15 Two-Hybrid Analysis of Protein-Protein Interactions in Yeast

15 Two-Hvbrid Analvsis of ProteiIi-protein InJteractions in Yeast John Rosamond

School of Biological Sciences, University of Manchester, Manchester, UK

CONTENTS Introduction Background DNA components of the two-hybrid system Interaction screening Future developments

List of Abbreviations HA 3-AT UAS

Hernagglutinin 3-amino- I ,ZCtriazole Upstream activating sequence

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INTRODUCTION

Protein-protein interactions underpin a wide range of complex cellular processes and in many cases make a critical contribution to the regulation of those processes. Consequently, a key question in the study of the cellular function of any novel protein is to identify other proteins with which it interacts. There are many physical, biochemical and immunological methods available to identify and quantify such interactions (Phizicky and Fields, 1995). These have recently been complemented by geneticbased methods of which one, the yeast two-hybrid system (Chien et al., 19911, was developed as a sensitive genetic tool to identify proteinprotein interactions in vivo. In this chapter, I will describe the background to the two-hybrid system, the properties of the basic elements used to define interactions using the two-hybrid system and briefly review .some of the potential future developments of this technology. METHODS IN MICROBIOLOGY, VOLUME 26 ISBN 0-12-5215264

Copyright 0 1998 Academic Press Ltd All rights of reproduction in any form reserved

Initiation of transcription in yeast, as in most eukaryotes, requires the concerted action of several molecular processes. All yeast genes are preceded by a loosely conserved TATA-box that influences both the start site for transcription and the intrinsic basal level of gene expression. In addition to the TATA-box, other cis-acting elements include upstream activating sequences (UAS), which act as binding sites for specific transcription factors. Interaction of a transcription factor with its cognate UAS acts to enhance expression of the adjacent structural gene. The two-hybrid system takes advantage of the specificity of this interaction between a transcription factor and its UAS as well as relying on other properties of the transcription factor itself. Eukaryotic transcription factors are modular proteins containing discrete, highly defined functional domains within the protein (Keegan et al., 1986; Hope and Struhl, 1986; Ma and Ptashne, 1987). Experiments with the GAL4-encoded transcription factor Gal4p established at least two, separable domains: one domain (the DNA-binding domain) was required to bind the protein to the DNA at the specific UAS (UAS,) upstream of the genes regulated by GAL4; the second domain (the activation domain) was required to activate transcription, probably by direct interaction between that domain of Gal4p and the transcriptional apparatus itself. Both domains of Gal4p are required for effective transcriptional regulation so that a protein containing only the DNAbinding domain or the activation domain will not activate transcription. However, several experiments have demonstrated that the two domains need not be localized within the same protein molecule providing they could be brought into an appropriate configuration at the UAS. Using a Gal4p derivative that contained the DNA-binding domain and a region that binds Gal8Op (but lacked the activation domain), Ma and Ptashne (1987) showed that this protein was unable to activate the transcription of a reporter gene containing an upstream UAS,. However, when the protein was co-expressed in yeast with a Gal8Op protein fused to the Gal4p activation domain, interaction between the Gal80p activationdomain fusion and the Gal4p DNA-binding protein at UAS, resulted in transcription of the adjacent reporter gene. In analogous experiments, Fields and Song (1989) showed that transcriptional activation of a reporter gene in yeast could be used specifically to monitor the association between two proteins if one was expressed as a fusion with a DNAbinding domain and the other as a fusion with an activation domain. Using Snflp fused to the Gal4p DNA-binding domain and Snf4p fused to the Gal4p activation domain, they showed that the known interaction between Snfl p and Snf4p could effectively reconstitute functional Gal4p transcription factor activity and direct the expression of a lucZ reporter with an upstream UAS,. These observations served as the basis for the two-hybrid system to detect protein-protein interactions in viva As shown in Figure 1, the assay of a simple reporter gene can be used as an output to measure the interaction between two known proteins fused independently to DNA-binding and activation domains (Iwabuchi et ul.,

I

Reporter Gene

Figure 1. Schematic representation of the two-hybrid system in which protein A, fused to the DNA-binding domain (BD), interacts with protein B fused to the transcriptional activator domain (AD), to reconstitute active transcription factor activity from the UAS adjacent to a reporter gene.

19931, or to screen for interactions between a known protein in one fusion and a genomic or cDNA library as the other fusion. All current versions of the two-hybrid system that have been developed in several laboratories are based on these experimental findings. Although the several versions differ in their specific components, all comprise the same three basic constituents: yeast vectors for the expression of DNA-binding domain fusions, usually referred to as “bait” fusions; vectors for the expression of cDNA or genomic D N A fused to a transcription activator domain, usually referred to as “prey” fusions; and 0 yeast strains carrying reporter gene(s) with appropriate UAS sites for the DNA-binding domain.

The basic protocol to test for interactions between two proteins can be summarized as follows: (a) constructing a plasmid encoding a protein fusion between a DNAbinding domain and a protein under test; (b) constructing a plasmid encoding protein fusions between an activation domain and either the second protein under test or a gene library; (c) introducing both plasmids into a yeast cell carrying an appropriately regulated reporter gene; and (d) assaying the transformed cells for reporter gene activity. The following sections describe the properties of several specific components for each of these steps and illustrate the relative merits and demerits of the various alternatives. 257

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DNA COMPONENTS OFTHE TWO-HYBRID SYSTEM

A. DNA-Binding Domain Vectors All DNA-binding domain vectors for the two-hybrid system are based on either Gal4p or LexAp. A list of potential vectors is given in Table 1 with their relevant properties, selectable markers, utilizable restriction sites and the reading frame from the DNA-binding domain through the cloning sites. The Gal4p DNA-binding domain is the N-terminal region of the protein comprising residues 1-147. This portion of the Gal4p protein is sufficient to ensure both the efficient localization of fusion proteins to the yeast cell nucleus and to maintain high-affinity binding to the well-characterized binding site for Gal4p. In the plasmids pAS2 and pGBT9, the DNAbinding-domain fusion is constitutively expressed from the yeast ADHZ promoter at a high level (pAS2)or at a relatively low level from a truncated version of the promoter (pGBT9). This is an important consideration in cases where the fusion protein is toxic to the cell, in which case pGBT9 can be used more effectively. However, this has to be balanced against the observation that some interactions will only be detected with the higher levels of expression obtained with pAS2. As with many aspects of the twohybrid system, there is no immediately obvious way to predict from the outset which vector will be better for any particular fusion. In addition to the Gal4p DNA-binding domain, pAS2 also contains an HA-epitope tag that enables the expression of fusion proteins to be monitored by Western blotting using commercially available antibodies, and the dominant CYH2 gene that confers sensitivity to cycloheximide. This can be used after screening to select for loss of the “bait” plasmid to test for direct transcriptional activation by the activation-domain plasmid alone. Although these two genetic determinants serve a useful purpose, they do introduce some unwanted complications. For example, the HA-epitope tag can act to increase non-specific interactions with some activation-domain fusions, so that many DNA-binding domain fusions derived from pAS2 have an associated low level of endogenous reporter gene activity. In addition, the CYH2 gene can be toxic when overexpressed. When mating is used to produce co-transformants (see below), using pAS2 reduces the efficiency of diploid formation by 5-10 fold relative to vectors lacking CYH2. A further potential difficulty in using vectors based on GAL4 can occur when using DNA fragments from an organism that can complement gal4 and gal80 mutations in the host yeast strain, which will result in a significant number of false positives. In these cases, vectors such as pEG202 (Gyuris et al., 1993; Zervos et al., 1993) provide an alternative in the form of the LexA coding sequence. LexA does not have a nuclear localization signal, but is expressed at high level from the yeast ADHl promoter and so enters the nucleus and can occupy LexAp binding sites (LexA,) upstream of reporter genes. There appear to be no native yeast proteins that can interact with LexA,, so that background levels of reporter gene activation are minimal. 258

Table I. Properties of DNA-binding domain vectors Plasmid

DNA-binding domain

Markers

Multiple cloning sites

Reference

Genbank accession

pAS2

GAL4 (1-147)

U30497

GAL4 (1-147)

Bartel et al. (1993)

U07646

pEG202

LexA (1-202)

ampRHIS3

NdeI SfiI NcoI SmaI BamHI SalI EcoRI SmaI BamHI SalI PstI EcoRI BamHI SalI NcoI Not1 XhoI

Harper et al. (1993)

pGBT9

ampRTRPl CYH2 HA-tag ampRTRPl

Gyuris et al. (1993)

N/A

Reading frames through the multiple cloning sites are as follows: pAS2 Gal4 (1-147) CAT ATG GCC ATG GAG GCC CCG GGG ATC CGT CGA C pGBT9: Gal4 (1-147) GAA l T C CCG GGG ATC CGT CGA CCT GCA G pEG202 LexA (1-202) GAA TTC CCG GGG ATC CGT CGA TGG CGG CCG CTC GAG N / A = not available.

B. Activation-Domain Fusion Vectors Several different vector systems have been developed to express fusion proteins from genomic and cDNAs with a transcriptional activation domain at the amino terminus of the fusion protein (Table 2). Because activation domains are largely interchangeable, various proteins have been used, although most vectors are based on the strong Gal4p activator (residues 768-8811, the equally strong activator from the herpes simplex virus VP16 protein or the weak bacterial activator B42. In most cases, expression of the activation-domain fusion is constitutive from the ADHZ promoter (high level in pACT2, low level in pGAD424). In pJG4-5, B42 expression is galactose-inducible via the GAL2 promoter; this vector is designed specifically for use as a partner with the LexA vector pEG202 (Table 1).

C. Activation-Domain Libraries As with any genetic screen, the overall success is critically dependent on the quality and complexity of the gene library being screened. Additionally, it goes without saying that the easiest way to get a library for two-hybrid screening is to pick up the telephone and ask a colleague if they have what you need! A large number of libraries have been constructed and used to screen for interaction; from simple unicellular organisms such as yeast to complex multicellular eukaryotes such as plants and humans. Many of these libraries are now commercially available (e.g. from Clontech Inc.). In some cases, these libraries have been made by partial digestion of genomic DNA with a restriction enzyme such as Sau3A and ligated into a compatible restriction site (e.g. BamHI or BglII) in one of the activation-domain vectors. Unlike conventional gene libraries, this is an ineffective way to produce fusion libraries; for an enzyme such as Sau3A, whose recognition site occurs on average every 256 bp, an in-frame fusion will occur about every 1.5 kb throughout the genome. Several strategies have been used to improve the quality of a library by increasing the frequency of in-frame fusions. One route is to engineer the activation-domain vector to produce derivatives in which the frame of the unique cloning site is altered by one or two nucleotides with respect to the activation-domain coding sequence. This, in effect, requires the construction and screening of three separate libraries with a concomitant increase in the number of transformants to be analysed, but significantly increases the probability of productive interactions. An extension of this approach has been to combine it with the use of several different enzymes to generate the genomic fragments (James et al., 1996).Genomic DNA for library construction was isolated from a yeast strain deleted for the GAL4 and HIS3 genes, then digested partially with one of five enzymes ( A d , HinPI, MaeII, MspI and TaqI). Each of these enzymes produces fragments whose termini are compatible with ClaI, which enabled their cloning into derivatives of pGAD424 containing the CZaI site in a different reading frame with respect to the GAL4 coding sequence. This increased the frequency of in-frame fusions to about one every 97 bp.

Table 2. Activation-domain vectors

-

h,

Plasmid

Activation domain

Markers

Multiple cloning sites

Reference

Genbank accession

pACT2

Gal4 (768-881)

ampRLEU2

Li et al. (1994)

U29899

pGAD424

Gal4 (768-881

ampRLEU2

Bartel et al. (1993)

U07647

pJG4-5

842

ampRTRPl

NdeI Sfir NcoI SmaI BamHI EcoRI XhoI EcoRI SmaI BamHI SalI PstI EcoRI XhoI

Gyuris et al. (1993)

N/A

OI

Reading frames through the multiple cloning sites are as follows: pACT2: Gal4 (768-881)-HA tag CAT ATG GCC ATG GAG GCC CCG GGG ATC CGA ATT CGA AGC TCG AGA GAT CT pGAD424 Gal4 (768-881) GAA TTC CCG GGG ATC CGT CGA CCT GCA G pJG4-5 B42-HA tag GAA 7TC N/A, not available.

An alternative and complementary approach makes use of a technique that has been widely used to generate DNA fragments for library construction as part of large-scale sequencing projects. Rather than cutting DNA with restriction enzymes, high-molecular-weight DNA was initially fragmented by hydrodynamic shearing or by ultrasonication to produce molecules with a defined, limited size range, the precise value of which can be controlled by varying the shearing parameters (Povinelli and Gibbs, 1993). The termini of these fragments can be repaired by a combination of Mung bean exonuclease, T4 DNA polymerase and the Klenow fragment of DNA polymerase to generate blunt-ended molecules that can either be cloned directly into the activation-domain vector, or converted to cohesive termini with short oligonucleotides before cloning. This latter option provides yet a further refinement that increases library quality by reducing the frequency of non-recombinant molecules. By partially filling-in the cohesive ends of the vector with Klenow polymerase (e.g. by converting the BamHI cohesive sequence GATC to GA by treating with Klenow polymerase, dATP and dGTP) and by using oligonucleotides at the ends of the genomic fragments that have an unpaired complementary sequence (in this case TC),recombinant formation can be enhanced while simple vector religation is virtually abolished. This type of approach has been used to construct libraries within the EUROFAN project (see Chapter 11, the systematic project for the functional analysis of the yeast genome, and for the EC-funded TAPIR programme (Two-hybrid analyses of proteins involved in W A metabolism) to provide an exhaustive screen of functional interactions of proteins involved in RNA processing in yeast, where the libraries are estimated to contain an in-frame fusion equivalent to every 24 bp through the yeast genome (Fromont-Racine et al., 1997).

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INTERACTION SCREENING

A. Yeast Strains Several yeast strains have been developed to meet the specific requirements of hosts for two-hybrid screening and the properties of some of these are given in Table 3. In general, all strains contain: 0 0

0

mutations in the chromosomal GAL4 and GAL80 genes; auxotrophic markers for transformation; reporter genes downstream of appropriate transcription factor binding sequences.

In many of the strains used originally for two-hybrid screening, such as GGYl::171 (Gill and Ptashne, 1987; Table 3), interaction between two proteins was reported solely by transcription of the GAL1-ZacZ reporter, which required one to assay for P-galactosidase activity in transformed 262

Table 3. Yeast strains used for two-hybrid screening Strain

Genotype

Reporter

Transformation markers

Reference

GGYl::171

MATa leu2-3,112 his3-200 met tyrl ura3-52 ade2 gal4A gal8OA URA3::GALl-lacZ

lacZ

his3 leu2

Gill and Ptashne (1987)

Y187

MATa ura3-52 his3-200 ade2-101 trpl-901 leu2-3,112 met gal4A gal8OA URA3::GALl,,-GALl,,,,-lacZ

lacZ

trpl leu2

Harper et al. (1993)

HF7c

MATa ura3-52 his3-200 ade2-101 lys2-801 trpl-901 leu2-3,112 gal4-542 ga180-538 LYS2::GALlUA,-GAL1,,,,-HZS3

HIS3 lacZ

trpl leu2

Feilotter et al. (1994)

MATa ura3 his3 trpl L.exAw,,,, -LEU2

LEU2

his3 trpl ura3

Estojak et al. (1995)

N W OI

UZU3::GAL4,,,,-CYClTAT,-lacZ

EGY48

cells (see below). Although this remains an essential component of most screens, it was unnecessarily tedious and wasteful for high-complexity library screens. This has been improved by incorporating a second reporter gene such that interactions in vivo convert an auxotrophic strain to prototrophy. The markers for this are typically GALZ-HIS3 or LexALEU2, and these greatly ease the complexity of any screen because potential interacting clones can be identified first by selection on medium lacking histidine or leucine. Although the use of auxotrophic and lacZ reporters decreases the incidence of false positives, the expression of GALZ-HIS3 can be leaky so that some of the strains can in fact be phenotypically prototrophic for histidine. This can be reversed by including the anti-metabolite 3-amino-l,2,4-triazole (3-AT; Yocum et al., 1984) in the medium although this in turn has several disadvantages, one of these being that the optimum 3-AT concentration will vary for each individual DNA-binding domain construct. Alternatively, the strains HF7c and CG1945 (Table 3) can be used without 3-AT, because these contain a GAL4dependent HIS3 reporter with a tightly regulated promoter such that these strains are phenotypically auxotrophic for histidine (Feilotter et al., 1994). Auxotrophic selection for HIS3 or LEU2 provides a useful first-step screen for interactions, but the lacZ reporter provides an essential role in quantifying the interaction. LacZ expression is routinely measured by assaying P-galactosidase activity for which there are several published protocols (Transy and Legrain, 1995). Usually this is done by assaying hydrolysis of the chromogenic substrate X-gal by replica-plating His' (or Leu') colonies either directly onto medium containing X-gal or onto filters before lysis, and assay in situ. While the X-gal medium provides a simple, convenient method that is amenable to large-scale screens, it is less sensitive than the filter-lift method and can give more problems with background color development. The time taken for color development is also considerably more variable and generally longer (up to 6 days) on medium. However, neither of these methods provides a direct quantitative assay of P-galactosidase activity. This can best be done by liquid assay of whole-cell extracts using o-nitrophenyl-P-D-galactopyranoside, chlorophenol red-P-D-galactopyranosideor chemiluminescent substrates (Campbell et al., 1995).

B. Screening Methods The two-hybrid screen requires co-expression of two fusion proteins within a single yeast cell (Figure 21, and there are three ways to engineer that: sequential transformation of the DNA-binding- and activation-domain constructs; co-transformation of both constructs; transformation of independent strains by the two constructs, then mating the transformants to form a diploid in which the interaction is assayed.

264

A D fusion plasmid

13D fusion plasmid

0

Transform

I

Co-t ransform

(-0:'I.1 Mate Mate

Co-transformation (introducing both DNA-binding-domain and activation-domain fusions in a single transformation) and sequential transformation (transforming with the DNA-binding-domain construct then subsequently transforming with the activation-domain fusion) using published transformation protocols (Gietz et al., 1992; see Chapter 4) are both satisfactory for most applications to study direct interactions involving the domains of two known proteins, where both plasmid constructs contained specific DNA sequences. Both methods can be, and have been, used successfully to screen libraries for novel interacting partners. However, both are inherently inefficient in generating the large numbers of co-transformants needed exhaustively to screen complex libraries, for which yeast mating provides an excellent solution (Finley and Brent, 1994, 1995).In this method, the DNA-binding-domain fusion and the activation domain fusion are transformed initially into different strains, one MATa and the other M A T a . These cells can be mated and the interaction assayed in the resulting diploid cell, an effective means of producing co-transformants that has been used to examine specific interactions between sites of proteins, to screen libraries and to define complete sets of interactions amongst the proteome of a small genome (Bartel et al., 1996).

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FUTURE DEVELOPMENTS

The impact of the two-hybrid method for defining and characterizing protein-protein interactions has been remarkable and although it represents 265

only one of the many available methods for studying such interactions, its application is enhanced considerably by the explosion in genome sequence information. Despite this though, the method in its current form remains confined to identifying those interactions that can occur between fusion proteins within the context of the yeast nucleus. Not surprisingly then, analogous systems to analyse interactions in other cell types are already being developed, including one for mammalian cells (Luo et al., 1997). Although this is conceptually similar to the yeast system, requiring the co-expression of fusion proteins to regenerate nuclear transcription factor activity to direct the expression of a reporter gene, it offers a useful starting point to develop an homologous system for the analysis of human gene products that are appropriately posttranslationally modified. In addition, by using reporter systems that rely on other physical properties resulting from protein-protein interaction, rather than transcription factor activity, it will be possible to screen for direct interactions in cell compartments other than the nucleus. The two-hybrid method in its original format is constrained in detecting only direct binary interactions. This is a major limitation and a significant advance has been the development of analogous systems for the detection of ternary interactions (so-called three-hybrid methods). These allow the detection of interactions in which the DNA-binding-domain fusion protein does not directly contact the activation-domain fusion protein without an intermediate effector protein, or in which a protein will only bind to a complex site formed as a result of the interaction between two other proteins (Zhang and Lautar, 1996). The potential of this development is enormous, because the effector molecule that docks the DNAbinding- and activation-domain fusions together at the UAS does not itself need to be a protein but can instead be a small synthetic molecule or even a molecule of RNA (SenGupta et al., 1996; Licitra and Liu, 1996). The recent use of a three-hybrid assay to screen for interactions in mammalian cells can only enhance the range of applications for this technology (Liberies et al., 1997). Finally, we are currently witnessing extraordinary advances in genome research, with complete microbial sequences already in the public databases and the prospect of the genome of at least one multicellular organism being completed imminently. When combined with chip-based technologies for gene identification and analysis (see Chapter 81, the prospect of using two-hybrid technology as a tool to define the complete interaction set for the proteome of an organism becomes an intriguing possibility. It is probably significant that this approach has already been applied successfully to a small bacteriophage genome (Bartel et al., 1996) and is currently being used to define complete interaction sets for complex cellular processes such as pre-mRNA splicing in yeast. These analyses serve not only to confirm the interactions defined previously by other technologies, but also to open up entirely new and unexpected interactions that serve to highlight novel overlaps between discrete cellular processes.

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