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M E T H O D S I N M O L E C U L A R B I O L O G YTM
Two-Hybrid Systems
Methods and Protocols
Edited by
Paul N. MacDonald
Department of Pharmacology, School of Medicine
Case Western Reserve University
Cleveland, OH
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Preface
Many, if not all, essential biological processes require selective interactions
between proteins. Complex signaling systems require sequential, ordered
protein–protein interactions at essentially all levels of the signaling cascade. For
example, peptide hormones interact with selective membrane receptor proteins,
and autophosphorylation of the receptor then recruits other key regulatory
proteins that initiate kinase cascades in which each phosphorylation event
requires selective recognition of the protein substrate. The ultimate signaling
effect, in many cases, is the regulation of RNA polymerase II-directed transcrip-
tion in the nucleus, a process that involves numerous, multiprotein complexes
important for transcription initiation, elongation, termination, and reinitiation.
Defining, characterizing, and understanding the relevance of these protein–
protein interactions is an arduous task, but substantial inroads have been made
over the past 20 years. The development of more recent methodologies, such as
mammalian expression systems, immunopurification schemes, expression
cloning strategies, surface plasmon resonance (BiaCore), and nanosequencing
technologies, has contributed a wealth of new insights into these complex
multiprotein mechanisms and clearly accelerated the discovery process.
Arguably, the yeast two-hybrid system has been one of the predominant and most
powerful tools in this discovery process.
On a personal note, my specific interest in the yeast two-hybrid system
developed in a manner probably not terribly different from that of many other
investigators who were interested in the early 1990s in identifying and charac-
terizing interactions between two proteins.While working in the laboratory of
Mark R. Haussler, our interests centered on the vitamin D receptor (VDR), a
member of the nuclear receptor family, and the mechanisms involved in VDR
binding to DNA. Specifically, I was interested in identifying a nuclear factor
that interacted with and conferred high-order binding of the VDR to DNA.
We and other larger groups in the nuclear receptor field chose a tradi-
tional biochemical approach that focused on purifying and identifying the
unknown nuclear accessory factor. Other laboratories used expression
cloning strategies with purified radiolabeled proteins to screen cDNA
expression libraries for clones encoding the interacting factor.Both approaches
were comparatively large efforts at the time, requiring a tremendous number
v
vi Preface
Fig. 1. The number of publications over the past 10 years that were found in a
search of PubMed using “two-hybrid” in the search window. The year 2000 value is
projected based on the number of references found at the time of the search (Septem-
ber, 2000) and the number of remaining months in the year.
of person-hours.Both approaches eventually resulted in the successful identi-
fication of the factor as retinoid X receptor, a common heterodimeric partner
for many of the class II nuclear receptors. Unfortunately, we were not one of
the groups to first report the identification of RXR as the partner. Our smaller
effort was, in no uncertain terms, “scooped.”
At about this same time, reports from the Fields laboratory on the successful
use of the yeast two-hybrid system began to emerge and more beneficial yeast
strains and vectors were being developed.The power of the system was inspiring
to anyone working on trying to identify protein interaction partners. Here was a
simple, direct screening assay that could uncover novel factors that interacted
with your protein of interest. Millions of cDNAs could be screened in a single
experiment, in a relatively short time, and with comparatively less effort.
Following the initial screen, the cDNA clones encoding the putative interactors
were already in hand and they could be directly sequenced and identified. The
playing field seemed somehow leveled a bit by the two-hybrid system.More than
twelve years have passed since the original description of the yeast two-hybrid
system was reported, and few would disagree that this system has had a
Preface vii
tremendous impact on virtually every field of modern biology. Continuous
refinements and novel innovations of the original systems over the past decade
have only strengthened the utility of the approach. As illustrated in Fig. 1, it is
obvious that many groups continue to adopt the two-hybrid system as a new
approach in their laboratories and this trend will only continue to expand in the
future as the era of functional genomics unravels over the next century.
Therefore, the overall goal for Two-Hybrid Systems: Methods and
Protocols is to introduce the yeast two-hybrid system to students, research
assistants, research associates, and other more senior investigators considering
this as a new approach in their laboratories and research projects. Toward this
end, I have assembled a collection of detailed descriptions of basic protocols and
a compendium of experimental approaches in different biological systems that
I hope reflects the utility of the system and its variations in modern biomedical
research.My hope is that this will also serve as a useful reference for those labo-
ratories that have extensive experience with the two-hybrid system. Thus,
I invited several authors to discuss in more general terms some of the problems
and strategies involved in the yeast two-hybrid assay as well as some of the alter-
native systems that have evolved from the original system that may prove useful
to those more experienced two-hybrid laboratories.
Two-Hybrid Systems: Methods and Protocols is divided into four main
sections. The first section is a compendium of general methodologies that are
used in the two-hybrid system. Here, the reader will find in-depth discussion
and detailed methodologies that serve as the foundation on which successful
yeast two-hybrid experiments rest. Since many laboratories beginning two-
hybrid approaches have not worked with yeast to a significant extent, this first
section begins with a general introduction to handling yeast, a detailed
compendium of media formulations, as well as an overview of the common
strains of yeast and plasmid vectors that are used for two-hybrid work.
This section ends with three chapters that describe the basic methodologies
involved in introducing plasmids into yeast, interaction assays, and recov-
ering the plasmids from yeast. This first section was intentionally designed to
be somewhat repetitive in nature with components of the subsequent applica-
tion chapters. The intent was to provide more in-depth methodological detail
and variations of these fundamental techniques that serve as the backbone of
any two-hybrid assay as well as to illustrate how these techniques are incorpo-
rated into individual applications. One well-known, recurring drawback of
the two-hybrid system is the potential for artifacts and false positives. Thus,
Section II provides a discussion of the various classes of false positives and
the common mechanisms through which false-positives arise. This section
also includes two chapters that focus on general strategies and detailed
viii Preface
protocols to confirm the authenticity of the interaction using in vitro protein–
protein interaction assays. Part III includes four application chapters that
describe how the yeast two-hybrid system was applied in various systems to
identify interacting partners in important biological systems including the Smad
and nuclear receptor pathways. Finally, Part IV describes various
alternative strategies that have arisen out of the original yeast two-hybrid
paradigm. These alternative strategies include the one-hybrid, split two-
hybrid, three-hybrid, membrane recruitment systems, and mammalian systems.
These alternative systems serve to illustrate the flexibility and refinements
that are possible with the basic two-hybrid approach.
The authors and I hope that Two-Hybrid Systems: Methods and Protocols
will prove a valuable addition to any laboratory that is interested in studying
macromolecular interactions between proteins.
I would like to express my sincere gratitude to all the authors for their
valuable, insightful contributions and for their patience in seeing this project
to fruition.This book is a testament to their breadth of knowledge on the topic
and the power of the two-hybrid approach. It is evident that both the basic
system, as well as its many variants, will continue to play a predominant role
in the characterization and identification of protein–protein interactions in the
genomic and proteomic arenas of the 21st century.
Paul N. MacDonald
Contents
Preface...........................................................................................v
Contributors..................................................................................xi
PART I GENERAL METHODS
1 The Two-Hybrid System: A Personal View
Stanley Fields and Paul L. Bartel..............................................3
2 Growth and Maintenance of Yeast
Lawrence W. Bergman................................................................9
3 Media Formulations for Various Two-Hybrid Systems
Michael Saghbini, Denise Hoekstra, and Jim Gautsch........15
4 Yeast Two-Hybrid Vectors and Strains
Philip James...............................................................................41
5 High-Efficiency Transformation of Plasmid DNA into Yeast
Robin A. Woods and R. Daniel Gietz......................................85
6 Qualitative and Quantitative Assessment of Interactions
Monica M. Montano...................................................................99
7 Strategies for Rescuing Plasmid DNA
from Yeast Two-Hybrid Colonies
Alyson Byrd and René St-Arnaud.........................................107
PART II FALSE POSITIVES
8 Two-Hybrid System and False Positives:
Approaches to Detection and Elimination
Ilya G. Serebriiskii and Erica A. Golemis.............................123
9 Confirming Yeast Two-Hybrid Protein Interactions
Using In Vitro Glutathione-S-Transferase Pulldowns
Dennis M. Kraichely and Paul N. MacDonald......................135
10 Two-Hybrid Interactions Confirmed
by Coimmunoprecipitation of Epitope-Tagged Clones
Louie Naumovski.....................................................................151
ix
x Contents
PART III APPLICATIONS
11 Smad Interactors in Bone Morphogenetic Protein Signaling
Xiangli Yang and Xu Cao........................................................163
12 Protein Interactions Important in Eukaryotic
Translation Initiation
Katsura Asano and Alan G. Hinnebusch..............................179
13 Steroid Receptor and Ligand-Dependent Interaction
with Coactivator Proteins
Sergio A. Oñate........................................................................199
14 Interaction of Cellular Apoptosis Regulating Proteins
with Adenovirus Anti-apoptosis Protein E1B-19K
Thirugnana Subramanian and G. Chinnadurai....................211
PART IV ALTERNATIVE STRATEGIES
15 Mammalian Two-Hybrid Assays: Analyzing Protein-Protein
Interactions in the Transforming Growth Factor-β
Signaling Pathway
Xin-Hua Feng and Rik Derynck..............................................221
16 One-Hybrid Systems for Detecting Protein-DNA Interactions
Mary Kate Alexander, Brenda D. Bourns,
and Virginia A. Zakian.........................................................241
17 The Split-Hybrid System: Uncoding Multiprotein Networks
and Defining Mutations That Affect Protein Interactions
Phyllis S. Goldman, Anthony J. DeMaggio,
Richard H. Goodman, and Merl F. Hoekstra....................261
18 Three-Hybrid Screens: Inducible Third-Party Systems
Björn Sandrock, Franck Tirode, and Jean-Marc Egly.........271
19 Three-Hybrid Screens for RNA-Binding Proteins:
Proteins Binding 3' End of Histone mRNA
Zbigniew Dominski and William F. Marzluff.........................291
20 Membrane Recruitment Systems for Analysis
of Protein–Protein Interactions
Ami Aronheim..........................................................................319
Index ..........................................................................................329
