cellular metabolism is comparable to that of other modifi- tions such as phosphorylation, acetylation and methylation. The small ubiquitin-related modifier SUMO uses a conjugation and de-conjugation system closely related to that of ubiquitin itself; yet, the functions of the SUMO system are highly diverse and largely independent of the ubiquitin system. SUMO modification controls the activity of tr- scription factors and can influence protein stability, but it also contributes to nucleo-cy- plasmic transport, chromosome segregation and DNA damage repair. As a consequence, the SUMO system pervades virtually all areas of basic molecular and cell biology, and scientists from different backgrounds, including medical researchers, are likely to enco- ter SUMO in the course of their studies. This volume, SUMO Protocols, therefore aims at presenting a collection of methods relevant to SUMO research in order to make these tools available to biochemists, molecular and cell biologists as well as research-oriented clinicians not yet familiar with the system. In contrast to the ubiquitin system, which has been the subject of several reviews and methods collections, no practical compendium entirely devoted to SUMO has been p- lished.

• Escherichia coli
• Proteomics
• Vivo
• enzymes
• molecular biology
• regulation
• saccharomyces cerevisiae
• transcription

From the reviews:

"The modification of proteins by small ubiquitin-like modification (SUMO) is relevant to virtually all areas of molecular and cell biology. SUMO Protocols is a quite comprehensive volume describing various in vivo and in vitro techniques … . The target audience is likely to be postdoctoral fellows and PhD students in laboratories not only dedicated to SUMO pathways but those beginning their initial forays in the field … and regulated by SUMO. … for those pursuing SUMO-specific modification this is a worthwhile addition to their bookshelves." (Peter O’Hare, Microbiology Today, July, 2009)

• London Research Institute Clare Hall Laboratories, Cancer Research UK, Herts, UK

  Helle D. Ulrich

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