two-hybrid dual bait system to discriminate specificity of

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A Two-hybrid Dual Bait System to Discriminate Specificity ofProtein Interactions*

(Received for publication, December 29, 1998, and in revised form, March 17, 1999)

Ilya Serebriiskii, Vladimir Khazak, and Erica A. Golemis

From the Division of Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

Biological regulatory systems require the specific or-ganization of proteins into multicomponent complexes.Two hybrid systems have been used to identify novelcomponents of signaling networks based on interactionswith defined partner proteins. An important issue in theuse of two-hybrid systems has been the degree to whichinteracting proteins distinguish their biological partnerfrom evolutionarily conserved related proteins and thedegree to which observed interactions are specific. Weadapted the basic two-hybrid strategy to create a noveldual bait system designed to allow single-step screeningof libraries for proteins that interact with protein 1 of

interest, fused to DNA binding domain A (LexA), but donot interact with protein 2, fused to DNA binding do-main B ( cI). Using the selective interactions of Ras andKrev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model,we systematically compared LexA- and cI-fused baitsand reporters. The LexA and cI baitr reporter systemsare well matched for level of bait expression and sensi-tivity range for interaction detection and allow effectiveisolation of specifically interacting protein pairs againsta nonspecific background. These reagents should proveuseful to refine the selectivity of library screens, to re-duce the isolation of false positives in such screens, andto perform directed analyses of sequence elements gov-erning the interaction of a single protein with multiplepartners.

To understand and manipulate the function of a particular

protein of biological interest, it is generally useful to identify

other proteins with which it associates. Although identification

of protein interactions initially proceeded almost solely by tech-

nically difficult biochemical methods, in recent years yeast

two-hybrid systems (1) have developed as a powerful genetic

tool to rapidly select previously uncharacterized proteins that

specifically interact with a target protein of interest from a

suitable library (25). In this schema, a protein of interest is

synthesized in yeast as a fusion to a DNA binding domain

(DBD),1 which is typically the bacterial repressor protein LexA

or the amino-terminal end of the yeast transcription factor

GAL4. Interaction of this DBD protein fusion (a bait) with a

transcriptional activation domain-fused partner protein (either

a defined partner or a novel protein screened from a library)

allows the activation of reporter genes (lacZ, HIS3, LEU2)

responsive to the cognate DBD. More recently, interest has

focused on expanding the utility of two-hybrid systems to en-

able the detection of interactions between proteins and RNA (6,

7), proteins and nonprotein ligands (8), proteins and peptides

(9, 10), and proteins and multiple partners (11, 12). A second

thrust has been to enable whole-genome applications (1315),

leading to the generation of maps of protein interaction net-

works with the potential to complement the vast resource of

sequence information now being developed as part of the Ge-

nome Project. Finally, there has been interest in developing

two-hybrid systems as tools in high throughput drug discovery

screening strategies to identify agents regulating the activity of

biologically important target proteins.

As two-hybrid technologies have evolved to more complex

applications, a question of mounting importance has been the

degree to which library screens performed in these systems

yield partners specific for the utilized bait, as opposed to pro-

teins of broad interaction capability (false positives). Al-

though the large number of published two-hybrid papers indi-

cates that many specific partners are obtained, a recent survey

has suggested that the majority of library screens isolate at

least some cDNAs that are nonspecific.2

As a related issue, it isclear that many biologically important proteins are organized

into families of evolutionarily related members that conserve

substantial sequence similarity (e.g. Refs. 1719). Thus, the

degree to which two-hybrid systems isolate proteins partners

absolutely specific for individual baits, rather than those that

interact generally with a class of protein (familial positives),

is also an issue. Although existing two-hybrid systems allow

discrimination of uniquely specific interactors from false posi-

tives or familial positives through use of various methods of

specificity testing performed subsequent to a screen (20), these

methods are frequently laborious, particularly when many pos-

sible interactors must be tested. For this reason, it has been of

considerable interest to devise a method to eliminate such

clones before selection.In this report, we describe a novel adaptation of the two-

hybrid system designated the dual bait system. This system

incorporates controls for false positive or nonspecific interac-

tions in a single step and allows the simultaneous assay of a

protein interaction with two related or unrelated partners in a

single cell, which should also be useful for a variety of high

throughput and genome-oriented studies. We demonstrated

that these reagents are effective at selectively identifying two

* This work was supported by an award from the Merck GenomeResearch Institute, by National Institutes of Health Core GrantCA06927, and by a Small Business Innovative Research program grantsubcontract from Invitrogen. The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked advertisement in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

Current address: Small Molecule Therapeutics, 11 Deer Park Dr.,Monmouth Junction, NJ 08852.

To whom correspondence should be addressed: Fox Chase CancerCenter, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-2860; Fax: 215-728-3616; E-mail: [email protected].

1 The abbreviations used are: DBD, DNA binding domain; X-Gal,5-bromo-4-chloro-3-indolyl -D-galactopyranoside; PCR, polymerasechain reaction; AD, activation domain; X-Gluc, 5-bromo-4-chloro-3-in-dolyl-D-glucuronic acid, sodium salt; Magenta-Gal, 5-bromo-6-chloro-

3-indolyl-D-galactopyranoside.2 I. Serebriiskii and E. A. Golemis, E. A. (1996) http://www.fccc.edu/

research/labs/golemis/interactiontrapinwork.html.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 24, Issue of June 11, pp. 1708017087, 1999 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org17080


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discrete sets of interacting proteins against an extensive back-

ground population of nonspecifically interacting proteins, sup-

porting the idea of reagent appropriateness for large scale

genomic applications.

EXPERIMENTAL PROCEDURES

Molecular Biology and Genetic TechniquesDH5 Escherichia coli

was grown on LB medium (21)where appropriate, and antibiotics wereadded at the concentrations recommended by the suppliers. Standard

DNA manipulation techniques were as described in (21). Yeast weregrown on yeast-peptone-glucose or minimal medium and manipulatedusing standard techniques (22). Two-hybrid experiments and -galac-tosidase assays were performed as described (23), with six independent

colonies assayed for each value presented; for sensitive plate-basedX-Gal and X-Gluc assays, the procedure of Duttweiler (24) was used.

Dual Bait System ReagentsRelevant properties of all strains andplasmids are described in the text. The bacteriophage repressor pro-

tein cI (25) was used as the basis of reagent development, as its size,structure, and DNA binding properties suggested it might behave com-parably as a DNA binding domain to the pre-existing two-hybrid system

DBD LexA (2628).cI-responsive LacZ ReportersA 68-base pair fragment of the bac-

teriophage genome (LAMCG, nucleotides 3795038018) containing 3

naturally occurring cI operators was amplified, and XhoI ends wereadded by PCR. The resulting product was digested with XhoI andinserted into theXhoI site of the plasmid LR11 (parent of all currently

utilized interaction trap lexAop-lacZ reporters (20)) in either orienta-tion upstream of a basal GAL1 promoter directing expression of thelacZ gene. The resulting plasmids pcIop-LacZA and pcIop-LacZB have

a 2-m origin of replication and use a URA3 marker for selection inyeast; they differ only in the orientation of the cI operator cassette.

cI-responsive LYS2 Yeast StrainsAn EcoRI fragment containing a

minimal GAL1 promoter, cI operator cassette, and the translationalstart of the GAL1 gene was excised from cIop-lacZA and inserted intopRFiLYS8 (a gift of R. Finley) to generate pCIL-1. In this construct, theGAL1 promoter-cI operator cassette directs the expression of a fusion

protein in which the first 31 amino acids of the GAL1 gene are fused toLYS2-coding sequences. The yeast strain RFY206 (13) (MATa trp1ura352 his3200 leu23 lys2 201 trp1::hisG) was transformed with

ApaI-digested pCIL-1 (targeting integration to URA3 gene), and stableintegrants were selected on ura- dropout medium. One of these strains,SK01 (MATa trp1 ura352 his3200 leu23 lys2201 trp1::hisG URA3:

cIop-LYS2) was taken for subsequent characterizations.

The yeast strain SK01was crossed to EGY48 (MAT trp1 ura3 his3lexAop-LEU2) (4), and the resulting diploid was sporulated. The strain

SK10, with the genotype (MATa trp1 ura3 his3 lexAop-LEU2 lys2201URA3:cIop-LYS2) was obtained following tetrad dissection.

As a separate approach, as SK10 proved refractory to deletion of theURA3 gene, the PstI (475045)-XbaI (474175) fragment of Saccharomy-

ces cerevisiae chromosome II, located 295 base pairs upstream of thewild-type LYS2 gene, and a PstI-HindIII fragment of pcIL-1, encom-passing the cI-responsive cassette, were assembled on a pUC-based

plasmid to yield pcIL-2. pcIL-2 reconstructed about 6 kilobases of yeastgenomic sequence with the native LYS2 promoter replaced by GAL1promoter/cI operator sequence. This construct was used for integrative

transformation of the EGY48 (4) and EGY191 (23) yeast strains, fol-lowed by Lys selection (ability to grow on 0.2% DL() aminoadipic acid(a toxic metabolite of the LYS2 protein (29)) as the sole source of

nitrogen. 24 stable integrants in each of the parent strains were se-lected and confirmed to be Lys-Leu-His-Ura-Trp-; representative inte-grants into each parent have been designated SKY48 and SKY191 and

have the genotype (MAT trp1 ura3 his3 lexAop-LEU2 cIop-LYS2),with 6 or 2 lexA operators upstream of LEU2, respectively.

cI-responsive gusA ReportersThe novel yeast vector pRG00 was

used as the basis of cIoperator-gusA reporter vector construction. Thedetailed map and sequence files for pRG00 are available upon request;this vector contains the colE1 origin of replication and kanamycinresistance gene for selection in bacteria, a 2-m origin and URA3 gene

for selection in yeast, and the complete coding sequence for E. coli-glucuronidase (gusA; a gift of J. Vossen, University of Amsterdam).

An EcoRI fragment containing a minimal GAL1 promoter, cI operator

cassette (containing three naturally occurring cI operators), and thetranslational start of the GAL1 gene was excised from cIop-lacZA andrecloned immediately upstream of the gusA sequences in pRG00 to

create the plasmid pRG2 (cIop-gusA).cI Fusion Bait VectorsA DNA fragment containing the complete

coding sequence (with no stop codon) for bacteriophage cI repressor

protein (LAMCG, nucleotides 3723037940) was amplified by PCR and

cloned into the plasmid pUC19 to yield pUC-cI. Separately, a HIS3-

containing fragment of pEG202 was removed by AatII-ClaI digestion

and replaced by a synthetic AatII-ClaI linker, to create pGK202. Sub-

sequent HindIII digestion, fill-in reaction, and EcoRI digestion were

used to remove the lexA gene from pGK202, followed by replacement

with the cI gene on a BglII (filled-in)-EcoRI fragment excised from

pUC-cI. The resulting plasmid pGK302, was digested with BamHI and

AatII and ligated to a BamHI-AatII fragment of pEG202 to create

pGKS3, a pEG202 sibling with the cI gene exactly replacing the lexA

gene. pGKS3 has a 2-m origin of replication, carries aHIS3 marker foryeast selection, and was used in control experiments.

Subsequently, a BsaBI-EcoRI fragment of pGKS3 (encompassing cI)

was used to replace the lexA gene in the plasmid pLexZeo (Invitrogen),

which had been digested with HpaI-EcoRI. The resulting plasmid,

pGKS6, used the ADH1 promoter to express a cI fusion. It had a 2-m

origin of replication and used Zeocin (Invitrogen) resistance for selec-

tion in yeast and bacteria. Expression of proteins was assayed by

standard lysis of cells expressing appropriate constructs (20) followed

by SDS-polyacrylamide gel electrophoresis and Western analysis with

antibodies to Krev-1 (Transduction Labs, Inc.), LexA, or cI repressor (agift of G. Kalmar).

cI Fusion Bait/cIop-gusA Reporter Dual Purpose VectorsTo allow

introduction of all system components into a single yeast strain, a

combined cI bait expression/cIop-gusA reporter with a single selectible

marker (ZeoR) was constructed. To create this plasmid, pGKS8, the

3cIop-gusA reporter cassette was excised from pRG2 as a KpnI-KpnI

fragment and inserted into the BsrGI site of pGKS6, destroying bothrestriction sites. The map of pGKS8 is available on request; this plas-

mid retained EcoRI, SacI, BglII, PvuII, KpnI, SacII, and NotI sites for

the insertion of coding sequences for expression as cI fusion proteins.

Baits and PreysTo create transcriptionally activating DBD-fusedbait plasmids, the full-length Krit1 gene (30) was inserted into EcoRI-

XhoI-digested pGKS3, pGKS6, pGKS8, and pEG202, as noted in the

text. Nonactivating bait fusions were constructed by cloning the full-

length Krev-1 gene (31) into the EcoRI-XhoI sites of pGKS3 or into

EcoRI-SacII sites of pGKS8 and by cloning the Ras gene into the

EcoRI-XhoI sites of pEG202. Activation domain fusion plasmids were

obtained by cloning Krit1 (full-length) and Raf1 ( amino acids 156)

genes into the EcoRI-XhoI sites of the plasmid pJG45 (4) and RalGDS

(amino acids 767848) (a gift of A. Vojtek) into BamH-EcoRI sites of

pYesTrp2 (Invitrogen).

Assaying Reporter Activation Activation ofLacZ reporters was as-sessed qualitatively by on-plate overlay assays (24) using the substrates

X-Gal or Magenta-Gal (Diagnostic Chemicals Ltd) and quantitatively

using -galactosidase assays as described in (21). Activation of gusA

reporters was assessed qualitatively using the same overlay procedure

as for LacZ but with X-Gluc (Diagnostic Chemicals Ltd) as a substrate.

A quantitative assay was performed as for a -galactosidase assay but

using 4-nitrophenyl--D-glucuronic acid instead of 2-nitrophenyl--D-

galactopyranoside as the substrate. For both assays, determination was

made in a plate reader; in a standard procedure, 100 l of exponential

phase yeast cultures were harvested by centrifugation in a microtiter

plate. Yeast pellets were resuspended in 50 l of Z-buffer, and the A590

was determined in a plate reader, after which the plate was frozen at

70 C. After thawing, 50 l of 4-nitrophenyl--D-glucuronic acid (2

mg/ml) or 2-nitrophenyl--D-galactopyranoside (2 mg/ml) in Z-buffer, as

appropriate, was added to each well. Immediately, the A405

was meas-

ured, and the plate was then placed in a 30 C incubator. Additional

A405

readings were taken between 5 and 45 min, depending on the

intensity of the reaction. To calculate -glucuronidase or -galactosid-ase activity, the following formula was used:

A405tA405

0 1000

A590vt(Eq. 1)

where v is volume in ml, t is time, and (A405

t A405

0 ) is a net increase of

optical density at 405 nm between zero time and time t, and A590

is

optical density at 590 nm. Activation of LYS2 or LEU2 reporters was accomplished by streaking

or replica-plating yeast to plates lacking leucine or lysine and monitor-

ing growth over 4 days. In assessing activation of the LYS2 reporter by

bait-prey combinations (as in Fig. 5), this was done either on medium

lacking uracil, histidine, tryptophan, and lysine in the presence of

zeocin, or alternatively, on medium lacking only uracil, histidine, andlysine in the absence of zeocin, with the selection for growth on lysine

medium providing positive selective force for the retention of cI bait and

A Selective Two-hybrid Dual Bait System 17081


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activation domain-fused prey. Qualitatively identical patterns of pro-

tein interaction are obtained with both media; however, because ofminor nonspecific growth inhibitory effects of zeocin, growth is faster(24 h to point of large colonies) with zeocin omitted.

For mixing experiments, yeast were pregrown in medium selectivefor plasmid retention and inducing for activation domain fusion expres-sion (ura-his-trp-galactose/raffinose zeocin), diluted as described inresults, and plated to selective medium; colony outgrowth was moni-

tored over 4 days.

RESULTS

Outline of StrategyThe general approach taken with a dual

bait selection strategy is outlined in Fig. 1. In the interaction

trap two-hybrid system (Fig. 1A (4)), a LexA-fused bait (ex-

pressed from plasmid pEG202 or a derivative) interacts with a

galactose-inducible B42 acid blob activation domain-fused

partner (expressed from plasmid pJG45) to induce the expres-

sion of two reporter genes under transcriptional control of lexA

operator (op) sites. These are (lexAop)n

-lacZ (borne on plasmid

pSH1834 (n 8), pJK103 (n 2), or pRB1840 (n 1)) and an

integrated (lexAop)n

-LEU2 (in yeast strain EGY48 (n 6) or

EGY191(n 2)).

In the dual bait system described here, three further compo-

nents are added (Fig. 1B). The first of these is a cI-fused

alternate bait, expressed from the novel ZeoR, 2-m plasmid,

pGKS8. The second is a (cIop)n

-gusA (-glucuronidase) (n 3)

reporter gene cassette, additionally borne on the plasmid

GKS8. The third is an additional integrated reporter system in

which cI operators direct the expression of the LYS2 gene;

(cIop)n-LYS2 (n 3) in the yeast strains SKY48 or SKY191

(derivatives of EGY48 and EGY191, respectively). These re-

agents can be utilized in multiple ways to enhance measure-

ment of protein interactions over currently existing

approaches.

As a first example, in a library screen, if an activation do-

main-fused interacting protein associates uniquely with a

LexA-fused primary bait but not with a cI-fused alternate bait,

SKY48 or SKY191 yeast containing the appropriate bait and

reporter constructs would turn blue on medium containing

X-Gal but not on medium containing X-Gluc, and grow on

medium lacking leucine, but fail to grow on medium lacking

lysine; in contrast, promiscuously interacting clones would be

revealed by their growth on medium lacking both leucine and

lysine and blue color with both X-Gal and X-Gluc. False posi-tives would be eliminated simultaneously with isolation of true

positive clones. As a second example, in targeted examination

of the interaction of a single activation domain-fused protein

with two defined partners (for example, interaction of activa-

tion domain-fused cyclin D with LexA-fused CDK4 and cI-fused

CDK6), a randomly mutagenized pool of activation domain-

fused partners could be screened to identify mutations that

disrupt interaction with either one or both of the partner pro-

teins. As a third example, one source of interest in two-hybrid

systems is their use in drug screening approaches to identify

compounds that disrupt interactions between discrete pairs of

interacting proteins (8, 32, 33); dual bait reagents would apply

a simultaneous control to the specificity of such interactions.

Parallel Performance of LexA and cI Expression and ReporterSystemsGiven that assessment of protein interactions in two-

hybrid systems is a factor of bait expression levels (34) and

stringency of reporter systems (23), for these hypothetical uses

to be practicable, the two-bait-reporter combinations utilized in

the dual bait system must express respective baits to similar

levels and possess similar sensitivities to transcriptional acti-

vation. Therefore, an initial step was to carefully measure

these parameters. To this end, we constructed equivalent

pEG202 (LexA) and pGKS3 (cI) fusions to the protein Krev-1/

rap1A (31), a human Ras-family GTPase. These and parent

vectors were transformed in parallel into EGY48 yeast, and

expression of the synthesized proteins was assayed by Western

analysis using antibodies to Krev-1, LexA, or cI (Fig. 2). Ex-

pression of the two Krev-1 fusion constructs was found to becomparable in 4 randomly chosen colonies, with slightly higher

levels (23-fold) in the cI constructs. Furthermore, expression

of the fusion protein was in each case similar to the matching

unfused DNA binding domain, indicating that cI tolerated at-

tachment of a fusion domain without loss of stability. Finally,

essentially identical expression levels were observed using

pGKS6-Krev-1, a ZeoR instead of HIS3 version of pGKS3 (not

shown), indicating the selectable marker could be exchanged

without gross alteration of plasmid copy number.

We next determined the degree to which activation through

cI operators was comparable with activation through lexA op-

erators. As a conservative first step, we constructed analogous

fusions of pGKS3 and pEG202 to Krit1 (a Krev-1-interacting

protein (30) that fortuitously functions as a transcriptional

FIG. 1. Interaction trap and dual bait systems. A, an activation(Act.) domain-fused prey interacts with a LexA-fused bait to drivetranscription of lexAop-responsive LEU2 and lacZ reporters. B, anactivation domain-fused prey interacts with a LexA-fused bait to drivetranscription of lexAop-responsive LEU2 and lacZ reporters but doesnot interact with a cI-fused bait and, thus, does not turn on transcrip-tion of cIop-responsive LYS2 and gusA reporters. Note, as shown here,that cI bait is drawn as representing a nonspecific partner; the systemcan also be configured so prey interacts with both baits; see text.

A Selective Two-hybrid Dual Bait System17082


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activator of moderate strength) and assayed activation of the

closely related cIop- and lexAop-lacZ reporters. Parallel trans-

formations were performed with pGKS3-Krit1 (cI-Krit1) plus

cIop-lacZA and cIop-lacZB, which contained the three natu-

rally occurring cIoperators OR1-OR3 of phage in either orien-

tation (25). In addition, for comparison, yeast were transformed

with pEG202-Krit1 (LexA-Krit1) plus pRB1840, pJK103, or

pSH1834 (1, 2, or 8 lexAop-lacZ) (23), and as a negative

control, with pEG202-Krit1 plus cIop-lacZA. -Galactosidase

assays were used to measure activation of the lacZ reporters

(Fig. 3). In these tests, the cI-Krit1 fusion protein activated the

two cIop-lacZ constructs to equivalent levels, which were

closely comparable with that obtained using the combination of

LexA-Krit1 and pJK103. As a negative control, the LexA-Krit1

construct was also shown not to activate the cIop-lacZ report-

ers, as expected. Based on this result, we next used the cIop/

minimal promoter cassette to develop a cIop-gusA reporter,

pRG2, to be used in conjunction with the standard lexAop-lacZ

reporters. Yeast were transformed with the reporter and an

activating cI-Krit1 fusion protein or LexA-fused Ras (which

does not activate transcription) as a negative control, and gusA

transcription was assessed with a quantitative -glucuronidase

assay, analogous to a -galactosidase assay (see Experimental

Procedures); as with the cIop-lacZ reporters, a high degree of

specific activation was observed against a much lower back-

ground (6300 -glucuronidase units for cI-Krit1 versus 85 for

the negative control, a 75-fold difference), although overall the

gusA reporter was more sensitive than lacZ, as reflected in the

higher units (700 -galactosidase units for cI-Krit1). In plateoverlay assays, positive and negative were similarly clearly

distinct, with cI-Krit1 medium to dark blue, whereas the neg-

ative LexA-fused control was pale blue or white (not shown;

also, see below).

Finally, we compared direct activation of the LEU2 versus

LYS2 auxotrophy reporters, again using analogous LexA- and

cI-fused Krit1(Fig. 4). Using SKY48 and SKY191 as hosts, we

determined that LexA-Krit1 activates the LEU2 reporter of

these strains, whereas cI-Krit1 does not (Fig. 4, second panel

from top). Conversely, cI-Krit1 is capable of activating the

LYS2 reporter of SKY strains, whereas LexA-Krit1 is not (Fig.

4, third panel from top). Finally, each fusion activated the

appropriate lacZ reporter to comparable degrees, independent

of growth properties on Leu or Lys medium (Fig. 4, bottom

panel, middle two rows). Notably, positive growth dependent on

activation of theLEU2 andLYS2 reporters could be assessed in

similar time frame, with results detectable at 2448 h after

plating yeast on selective media. Based on visual estimation of

growth rate, the sensitivity of the cIop-LYS2 reporter in these

strains appears to be intermediate between that of the LEU2

reporters in EGY48 and EGY191.

Cumulatively, these results indicated that the cI- and LexA-

based expression and reporter constructs yielded results in a

similar sensitivity range, making them suitable for compara-

tive purposes. One point remaining was the development of

reagents suitable for expressing all baits and reporters in the

same strain, to allow simultaneous assay. SKY strains (MAT

trp1 ura3 his3 lexAop-LEU2 cIop-LYS2) utilize the LEU2 and

LYS2 markers for reporter genes. The activation domain fusion

expression plasmid (pJG45) uses a TRP1 marker; the lexAop-

LacZ reporter (pJK103) uses a URA3 marker; and the LexA

fusion bait plasmid (pEG202) uses a HIS3 marker; the cI fusion

bait plasmid (pGKS6) utilizes a ZeoR marker. To introduce the

cIop-gusA reporter, we took advantage of the fact that the

cIop-gusA cassette is only 2.6 kilobases, whereas the plasmid

backbone for the cI fusion plasmid pGKS3 is unusually small,

as the ZeoR marker is used for both bacterial and yeast selec-

tion. The cIop-gusA cassette was introduced into pGKS6, re-sulting in a new plasmid, pGKS8, which encompassed both

cI-bait and cIop-gusA reporter. Control experiments similar to

those outlined above demonstrated that this bait-reporter-com-

bined plasmid yielded results similar to those obtained with

the combination of pGKS3 and pRG2 (not shown). This con-

struct was used for the experiments described in the following

sections.

Specificity of the Dual Bait System in Controlled Two-hybrid

AssayThe major criterion for effective use of a dual bait

system is that it should effectively discriminate interactions of

a partner with related but distinct proteins. Ras and Krev-1

possess 56% amino acid identity and are known to interact with

an overlapping set of protein partners (3537). In experiments

described elsewhere, we determined that Raf preferentiallyinteracts with Ras by a two-hybrid system assay, whereas

Krit1 preferentially interacts with Krev-1 (30). The Ral gua-

nine nucleotide dissociation stimulator RalGDS interacts with

both Krev-1 and Ras (36). Neither Ras nor Krev-1 activates

transcription when expressed as a DNA binding domain fusion.

The strain SKY191 with the plasmid pSH1834 was used as

a host for transformation by pEG202-Ras (LexA-Ras) and

pGKS8-Krev-1 (cI-Krev-1). We then super-transformed the

SKY191/pEG202-Ras/pGKS8-Krev-1 combination in parallel

with each of the galactose-inducible expression plasmids

pJG45-Raf (AD-Raf), pJG45-Krit1 (AD-Krit1), or pYesTrp2-

RalGDS (AD-RalGDS) or with empty AD vector and assayed for

reporter activation/growth properties on selective medium. As

noted above, activation through a LexA fusion permits growthon Leu medium and production of LacZ (cleaves X-Gal, Magen-

ta-Gal, etc. to produce colored products); activation through a cI

fusion permits growth on Lys medium and production of-glu-

curonidase (cleaves X-Gluc, etc., to produce colored products).

All yeast grew on nonselective plates (ura his trp, glu-

cose, or galactose, Fig. 5, panel A). No strains grew on either

Leu or Lys plates when glucose was present as the carbohy-

drate source. However, under galactose induction, strains con-

taining pJG45-Raf were able to grow preferentially on Leu

medium (Fig. 5, panel F) but only minimally on Lys medium,

based on the association between Raf and LexA-Ras; con-

versely, strains containing pJG45-Krit1 grew well on Lys

medium but only weakly on Leu medium, based on the inter-

action between Krit1 and cI-Krev-1 (Fig. 5, panel E). Strains

FIG. 2. LexA and cI expression vectors synthesize comparablelevels of fusion protein. Whole cell extracts from yeast expressingeither pEG202-Krev-1 (LexA-Krev-1), pGKS3-Krev-1 (cI-Krev-1), or pa-rental vectors pEG202 or pGKS3 were examined by protein immunoblotwith the antibodies to Krev-1 (top panel); the blot was then stripped andreprobed with antibodies to LexA (bottom left) or cI (bottom right).Before transfer to membrane, the blot was stained with aqueous Coo-massie to confirm equivalent protein load in all lanes (not shown).



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containing pYesTrp2-RalGDS grew well on both Lys and Leu

medium (Fig. 5, panels E and F), whereas a negative control

(strains containing empty plasmid pJG45) did not grow on

any selective plates (Fig. 5, panels E and F). Interaction of

RalGDS with both baits could be also detected on the double

auxotrophic Lys-Leu plate, where this was the sole plasmid

combination resulting in growth (Fig. 5, panel G). Interactors

that associated with only the cI-fused bait or nonselectively,

with both the cI- and LexA-fused baits, could be counters-

elected by inclusion of-aminoadipate in the growth medium

as the sole source of nitrogen (Fig. 5, panel H).

The results of X-Gal and X-Gluc assay on the plates are in

good correspondence with the auxotrophic selection assay, with

Raf-Ras positive with X-Gal (Fig. 5, panel C), Krev1-Krit1

positive with X-Gluc (Fig. 5, panel B), and Ral-GDS positive

with both (Fig. 5, panel B and C). Note: using a complementary

set of color-producing substrates (Magenta-Gal X-Gluc), both

LacZ and GusA activities can also be assayed on a single plate

(Fig. 5, panel D). These results paralleled those previously

obtained using a conventional two-hybrid selection (30) andconfirmed that the dual bait system can be used to distinguish

interactions between two closely related potential partner pro-

teins. We note that although the -aminoadipate counterselec-

tion works well in the controlled situation shown above, over

time background colonies arose on the counterselected plates,

suggesting this particular application may be more useful in

targeted disruption of known interactions than in conjunction

with library screens.

Selection of Specifically Interacting Protein Pairs from a

Nonspecific PoolThe previous results demonstrated that di-

rect streaking of uniform populations of yeast containing pre-

determined combinations of baits, activation domain fusions,

and reporters yields expected results. A more rigorous test of

the power of the ability of these reagents to discriminate spe-cific interactions was performed using a mixing experiment, as

outlined in Fig. 6. Four populations of SKY191 yeast were

generated. Each expressed LexA-Ras and cI-Krev-1 and con-

tained lexAop-lacZ and cIop-gusA reporters and an activation

domain fused to either 1) Raf, 2) RalGDS, 3) Krit1, or 4)

nonspecific (a fragment of hsp90). 10100 colony forming units

(30300 cells), each of populations 13, were mixed together

with 2 106 cells containing the nonspecific control and par-

allel samples of the pooled cells plated to media selective for the

lexAop-LEU2 (ura-his-trp-leu-Zeo) or cIop-LYS2 (ura-his-trp-

lys-) reporters.

Approximately 50 colonies arose on each of these plates, in

good accord with the number anticipated based on the seed. Of

these, 24 were chosen from each of the Leu and Lys plates andtransferred to a master plate, then retested for growth on both

leu and lys medium, as well as activation of lacZ or gusA

reporters. 43 of the 48 analyzed colonies were specific for

growth on Leu or Lys medium, whereas 5 of the 48 total

colonies analyzed grew on both Leu and Lys medium. In tests

with the colorigenic reporters, 45 of the 48 displayed expected

patterns for lacZ and gusA; LEU colonies were blue with

X-Gal, LYS colonies were blue with X-Gluc, LEULYS

colonies were blue with both; the remaining 3 colonies were

white with both substrates. Finally, 5 colonies for each group

(LEU LacZ LYS-gusA, LEU LacZ LYS gusA,

LEULacZ-LYS gusA) were selected at random and used

for PCR using primers containing sequences complementary to

library vector sequence-flanking inserts to identify inserts

FIG. 3. Proportional activation oflacZ reporters by cI or LexA-fusedactivators. A, comparative activation oflacZ reporters, lexA-op versus cI. Valuesshown reflect proportional enhancementof activation in -galactosidase assays;the degree of activation obtained fromLexA-Krit1 through a 2 lexAop-lacZ re-porter is arbitrarily set to 1.

FIG. 4. Comparative activation of yeast reporter plasmids andstrains through LexA versus cI operators. Strains SKY48 andSKY191 were transformed with the pairwise combinations of eitherpEG202-Krit1 or pGKS3-Krit1, transcriptionally activating fusions toLexA and cI, respectively, and either pJK103 or cIop-lacZA, reporterswith lacZ transcriptionally responsive to LexA or cI operators, respec-

tively. Three independent transformants were replica-plated either onnonselective medium (top) or medium selecting for activation of LEU2(leucine, second panel), LYS2 (lysine, third panel), and LacZ (with

X-Gal, fourth panel) reporters.



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based on characteristic product size (Fig. 6, bottom). Sepa-

rately, each individual library plasmid was amplified to pro-

vide a size standard for Krit1, Raf, and RalGDS). As shown,

each of the selected LEULYS colonies contained Raf, each

of the LEU-LYS colonies contained Krit1, and each of the

LEULYS colonies contained RalGDS as predicted, based on

their reporter activation profile. In contrast, PCR from the

three colonies with an inappropriate pattern of activation (e.g.

LYS

but gusA

) revealed the presence of the hsp90 fragment(data not shown). In numerous tests of simple two-hybrid sys-

tem reagents, such as the interaction trap, we previously found

that a common source of false positives is selection of strains

mutated so as to transcribe the auxotrophic reporter (LEU2) in

the absence of contribution from either bait or prey; these false

positives are standardly detected because of their inability to

additionally activate the second chromogenic reporter (20). The

detection of the nonspecific hsp90 fragment in the LYS

gusA cells suggests that these colonies represent similar such

selected mutant strains and supports the idea that the pres-

ence of two distinct reporters continues to provide a useful

specificity for the system.

DISCUSSION

In this report, we describe the development and character-ization of novel dual bait reagents that can be used to study the

interaction of a protein with two distinct partners in a single

yeast cell. The cI repressor/cI operator system utilized in the

SKY yeast strains and cIop-LacZ plasmids is demonstrated to

function with a sensitivity range closely comparable with the

pre-existing LexA repressor/lexA operator system in the inter-

action trap, facilitating their combined use. In a model system

assaying the interaction of the related GTPases Ras and Krev-1

with their preferred partners Raf and Krit1 and their shared

partner RalGDS, the dual bait system clearly differentiates

higher affinity versus lower affinity interactions. In addition to

effectiveness in discriminating interactions in grids of yeast

streaked to selective plates, the discrimination observed is

sufficiently robust to allow the isolation of yeast containing

specifically interacting protein pairs against a vast excess of

noninteracting pairs. These properties support the idea that

these reagents will be useful in library screening and genome-

scale applications. The reagents described here offer the option

of performing two independent, simultaneous screens in a sin-

gle yeast, with one set of positives registering through lexAop

activation of LEU2 and lacZ and a second set through cIop

activation of LYS2 and gusA, both negatively controlled

against each other. Together, these developments have thepotential to greatly expand two-hybrid system contributions to

studies of biological interactions.

Several groups have recently described the use of two simul-

taneously expressed baits to identify mutations that selectively

affect interactions of an activation domain-fused protein with

one of two partners (3840). In each case, introduction of a

second bait-reporter system was obtained by eliminating one of

the two reporters used for the primary bait, greatly reducing

the screening power available to the system. However, in these

novel reagents, both baits retain two distinct reporters, greatly

facilitating the screening process. The value of having two

separate reporters is evident even in the mixing experiment

performed here (Fig. 6), as their use allowed the immediate

discrimination of the noninteracting background clones from

specific partners, which activated both reporters. The dual baitreagents described here can be similarly used for mutational

analysis and have been recently used to successfully identify

mutations in Pak1 kinase, which selectively reduce interaction

affinity for either of two partners, the Cdc42 or Rac GTPases.3

Finally, there is preliminary evidence that these reagents pos-

sess the power necessary to perform library screens in organ-

isms with complex genomes; as in several recent library

screens using the above reagents, cI- and the LexA-fused baits

have yielded specific partners.4

3 M. Reeder, J. Chernoff, E. A. Golemis, and I. Serebriiskii, unpub-lished data.

4 M. J. Russell, personal communication; V. Khazak, unpublished

data; Y. Z. Zhang and E. Golemis, unpublished data.

FIG. 5. Discrimination of interaction specificity by dual bait. All yeast colonies shown express LexA-Ras (from pEG202) and cI-Krev-1(from pGKS8) and contain lexAop-lacZ (pJK103) and cIop-gusA (pGKS8) reporters and integrated lexAop-LEU2 and cIop-LYS2 (SKY191)reporters. Yeast additionally contain the AD fusion vector ( top row), AD-Krit1 (second row), AD-RalGDS (third row), AD-Raf ( fourth row). Panel

A, growth on nonselective medium; panel B, growth on plate assay for -glucuronidase activity with X-Gluc; panel C, growth on plate assay for-galactosidase activity with X-Gal; panel D, dual assay for for -glucuronidase and -galactosidase activity with X-Gluc and Magenta-Gal; panelE, growth on plates lacking lysine; panel F, growth on plates lacking leucine; panel G, growth on plates lacking both leucine and lysine; panel H,growth on plates lacking leucine but containing -aminoadipic acid (AA) (counterselective for LYS2). Note: less yeast are generally plated whenassaying for -glucuronidase than for -galactosidase because of the increased sensitivity of the gusA reporter; similarly, when performing dualassays, an initial overlay is done with agar containing Magenta-Gal, followed by a subsequent addition of X-Gluc.



7/8

On a more basic level, the system allows considerable sav-

ings in the effort required by individual investigators wishing

to perform multiple two-hybrid screens without invoking thedual bait selectivity function. Instead of having to perform two

separate library transformations or matings (generally the

most laborious step in a screening process) and subsequent

selection of positive clones, only one such step is required for

any two baits. Furthermore, in the case where only one-half of

a dual bait screen of a previously untested cDNA library is

positive (e.g. if a LexA bait yields interactors but a cI bait does

not), the fact that positive interactors are obtained for at least

one bait will provide useful information toward the determina-

tion of whether the library utilized was of acceptable quality.

Finally, it has previously been noted that some proteins of

interest for cDNA library screening perform better with par-

ticular fusion domains (i.e. are productively utilized as LexA

fusions but not as GAL4 fusions; or vice versa (23)). In cases

where specific DNA binding domain/fusion domain problems

are suspected, an investigator could express a bait of interest

both as a LexA and as a cI fusion and screen with the bait in

both configurations to maximize chances of obtaining valid

positive interacting partners.

The dual bait reagents are built upon the interaction trap

form of two-hybrid system (4). cI and LexA are similar in size

(237 versus 202 amino acids) and structure (41) and use related

amino-terminal helix-turn-helix domains to bind palindromic

operator sites with similar Km values, ranging from 200 pM to20 nM for LexA (discussed in Estojak et al. (23)) similar to cI

(42). Because of these many similarities, it is clear that the

LexA and cI systems are well matched. However, in addition to

use in the current interaction trap shell, the cI add-on parts

of this system have been constructed to potentially supplement

any of the currently existing two-hybrid variants. Thus, the

reporter system developed in this study purposely uses a DNA

binding domain (cI), reporter genes (gusA and LYS2), and

plasmid marker (zeocin resistance) not in use in any other

two-hybrid-based system (2, 3, 5), including the recently de-

scribed membrane-based Sos recruitment system (16). Thus,

these reagents could readily be integrated with any of the other

screening systems operating on two-hybrid principles; in the

case of the Sos recruitment system, this raises the possibilitythat with minor modifications of the library vector, a single bait

could be simultaneously used to identify interactors using ei-

ther a membrane-based or a transcriptional activation-based

selection strategy, enlarging the potential pool of interacting

proteins obtained. An additionally useful feature of the gusA

reporter is that it is assayed using similar protocols even on the

same plates as the lacZ reporter standardly used in two-hybrid

systems, again contributing to ease of use. Finally, although

the dual bait reagents here described have been optimized for

use in conjunction with LexA fusions, parameters have been

previously established to test and vary sensitivity levels (23),

making merging of two-hybrid systems a relatively simple and

certainly useful effort that should contribute to efforts to un-

derstand complex protein-protein interactions on the genome

scale.

AcknowledgmentsWe thank Russ Finley, Anne Vojtek, Jack Vos-sen, and Gabe Kalmar for their gifts of reagents, Michelle Berman forexcellent technical assistance, Joe Hurley for yeast photography, andMelissa Reeder and Marijane Russell for their efforts in testing thedual bait system. We are grateful to Jonathon Chernoff, Randy Strich,Garabet Toby, Russ Finley, and Bob Perry for helpful critique of themanuscript.

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