Plant Proteostasis: Methods and Protocols

321 Pages·2016·11.988 MB·English

by  L. Maria Lois,  Rune Matthiesen

Methods in
Molecular Biology 1450
L. Maria Lois
Rune Matthiesen Editors
Plant
Proteostasis
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

Plant Proteostasis
Methods and Protocols
Edited by
L. Maria Lois
Development Program, CRAG (CSIC-IRTA-UAB-UB) Edifici CRAG-Campus UAB, Bellaterra
(Cerdanyola del Vallés), Barcelona, Spain
Rune Matthiesen
Computational and Experimental Biology Group, National Health Institute

Dr. Ricardo Jorge, IP, Lisbon, Portugal
Editors
L. Maria L ois Rune Matthiesen
Development Program Computational and Experimental Biology Group
CRAG (CSIC-IRTA-UAB-UB) Edifici CRAG- National Health Institute Dr. Ricardo Jorge
Campus UAB IP , L isbon , P ortugal
Bellaterra (Cerdanyola del Vallés)
Barcelona, Spain
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-3757-8 ISBN 978-1-4939-3759-2 (eBook)
DOI 10.1007/978-1-4939-3759-2
Library of Congress Control Number: 2016943033
© Springer Science+Business Media New York 2 016
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Prefa ce
Plant responses to environmental stimuli and developmental transitions are regulated by
complex regulatory networks that deliver the specifi c physiological outcome to assure plant
survival. These networks include transcriptional regulation but also sophisticated posttrans-
lational modifi cations that aim to regulate protein activity. In contrast to transcriptional
regulation, which involves de novo protein synthesis, posttranslational modifi cations mod-
ulate protein activity in short time periods facilitating rapid cell responses. The molecular
consequences of posttranslational modifi cations on the protein target are highly variable
and include changes in protein structure, subcellular localization, activity, partner interac-
tions, stability, or solubility.
Proteins can be modifi ed by a wide array of compounds that vary in their nature, size,
and conjugation mechanism. As such, reactive oxygen species induce protein o xidation
independently on enzymatic catalysis, while other posttranslational modifi cations involving
the addition of small organic groups (i.e., phosphate or methyl groups) are regulated by
enzymes dedicated to the addition or removal of the specifi c modifi er. Finally, one of the
most complex posttranslational modifi cation groups is represented by the ubiquitin (Ub)
and ubiquitin-like (Ubl) modifi ers, which are small proteins that are conjugated to protein
targets through a cascade of three enzymatic steps and deconjugated by specifi c
peptidases.
The branching complexity of post-translational modifi cations, together with their labile
nature and the need of custom-tailored molecular tools, make their analysis really challeng-
ing. In plants, the absence of well established commercial tools, the more complex plant cell
manipulation required for biochemical studies, and the gene amplifi cation displayed by
many members of these regulatory components, result in a higher diffi culty degree of bio-
chemical and genetic studies. The analysis of protein homeostasis is even more complex in
non-plants models since specifi c protocols and tools are poorly or not developed.
In this book, we have collected detailed protocols describing state-of-the-art approaches
that will facilitate the understanding of protein homeostasis in plant stress responses and
development. Some fi ndings made in this area of plant research could become valuable
molecular tools in selection processes for improving agronomic performance, but also for
contributing to address next challenges in agriculture such as precision horticulture.
Part I contains protocols focusing on the study of ubiquitin-dependent posttransla-
tional modifi cations. While Chapter 1 describes a protocol for studying a novel ubiquitin
conjugation mechanism independent of lysine residues, the other chapters focus on differ-
ent aspects of the classical ubiquitin-dependent protein degradation system. Chapter 2 pro-
vides methods for analyzing the in vivo dynamics of cullins, key components of RING E3
ligases catalyzing ubiquitin conjugation to substrates. Chapter 3 describes the study of
F-box proteins , another component of RING E3 ligases, as plant hormone receptor , which
has become a key step in triggering hormone signaling. As many posttranslational modifi ca-
tions, ubiquitination is a reversible modifi cation and Chapter 4 focuses on approaches for
the study of enzymes involved in ubiquitin removal from its substrate. Chapters 5 and 6
address the generation of substrates for analyzing the in vivo ubiquitin/proteasome system
v
vi Preface
and the N-rule pathway for protein degradation, respectively. Finally, Chapter 7 extends the
study of the N-rule pathway through methods for identifying E3 ligases.
Part II is dedicated to protocols focused on the study of Ubl posttranslational m odifi ca-
tions , including in vitro SUMO c hain formation (Chapter 8 ) , the kinetic analysis of SUMO
conjugation machinery (Chapter 9 ), and the in vitro analysis of SUMO p roteases involved
in SUMO maturation and SUMO removal from substrates (Chapter 10 ) . In addition,
Chapter 11 addresses the analysis of cellular distribution of SUMO conjugation machinery
members as a strategy to get insights into their in vivo role. Another Ubl modifi cation
involved in many aspects of plant stress responses and development is autophagy , and bio-
chemical and cell biology protocols for its study are described in Chaps. 12 and 1 3 .
The study of protein homeostasis requires a broad variety of protocols that go beyond
the analysis of enzymatic activities responsible for posttranslational m odifi cations , and some
of these protocols are comprised in Part III. A very useful and rapid approach to study
protein stability consists in the expression of recombinant protein in plant protoplasts as is
described in Chapter 14 . Another emerging fi eld in plant protein homeostasis is the study
of protein aggregate formation in response to environmental stress, and their purifi cation,
described in Chapter 15 , is the fi rst step into their analysis. Chapter 1 6 provides a protocol
for the study of another phenomenon occurring in response to stress consisting in protein
oxidation under reactive oxygen s pecies generation and the determination of proteasome
activity . When the aim is to identify global changes in protein homeostasis during physio-
logical responses, comparative proteomics based on iTRAQ are to be used (Chapter 1 7 ).
Chapter 18 describes methods for the study of protein binding to phosphatidylinositol as a
modulation mechanism of protein homeostasis. Also, organelle purifi cation is recom-
mended in order to reduce the complexity of the sample when performing proteomic stud-
ies in cell compartments, and Chapter 19 describes methods for the study of c hloroplast
proteome. Finally, Chapter 2 0 focuses on a general but also essential technique when trying
to determine fl uctuations in protein levels between samples, which is w estern blotting
normalization.
Finally, Past IV encloses protocols for the in silico analysis of different aspects of pro-
teostasis. Chapter 21 describes a protocol for identifying the genes encoding specifi c pro-
tein families and investigating their syntenic relationship. Chapter 22 focuses on methods
for performing phylogenetic analysis, as a means of inferring functional conservation in
different plant species. The last chapter (Chapter 23 ) describes the use of bioinformatics
tools for data mining, focusing on the SUMO gene n etwork .
We are thankful to the authors who have contributed to make this book possible. Also,
we thank John Walker, the series editor, for his advice and the colleagues at Humana Press
for producing this book. This book is based upon work from COST Action (PROTEOSTASIS
BM1307), supported by COST (European Cooperation in Science and Technology).
Barcelona, Spain L. Maria Lois
Lisbon, Portugal R une Matthiesen
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I UBIQUITIN CONJUGATION AND DECONJUGATION ANALYSIS
1 Approaches to Determine Protein Ubiquitination Residue Types. . . . . . . . . . . 3
Qian C hen , X iaoyuan Y ang , and Qi X ie
2 I mmunoprecipitation of Cullin-Ring Ligases (CRLs) in A rabidopsis
thaliana Seedlings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Anna F ranciosini and Giovanna S erino
3 Radioligand Binding Assays for Determining Dissociation Constants
of Phytohormone Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 3
Antje Hellmuth and Luz Irina A. Calderón Villalobos
4 Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes. . . . 3 5
Kamila K alinowska , Marie-Kristin N agel , and Erika I sono
5 F luorescent Reporters for Ubiquitin-Dependent Proteolysis in Plants . . . . . . . 4 5
Katarzyna Zientara-Rytter and Agnieszka Sirko
6 G eneration of Artificial N-end Rule Substrate Proteins
In Vivo and In Vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Christin N aumann , A ugustin C . M ot , and Nico D issmeyer
7 P eptide Arrays for Binding Studies of E3 Ubiquitin Ligases. . . . . . . . . . . . . . . 8 5
Maria K lecker and Nico D issmeyer
PART II U BIQUITIN-LIKE PROTEIN CONJUGATION, DECONJUGATION,
AND CELL IMAGING STUDIES
8 SUMO Chain Formation by Plant Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 9 7
Konstantin Tomanov , I onida Z iba , and Andreas B achmair
9 K inetic Analysis of Plant SUMO Conjugation Machinery . . . . . . . . . . . . . . . . 1 07
Laura Castaño-Miquel and L. M aria L ois
10 Expression, Purification, and Enzymatic Analysis
of Plant SUMO Proteases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Gary Yates , Anjil S rivastava , B eatriz O rosa , and Ari Sadanandom
11 Q uantitative Analysis of Subcellular Distribution of the SUMO
Conjugation System by Confocal Microscopy Imaging . . . . . . . . . . . . . . . . . . 135
Abraham Mas , Montse A menós , and L. M aria Lois
12 B iochemical Analysis of Autophagy in Algae and Plants
by Monitoring the Electrophoretic Mobility of ATG8. . . . . . . . . . . . . . . . . . . 151
María E sther P érez-Pérez , A scensión A ndrés-Garrido ,
and J osé L. Crespo
vii
viii Contents
13 Detection of Autophagy in Plants by Fluorescence Microscopy . . . . . . . . . . . . 1 61
Yunting Pu and Diane C . B assham
PART III PROTEOMIC ANALYSIS AND OTHER POST-TRANSLATIONAL
MODIFICATION STUDIES
14 Protocols for Studying Protein Stability in an Arabidopsis
Protoplast Transient Expression System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 75
Séverine P lanchais , Laurent Camborde , and Isabelle J upin
15 D etection and Quantification of Protein Aggregates in Plants . . . . . . . . . . . . . 1 95
Marc Planas-Marquès , Saul L ema A. , and N úria S. C oll
16 D etermination of Protein Carbonylation and Proteasome
Activity in Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 05
Qiong X ia , H ayat E l-Maarouf-Bouteau , Christophe Bailly ,
and Patrice M eimoun
17 I sobaric Tag for Relative and Absolute Quantitation (iTRAQ)-Based
Protein Profiling in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Isabel C ristina V élez-Bermúdez , Tuan-Nan W en , P ing Lan ,
and Wolfgang S chmidt
18 U se of a Phosphatidylinositol Phosphate Affinity Chromatography
(PIP Chromatography) for the Isolation of Proteins Involved
in Protein Quality Control and Proteostasis Mechanisms in Plants. . . . . . . . . . 223
T . Farmaki
19 In Vivo Radiolabeling of A rabidopsis Chloroplast Proteins and
Separation of Thylakoid Membrane Complexes by Blue Native PAGE. . . . . . . 233
Catharina Nickel , T homas B rylok , and Serena Schwenkert
20 N ormalized Quantitative Western Blotting Based on Standardized
Fluorescent Labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Frederik F aden , L ennart E schen-Lippold , and Nico D issmeyer
PART IV BIOINFORMATICS ANALYSIS
21 Sequence Search and Comparative Genomic Analysis of SUMO-Activating
Enzymes Using CoGe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Lorenzo Carretero-Paulet and V ictor A. A lbert
22 S tudying Evolutionary Dynamics of Gene Families Encoding
SUMO-Activating Enzymes with SeaView and ProtTest . . . . . . . . . . . . . . . . . 273
Lorenzo C arretero-Paulet and Victor A . A lbert
23 B ioinformatics Tools for Exploring the SUMO Gene Network . . . . . . . . . . . . 2 85
Pedro H umberto C astro , Miguel  ngelo Santos , Alexandre P apadopoulos
Magalhães , R ui Manuel Tavares , and Herlânder Azevedo
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 03
Contributors
VICTOR A. A LBERT • Department of Biological Sciences, S UNY-University at Buffalo ,
Buffalo, N Y , U SA
MONTSE AMENÓS • Confocal Microscopy Facility, CRAG (CSIC-IRTA-UAB-UB), E difi ci
CRAG-Campus UAB , B ellaterra (Cerdanyola del Vallés), B arcelona, Spain
ASCENSIÓN ANDRÉS-GARRIDO • Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo
Superior de Investigaciones Científi cas (CSIC), U niversidad de Sevilla , S evilla, Spain
HERLÂNDER A ZEVEDO • CIBIO, InBIO—Research Network in Biodiversity and
Evolutionary Biology, U niversidade do Porto, Campus Agrário de Vairão , V airão,
Portugal
ANDREAS BACHMAIR • Department of Biochemistry and Cell Biology, Max F. Perutz
Laboratories, U niversity of Vienna , Vienna, A ustria
CHRISTOPHE BAILLY • Institut de Biologie Paris-Seine, U nité Mixte de Recherche 7622 ,
Paris , F rance ; B iologie du Développement, Institut de Biologie Paris-Seine, Unité Mixte
de Recherche 7622, C entre National de la Recherche Scientifi que , P aris, F rance
DIANE C . BASSHAM • Department of Genetics, Development and Cell Biology and Plant
Sciences Institute, Iowa State University , Ames , I A , U SA
THOMAS BRYLOK • Botanik, Department Biologie I, Ludwig-Maximilians-Universität ,
Planegg-Martinsried, G ermany
LAURENT C AMBORDE • Laboratoire de Virologie Moléculaire, Institut Jacques Monod,
CNRS, Univ. Paris-Diderot, Sorbonne Paris Cité , P aris, France; Laboratoire de
Recherche en Sciences Végétales, CNRS, Univ Toulouse Paul Sabatier, Toulouse, France
LORENZO C ARRETERO-PAULET • Department of Plant Systems Biology , VIB, Ghent
University , G hent, Belgium
LAURA C ASTAÑO-MIQUEL • Development Program , CRAG (CSIC-IRTA-UAB-UB), Edifi ci
CRAG-Campus UAB , Bellaterra (Cerdanyola del Vallés), Barcelona, Spain
PEDRO H UMBERTO CASTRO • Biosystems and Integrative Sciences Institute (BioISI), Plant
Functional Biology Center , University of Minho, Campus de Gualtar , B raga, Portugal
QIAN C HEN • State Key Laboratory of Plant Genomics, National Center for Plant Gene
Research, Institute of Genetics and Developmental Biology, C hinese Academy of Sciences ,
Beijing, P . R. China ; Graduate University of Chinese Academy of Sciences , B eijing,
P. R. China
NÚRIA S . COLL • Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB) ,
Bellaterra, S pain
JOSÉ L. C RESPO • Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de
Investigaciones Científi cas (CSIC), Universidad de Sevilla , Sevilla, S pain
NICO DISSMEYER • Leibniz Institute of Plant Biochemistry (IPB) , H alle (Saale), G ermany ;
ScienceCampus Halle – Plant-Based Bioeconomy , H alle (Saale), G ermany
HAYAT EL-MAAROUF-BOUTEAU • Institut de Biologie Paris-Seine, Unité Mixte de Recherche
7622, S orbonne Universités , P aris , F rance ; Biologie du Développement, Institut de
Biologie Paris-Seine, Unité Mixte de Recherche 7622 , C entre National de la Recherche
Scientifi que , P aris , France
ix