SUMO: Methods and Protocols

319 Pages·2016·9.609 MB·English

by  Manuel S. Rodriguez

Methods in
Molecular Biology 1475
Manuel S. Rodriguez Editor
SUMO
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB , UK
For further volumes:
http://www.springer.com/series/7651

SUMO
Methods and Protocols
Edited by
Manuel S. Rodriguez
INBIOMED, San Sebastian, Spain
Editor
Manuel S . R odriguez
INBIOMED
San Sebastian
Spain
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6356-0 ISBN 978-1-4939-6358-4 (eBook)
DOI 10.1007/978-1-4939-6358-4
Library of Congress Control Number: 2016945171
© Springer Science+Business Media New York 2 016
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Cover illustration: The image depicts a C. elegans embryo in Metaphase of the fi rst mitotic division. Tubulin is seen in
green and SUMO in magenta. Image was acquired using 3D structured illumination with an OMX super-resolution
microscope.
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Prefa ce
The Small Ubiquitin-related MOdifi er (SUMO) molecules are implicated in the regulation
of multiple critical cellular functions and consequently associated with several pathologies.
Functions regulated by the different SUMO molecules are diverse and in most cases unre-
lated to those controlled by ubiquitin, with the enzymes regulating protein modifi cation
and de-m odifi cation by SUMO molecules being distinct to those regulating other members
of the ubiquitin family. However, recent discoveries indicate that SUMO-regulated func-
tions can be more interconnected with those regulated by ubiquitin than initially suspected.
In this volume of SUMO Methods and Protocols , leading experts propose basic and “state-
of-the-a rt” methodologies to explore biochemical, molecular, and cellular biology aspects
of some of the many processes regulated by protein SUMOylation . Chapters highlight
relevant aspects of the SUMO biology that should contribute to develop fundamental and
translational research in this area.
This volume is organized in four parts, which start with an historical overview of pro-
tein SUMOylation and a presentation of the methods included in this book. The fi rst part
also includes a review on chromatin regulation by dynamic SUMO modifi cations. The
second part of this volume focuses on in vitro techniques including biochemical methods
to study mechanistic aspects of protein SUMOylation. The third part includes protocols to
be used with cell cultures, which often are the fi rst approaches used in most laboratories.
The fi nal part includes methodologies adapted for the analysis in vivo using distinct model
organisms. This volume of S UMO Methods and Protocols has been written following the
highly successful Methods in Molecular Biology™ series format. Each chapter includes a
brief introduction to the subject, a list of necessary materials and reagents, a step-by-step
reproducible laboratory protocol, and a Notes section detailing tips on troubleshooting
and strategies to avoid known pitfalls. Unique and cutting edge, this SUMO Protocols
volume provides the necessary procedures for specialists as well as for researchers not famil-
iar with this vital system.
I would like to extend my deepest gratitude to all contributors of this book. Sharing
your know-how with the readers of this book is priceless. I would like to gratefully empha-
size the special efforts of Michael Matunis and Mark Hochstrasser who contributed with
outstanding reviews that provide relevant scientifi c background to this book. Special thanks
to the PROTEOSTASIS COST Action 1307 and the contribution of its members. We (the
SUMO community) are grateful to Humana Press for giving us the opportunity to assem-
ble this book and to John Walker for his help in the edition of this book.
San Sebastian, Spain Manuel S. Rodriguez
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I INTRODUCTION
1 Concepts and Methodologies to Study Protein SUMOylation:
An Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Michael J. Matunis and M anuel S . R odríguez
2 T he Regulation of Chromatin by Dynamic SUMO Modifications . . . . . . . . . . 2 3
Nicole R . W ilson and Mark H ochstrasser
PART II PROCEDURES TO STUDY PROTEIN SUMOYLATION IN VITRO
3 Reconstitution of the Recombinant RanBP2 SUMO E3 Ligase Complex . . . . 4 1
Tobias Ritterhoff , H rishikesh D as , Yuqing H ao , V olkan Sakin ,
Annette Flotho , Andreas W erner , and Frauke Melchior
4 P roduction and Purification of Recombinant SUMOylated Proteins
Using Engineered Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5
Frédérique Brockly , M arc Piechaczyk , and Guillaume Bossis
5 A Fluorescent In Vitro Assay to Investigate Paralog-S pecific
SUMO Conjugation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Nathalie E isenhardt , Viduth K. C haugule , and Andrea Pichler
6 I dentification and Characterization of SUMO-SIM Interactions . . . . . . . . . . . 7 9
Koraljka H usnjak , Jan K eiten-Schmitz , and S tefan Müller
7 R eal-Time Surface Plasmon Resonance (SPR) for the Analysis of Interactions
Between SUMO Traps and Mono- or PolySUMO Moieties . . . . . . . . . . . . . . 9 9
Wendy X olalpa , M anuel S . Rodríguez , and Patrick England
8 U sing Biotinylated SUMO-Traps to Analyze SUMOylated Proteins . . . . . . . . 1 09
Valérie Lang , Elisa D a S ilva-Ferrada , R osa Barrio ,
James D. Sutherland , and Manuel S. R odríguez
9 I n Vitro Characterization of Chain Depolymerization Activities
of SUMO-Specific Proteases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Julia Eckhoff and R. J ürgen D ohmen
PART III ANALYSIS OF PROTEIN SUMOYLATION USING CELL LINES
10 Detection of Protein SUMOylation In Situ by Proximity Ligation Assays . . . . 1 39
Umut S ahin , F lorence J ollivet , Caroline B erthier , H ugues d e Thé ,
and Valérie Lallemand-Breitenbach
11 I n Situ SUMOylation and DeSUMOylation Assays: Fluorescent Methods
to Visualize SUMOylation and DeSUMOylation in Permeabilized Cells . . . . . 151
Eri Yuasa and H isato S aitoh
vii
viii Contents
12 Analysis of SUMOylated Proteins in Cells and In Vivo
Using the BioSUMO Strategy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Lucia Pirone , Wendy X olalpa , Ugo Mayor , R osa Barrio ,
and James D. S utherland
13 L abel-Free Identification and Quantification of SUMO Target Proteins . . . . . 1 71
Ivo A . Hendriks and Alfred C . O. V ertegaal
14 The Use of Multimeric Protein Scaffolds for Identifying Multi-SUMO
Binding Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Elisa Aguilar-Martínez and Andrew D . Sharrocks
15 I solation of In Vivo SUMOylated Chromatin-Bound Proteins. . . . . . . . . . . . . 2 05
Tasneem Bawa-Khalfe
PART IV PROCEDURES TO STUDY PROTEIN SUMOYLATION IN VIVO
16 Identification of Substrates of Protein-Group SUMOylation . . . . . . . . . . . . . . 2 19
Ivan Psakhye and Stefan J entsch
17 Tools to Study SUMO Conjugation in Caenorhabditis elegans. . . . . . . . . . . . . 233
Federico Pelisch and Ronald T. Hay
18 Purification of SUMO Conjugates from Arabidopsis for Mass
Spectrometry Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Thérèse C. Rytz , Marcus J . Miller , and Richard D. Vierstra
19 Detection of SUMOylation in Plasmodium falciparum . . . . . . . . . . . . . . . . . . 283
Katherine H. Reiter and M ichael J . Matunis
20 S ystematic Localization and Identification of SUMOylation Substrates
in Knock-In Mice Expressing Affinity-Tagged SUMO1. . . . . . . . . . . . . . . . . . 291
Marilyn Tirard and Nils Brose
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 03
Contributors
ELISA AGUILAR-MARTÍNEZ • Faculty of Life Sciences, University of Manchester , M anchester, UK
ROSA BARRIO • CIC bioGUNE , D erio , B izkaia, S pain
TASNEEM BAWA-KHALFE • Center for Nuclear Receptors and Cell Signaling, Department of
Biology and Biochemistry, University of Houston , H ouston, T X , U SA
CAROLINE B ERTHIER • INSERM U944 - CNRS UMR7212 - Université Paris Diderot,
Sorbonne Paris Cité - Equipe labellisée par la Ligue Nationale contre le Cancer, IUH,
Hôpital St. Louis 1 , P aris , F rance ; C ollège de France , P aris, France
GUILLAUME BOSSIS • Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique
Moléculaire de Montpellier, CNRS UMR 5535 , U niversité de Montpellier , M ontpellier,
France
FRÉDÉRIQUE BROCKLY • Equipe Labellisée par la Ligue contre le Cancer, Institut de
Génétique Moléculaire de Montpellier, CNRS UMR 5535 , Université de Montpellier ,
Montpellier, F rance
NILS B ROSE • Department of Molecular Neurobiology, M ax Planck Institute
of Experimental Medicine , G oettingen, Germany
VIDUTH K. C HAUGULE • Department of Epigenetics, M ax Planck Institute
of Immunobiology and Epigenetics , Freiburg, Germany ; M RC Protein Phosphorylation
and Ubiquitylation Unit, College of Life Sciences, U niversity of Dundee , D undee, U K
HRISHIKESH DAS • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH),
DKFZ - ZMBH Alliance , H eidelberg, Germany
R. JÜRGEN D OHMEN • Institute for Genetics, Biocenter, U niversity of Cologne , C ologne ,
Germany
JULIA ECKHOFF • Institute for Genetics, Biocenter, University of Cologne , Cologne, G ermany
NATHALIE E ISENHARDT • Department of Epigenetics, M ax Planck Institute of Immunobiology
and Epigenetics , F reiburg, Germany
PATRICK ENGLAND • Plate-forme de Biophysique des Macromolécules et de leurs Interactions,
Institut Pasteur, CNRS , Paris , F rance ; CNRS-UMR3528 , P aris, F rance
ANNETTE FLOTHO • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH),
DKFZ - ZMBH Alliance , Heidelberg, G ermany
YUQING HAO • Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH),
DKFZ - ZMBH Alliance , H eidelberg, Germany
RONALD T. H AY • Centre for Gene Regulation and Expression, School of Life Sciences,
University of Dundee , Dundee, UK
IVO A. HENDRIKS • Department of Molecular Cell Biology , L eiden University Medical
Center , L eiden, Netherlands
MARK HOCHSTRASSER • Department of Molecular Biophysics and Biochemistry , Y ale
University , N ew Haven, CT, USA
KORALJKA H USNJAK • Institute of Biochemistry II, Goethe University Medical School ,
Frankfurt (Main), G ermany
STEFAN JENTSCH • Department of Molecular Cell Biology , Max Planck Institute
of Biochemistry , Martinsried, Germany
ix