Methods 58 (2012) 392–399

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Methods

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Studying protein complexes by the yeast two-hybrid system

Seesandra V. Rajagopala a,⇑, Patricia Sikorski a, J. Harry Caufield b, Andrey Tovchigrechko a, Peter Uetz b,⇑a J Craig Venter Institute, Rockville, MD 20850, USAb Center for the Study of Biological Complexity, Virginia Commonwealth University, P.O. Box 842030, 1015 Floyd Ave., Richmond, VA 23284, USA

a r t i c l e i n f o a b s t r a c t

Article history:Available online 24 July 2012

Communicated by Kenneth Adolph

Keywords:Y2HRibonucleotide reductaseDNA polymeraseMntR/PerR complexPhage lambdaProteasome

1046-2023/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.ymeth.2012.07.015

Abbreviations: AD, activation domain; AP/MS, affitrometry; DBD, DNA-binding domain; PPI, Proteiribonucleotide reductase; SAI, socio-affinity index;Y2H, yeast two-hybrid.⇑ Corresponding authors.

E-mail addresses: rajgsv@gmail.com (S.V. Rajagopa

Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometricanalysis. However, in most cases the structure and topology of the complexes remains elusive from suchstudies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactionsamong proteins in a complex. First we tested all pairwise interactions among the seven proteins of Esch-erichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Fourand seven interactions were identified in these two complexes, respectively. In addition, we review Y2Hdata for three other complexes of known structure which serve as ‘‘gold-standards’’, namely VaricellaZoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda.Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for adynamic mega-complex.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Proteins act by either interacting with other proteins or smallmolecules such as sugars. In fact, many enzymes that interact withtheir low molecular weight substrates also form protein com-plexes, such as fatty acid synthase, a complex of 12 chains [1]. Inthe past 15 years high-throughput methods have been developedto map both protein–protein interactions on a large scale, usingeither the yeast two-hybrid system for binary interactions [2–4]or affinity purification and mass spectrometry for protein com-plexes [5–8]. In principle, a two-hybrid screen using all the pro-teins of a complex should yield all the binary interactions withinthat complex, but this is rarely the case; in most cases only a fewinteractions are discovered [9,10]. On the other hand, the two-hybrid system has a certain preference for transient interactionsthat are lost during complex purification because of the necessarywashing steps [4]. In addition, two-hybrid screening data derivedfrom genome-scale projects is not complete or/and comprehen-sive. Thus, little overlap is often observed between the proteininteraction datasets generated by protein-complex purificationand two-hybrid studies [4].

ll rights reserved.

nity purification/mass spec-n–protein interaction; RNR,VZV, Varicella Zoster Virus;

la), peter@uetz.us (P. Uetz).

In order to infer direct interactions from complex purificationdata, either the matrix or spoke model has been applied to listsof co-purified proteins. More recent protein complex purificationstudies used the socio-affinity index (SAI) to infer the direct interac-tions between complex members [5–8]. A related strategy usesdata from complex purification data sets to identify interactingproteins [11]. These strategies are based on the observation thatcertain pairs of proteins are more frequently found in multiplepurifications than others, and are thus predicted to be closer oreven directly associated in the complex. Similarly, solutions havebeen presented to computationally identify complexes from Y2Hinteraction networks [12–15].

While these experimental and computational attempts to mapprotein–protein interactions have produced a massive amount ofdata, structural analysis of complexes and interacting proteinshas lagged behind. One of the goals of PPI studies is thus to identifycomplexes that are amenable for crystallization. Often, severalstrategies need to be combined to reconstruct the topology of com-plexes that cannot be crystallized, including proteomics, cryo-elec-tron microscopy and others.

Recently, we have developed multiple variants of the yeast two-hybrid system and shown that different two-hybrid systems detectmarkedly different subsets of interactions in the same interactome[16]. Ten different configurations of bait-prey fusions were re-quired to detect up to 67% of a set of gold-standard interactions,whereas individual vector pairs detected only 25% on average [17].

Here we describe and review a similar strategy, using yeasttwo-hybrid assays to map the interactions within several com-plexes. In addition, we analyze several well-characterized protein

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complexes, ranging from ribonucleotide reductase (four subunits)to spliceosomes (>200 proteins), and compare structural data topublished Y2H data. Our data shows that a majority of interactionsin a complex can be identified by systematic Y2H screening andthat Y2H assays often detect subcomplexes within a larger com-plex that may be amenable to crystallography while the wholecomplex may be not crystallizable.

2. Materials and methods

2.1. Yeast two-hybrid (Y2H) assays

All full-length ORF clones of two protein complexes (Tables 1and 2, Fig. 1) were selected from the Escherichia coli ORFeome col-lection [18]. The entry clones were transferred into prey and baityeast two-hybrid expression vectors using Gateway LR reactions(namely pGADT7g and pGBGT7g for N-terminal fusions andpGBKCg-atg for C-terminal fusions; the latter vector having anin-frame start codon for ORFs that do not have one) [19]. After bac-terial transformation, miniprep plasmid DNA of all two-hybridprey and bait clones were transformed into yeast two-hybridstrains MATa Y187 (prey clones) and MATa AH109 (bait clones).Yeast two-hybrid matrix screening was conducted as describedin [20]. In brief, a yeast strain expressing a single protein as a baitfusion was mated to individual preys of all other protein complexmembers (Fig. 1A and B). After mating, the colonies were trans-ferred to the Y2H protein–protein interaction selective medium,

Table 1DNA Polymerase III complex [7]. See Fig. 1 for interactions.

Name Locus Description

AsnB JW0660 Asparagine synthetase BDnaE JW0179 DNA polymerase III alpha subunitDnaX JW0459 DNA polymerase III/DNA elongation factor III, tau and

gamma subunitsHolC JW4216 DNA polymerase III, chi subunitHolD JW4334 DNA polymerase III, psi subunitHolE JW1831 DNA polymerase III, theta subunitTrmD JW2588 tRNA (guanine-1-)-methyltransferase

Table 2Protein–protein interactions of the Escherichia coli DNA polymerase III proteincomplex. Interactions between protein_A and protein_B have been curated from theliterature (‘‘References’’) or are the results of our own Y2H studies (vectors used aregiven after ‘‘Y2H:’’).

Protein_A Protein_B Interaction detectionmethod

Reference

DnaE AsnB Tandem affinity purification [5]DnaX DnaE Biochemical [61]HolC DnaE Tandem affinity purification [5]HolD DnaE Tandem affinity purification [5]HolC DnaX Molecular sieving and

biochemical[62,63]

HolD DnaX Molecular sieving [62,64]HolD HolC X-ray crystallography,

molecular sieving andbiochemical

[54,62–64]

DnaX DnaX Y2H: GBG-GAD This studyHolD DnaX Y2H: GBG-GAD This studyHolD HolC Y2H: GBG-GAD This studyHolC HolD Y2H: GBG-GAD and GBKC-

GADThis study

TrmD TrmD Y2H: GBG-GAD and GBKC-GAD

This study

AsnB AsnB Y2H: GBKC-GAD This studyDnaE DnaX Y2H: GBKC-GAD This studyHolC DnaX Y2H: GBKC-GAD This studyHolC HolC Y2H: GBKC-GAD This study

and the interacting protein pairs were identified by the resultingpositive yeast colony. The positive interactions show a clear colonygrowth at a certain level of 3-Amino-1,2,4-triazole, whereas nogrowth was usually seen in the negative control (auto-activation),i.e. the bait mated with the empty prey vector strain (Fig. 1B).

Three Dimensional Modeling: Homology models of the VZV R1(ORF19) and R2 (ORF18) proteins were produced with I-TASSERserver [21] and superimposed onto the 2BQ1 asymmetric holo-complex structure from PDB (http://www.rcsb.org). The final mod-el was rendered in Pymol (http://www.pymol.org).

3. Results and discussion

3.1. Bacterial complexes

3.1.1. The E. coli DNA polymerase III complexIn order to validate how well multiple variants of the two-hy-

brid system work for mapping the topology of protein complexes,we have selected the E. coli DNA polymerase III complex as anexample of a well-characterized protein complex (protein complex42 in [7]). Hu et al. affinity-purified this complex and identified se-ven subunits, namely, AsnB, DnaE, DnaX, HolC, HolD, HolE andTrmD. We did not include other proteins known to be more looselyassociated with DNA polymerase III such as the epsilon subunit(DnaQ) which catalyzes the 30 to 50 proofreading exonuclease activ-ity of the holoenzyme.

E. coli DNA polymerase contains two sub-complexes: the cata-lytic polymerase/exonuclease sub-complex (with alpha, beta, deltaand epsilon subunits), plus the DnaX complex, a heptamer that in-cludes the tau and gamma products of the dnaX gene and confersstructural asymmetry that allows the polymerase to replicate bothleading and lagging strands. We have curated all the known pro-tein–protein interactions between co-purified complex membersfrom several studies (Fig. 1C and Table 2).

In order to validate whether we can capture all the known inter-actions of the E. coli DNA polymerase III proteins, we subjected itsproteins to an Y2H matrix screening. The yeast two-hybrid assaywas able to detect 70% known interactions (seven out of 10 interac-tions, including homo-dimers) and was able to connect four protein(out of seven proteins) in the complex compared to five proteinsconnected by literature data (Fig. 1C). Some of the known interac-tions are not reproduced here, for example the theta (HolE) subunitof DNA polymerase III binds to the epsilon subunit (DnaQ) but notto the alpha subunit (DnaE) [22]. This binding appears to enhancethe interaction between alpha (DnaE) and epsilon (DnaQ) as wellas slightly stimulating epsilon activity [23]. Since DnaQ is not amember of the protein complex described here, the interaction be-tween HolE ? DnaQ ? DnaE has not been tested. These resultsdemonstrate that Y2H screening can detect majority of the directinteractions within a complex and aid the mapping of its topology.

The MntR complex is a largely uncharacterized protein complexthat has been identified as ‘‘complex 34’’ by AP/MS [7]. This com-plex consists of eight proteins of which five proteins have beencharacterized and three uncharacterized proteins (Table 3,Fig. 1D). Even though the complex contains several well-character-ized proteins the direct interaction and functional associationsamong the complex members are unknown.

The Y2H screening of all the members of this complex identifiedseven interactions (plus four homo-dimers) and based on theseinteractions we were able to precisely map the topology of thecomplex (Fig. 1B and D), confirming that these proteins actuallyform a complex. These protein–protein interactions should helpto characterize the function of unchartered proteins in the com-plex. For example, the interaction between the transcriptional reg-ulators MntR and PerR supports the predicted DNA-binding and

A B

C

D

Fig. 1. Mapping interactions within Escherichia coli protein complexes by Y2H. (A) The Escherichia coli DNA polymerase III complex as determined by AP/MS (complex 42 in[7]). (B) Yeast two-hybrid matrix screening of the DNA polymerase III complex with N and C-terminal fusion baits with N-terminal fusion prey clones. Growth of yeastindicates positive PPI; auto-activators (false positive PPI) are marked as red. (C) The topology of the protein complex based on protein–protein interactions obtained in (B) aswell as known direct interactions and structural information collected from the literature [52–59] and PDB. (D) Protein complex 34 from [7]. No published information isavailable on the topology of this complex. As in panels (A–C) Y2H protein–protein interaction data was used to map the topology of this complex.

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transcriptional activity of the latter. While PerR has been studied inBacillus subtilis, its ortholog in E. coli is poorly understood. PerR and

Table 3Protein complex 32 [7]. See Fig. 1 for interactions.

Name Locus Description

HycG JW2689 Hydrogenase 3 and formate hydrogenase complex, HycGsubunit

MetN JW0195 DL-methionine transporter subunit, (ATP)-bindingcassette (ABC) family

MntR JW0801 Regulator of manganese levels, transcriptional regulator ofmntH [23]

PerR JW0244 Predicted DNA-binding transcriptional regulatorWcaC JW2042 Predicted glycosyl transferase, colanic acid synthesis [24]YhjB JW3488 Two-component response regulator, predicted DNA-

bindingYjhC JW5769 Predicted oxidoreductase, maybe involved in sialic acid

metabolism [25]YraM JW3116 Conserved hypothetical protein

related members of the LysR family have been shown to interactwith other members of the family to form heterodimers, but thephysiological significance of this is unknown [24]. In order to studythe function of these complexes in more detail, the topology ofinteractions suggests several strategies to crystallize this complex,e.g. either as dimers (YjhC–YjhC), trimers (MntR–HycG–MetN orMntR–MetN–YjhC), tetramer (MntR–HycG–MetN–YjhC), or as het-ero-hexamer. Such crystallization experiments are now under way.

3.2. Previously published complexes

In addition to the new interactions in the previous section, wehave also re-visited the literature on several protein complexesthat have been studied by Y2H methods. They cover sizes rangingfrom ribonucleotide reductase, a small protein complex of foursubunits, to the spliceosome, one of the largest complexes in livingsystems with hundreds of proteins. These complexes are discussedin the order of increasing complexity.

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3.3. Ribonucleotide reductase

Ribonucleotide reductases (RNRs) convert ribonucleotides todeoxyribonucleotides and thus provide the raw materials forDNA synthesis. All living systems that are based on DNA as theirgenetic material, either encode RNRs, or must obtain their dNTPsfrom an outside source, as several parasites and endosymbiontsdo [25]. Most eukaryotes and some of their viruses encode so-called type I RNRs that are characterized by a a2b2 quarternarystructure [26].

RNRs are interesting complexes for several reasons (reviewed in[25–27]). First, they all appear to be homologous, yet show a vari-ety of quarternary structures and enzymatic mechanisms. For in-stance, class I RNRs form a2b2 quarternary structures while classII enzymes are either a monomers or a2 dimers. Second, differentspecies use different cofactors (e.g. thioredoxin vs. formate), areaerobic or anaerobic, and have different modes of interaction.Third, they are highly dynamic complexes that undergo conforma-tional changes during a reaction cycle, which also affects their

A

B

= R2 = ORF18 (306 aa)

= Gal4 DBD or AD

= R1 = ORF19 (775 aa)

N

N C

N C

OR

Fig. 2. The ribonucleotide reductase (RNR) complex of Varicella Zoster virus (VZV). (A) MRNR (2BQ1 [32]). VZV ORF18 = R2 (Uniprot P09247), ORF19 = R1 (P09248) with chains irainbow from N-terminal (blue) to C-terminal (red), with terminals labeled as NT and CT,peptide taken from 2BQ1 (thick red tube on the picture) where it is in contact with the grin 2BQ1. The C-terminal of R1 (I) and the N-terminal of R2 (II) are shown as thick tubes ithe symmetric homo-dimers in this view and thus not labeled. (B) Interactions amongdomains (AD) are shown as green boxes that are either fused to the N or C-terminus offragments (and each fragment was tested as N- and C-terminal fusion to DBD and AD).

interactions. Fourth and last, RNRs and their subunit interactionsare affected by allosteric regulators such as dGTP.

In the course of our studies on human herpesviral interactomes[28,29], we have analyzed RNRs of several viruses. However, onlythe RNR of Varicella Zoster Virus (VZV) was analyzed with multipleY2H vectors (Fig. 2, [16]) which allowed us to draw some moregeneral conclusions about interactions that can be detected withinprotein complexes. First, under favorable conditions, majority ofinteractions in a complex can be detected by Y2H assays. In thecase of VZV RNR, all interactions among the four RNR subunitswere found: R1–R1, R1–R2, and R2–R2. Second, full-length pro-teins not necessarily work in such assays. Here, the full-lengthR2 protein neither interacted with R1 nor with itself. However,fragments clearly worked in multiple orientations. As is shown inFig. 2B, N- and C-terminal fragments of R2 interacted in a homo-di-meric fashion (ORF18C–ORF18C), as pseudo-homo-dimers(ORF18N–ORF18C), as well as true hetero-dimers (ORF18C–ORF19). Third, and finally, the N- and C-terminal fusions of theGal4 DNA-binding and activation domains had very distinct

ORF18C 134 aa

N

C

C

F18N = 151 aa

odel of the VZV RNR complex based on crystal structures of Salmonella typhimuriumn each homo-dimer numbered as I and II. R1 chain II and R2 chain II are colored inrespectively. The C-terminal of the R2 (II) chain was replaced with the correspondingoove in the R1 (I). The remaining part of R2 (II) CT is hidden because it is disorderedn regions disordered in 2BQ1. The remaining terminals are behind the structures ofRNR subunits detected in Y2H assays. The Gal4 DNA-binding (DBD) and activationthe RNR proteins. ORF18 was tested as full-length protein and as N- or C-terminalY2H data in (B) from [16].

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patterns of interaction. For instance, while an N-terminal fusion ofORF18C interacted with almost all other proteins, both N- and C-terminal fusions of ORF18N interacted very specifically only withORF18C.

These observations are largely supported by the crystal struc-ture of the RNR holocomplex (Fig. 2A) [30,31], even though the bio-logical complex appears to be a dynamic assembly of proteins inwhich the R2 subunits moves and rotates [32]. This fact has im-paired crystallization of the biological complex and it is still not en-tirely clear how the subunits move during a catalytic cycle in vivo.

Our homology model superimposed on the available assymetrictemplate (Fig. 2A) shows C- and N-terminals either located com-pletely away from the interface areas or having likely disordered/flexible ends, thus leaving enough room for accommodating theY2H fusion constructs in all cases.

3.4. The proteasome

The proteasome is a complex assembly of proteins found withinall eukaryotic cells and some bacterial species. In its fully-assem-bled state, this protein complex recycles peptides within the cellby degrading misfolded or otherwise unnecessary proteins. Each

A B

C

Fig. 3. The proteasome. (A) Structure of the 26S proteasome from Schizosaccharomyces poshown in red, the AAA-ATPase hexamer in blue and the Rpn subunits in gold. (B) Interactilink’’). Four interactions between 19S proteins and beta subunit proteins are omitted for care derived from 3 independent studies on the proteasomes of three different species (Scin two species (Sc = Saccharomyces cerevisiae, Sp = Schizosaccharomyces pombe) [38,4crystallography, and molecular modeling and used as ‘‘gold-standard’’ interactions, showand the fraction that is seen in a structural model (red) that serves as ‘‘gold standard’’. Nureproduced by permission from PNAS.

full 26S proteolytic complex is formed by a 20S core particle (CP)and at least one regulatory particle (RP), including six AAA-ATPasesubunits and thirteen non-ATPase subunits. In eukaryotes, each ofthese 26S proteasome particles may be found in the cytoplasm andthe nucleoplasm [33]. Several sets of yeast two-hybrid analyseshave been used alone and in concert with other methods to clarifythe protein interactions required for assembly of a functional pro-teasome. Work by Cagney et al. [34] employed a yeast two-hybridapproach to search for interactions between 31 known proteasomeproteins and an array of nearly 6000 different Saccharomyces cere-visiae proteins. This genome-wide screen revealed 55 potentialprotein–protein interactions, more than a third of which involvedonly proteasome components rather than non-proteasomal pro-teins. The specific pairs of interactions found in these screens dem-onstrated how many proteins of the 19S and 20S subcomplexesspecifically interact with other proteins within those subcomplex-es (Fig. 3A and B). One interaction between a 20S a ring protein,Pre8, and a 20S b ring protein, Pup1, was observed, confirmingstructural arrangements seen in the 20S crystal structure [35].Some interactions did occur between subunits, as comparisonswith the crystal structure of the 20S core showed. Out of 14 inter-acting protein pairs in the 20S subcomplex, just 3 involved proteins

mbe as determined by cryo-EM density map (adapted from [41]). The core particle isons among proteasome subunits as determined by Y2H and cross-linking assays (‘‘X-larity (a3-Rpt2, a4-Rpt2, a6-Rpt5, a7-Rpt5; see Supplementary Table 1). Y2H results= yeast [34], Ce = C. elegans [36], Hs = human [37]), crosslinking has been carried out1]. Structural and modeling results are derived from cryo-EM mapping, X-rayn as grey bars [41,60]. (C) Number of interactions in Y2H and cross-linking studies

mbers in red areas are the fraction of PPIs found in the ‘‘gold-standard’’ structure. (A)

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not predicted to be neighbors in the crystal structure. As Cagneyet al. studied yeast proteasomal proteins in an Y2H system; it isquite likely that endogenous proteasomal proteins bridged thetwo hybrid proteins. This background may have contributed toartifactual interactions not seen in the 20S crystal structure. Useof the Y2H system may therefore be better suited to the study ofheterologous protein pairs from sources other than yeast.

Work by Davy et al. [36] used the Y2H system to generate inter-action data for 30 proteasome subunits from Caenorhabditis ele-gans. Each of the proteins assayed in this study are orthologousto those of S. cerevisiae. Though this study found many interactionscorrelating with those seen in with yeast proteasome proteins, anumber of new interactions were also observed (Fig. 3). Amongthese are four interactions between 19S subunit proteins and three20S a subunits.

An independent study of the human proteasome [37] also re-vealed protein–protein interactions by Y2H, finding 114 potentialinteracting pairs. Many of these interactions were found to bestructurally relevant. Interactions between the a and b rings ofthe 20S core were observed, confirming observations by Cagneyet al. (Fig. 3B, Supplementary Table 1). Subsequent studies haveconfirmed many of the interactions revealed by Y2H assays usingother methods. A mass spectrometry-based approach [38] gener-ated 64 potential proteasome-associated protein–protein interac-tions beyond those seen between proteasome subunit proteins.

Unfortunately, the fragile nature of the 26S proteasome has ren-dered it difficult to crystallize for extensive X-ray crystallographicanalysis. The 20S core particle crystal structure prepared by Grollet al. [35] provides an example of many potential protein interac-tions on the basis of the barrel-like structure of this subunit, but itprovides no evidence for protein interactions beyond those of thecore particle. Combined data from crystallography, cryoelectronmicroscopy, and protein co-purifications have been used to clarifyproteasomal structural organization predicted by Y2H results [39–41]. One such model prepared by Lasker et al. displays the overallarchitecture of the 26S proteasome holoenzyme. While the overallstructure is based on cryo-EM mapping and molecular modeling(including a model prepared by Förster et al. [42]), the specificlocations of each protein are based on crystal structures, residue-specific lysine crosslinks, and known protein–protein interactions,including those found though Y2H screens.

In summary, the three Y2H studies found a total of 183 PPIs,while structural studies revealed a total of 38 PPIs. No Y2H studyfound more than 40% of the interactions assumed to take placein the proteasome, but all three studies together found 79% of allinteractions because each study found a different subset (Supple-mentary Table 1). This is certainly due to the fact that proteinsfrom different species as well as different Y2H systems were used.

Clearly, Y2H screens produced a substantial number of falsepositives in these studies, (as shown by interactions not seen inthe proteasome structure), but some of them may be truly physio-logical interactions (i.e. taking place in vivo), given that the a and bsubunits of the proteasome are closely related proteins that prob-ably interact in more than the canonical combinations even withinthe assembled proteasome (Fig. 3).

Notably, cross-linking studies found a total of 32 interactions, ofwhich nine (32%) were seen in the structure. However, these stud-ies may have not been as comprehensive as the Y2H screens, but itis remarkable that they also produced a number of false positivesthat is similar to those found in Y2H screens.

3.5. Phage Lambda

Bacteriophage lambda virion consists of �14 different proteinsand a total of �1075 subunits. After its discovery in 1960 hundredsof studies have revealed its structure in great detail and we know

now that its subunits are connected by at least a dozen differentprotein–protein interactions (Fig. 4A and B). Interestingly, not evenin this extremely well-studied phage all interactions are known forsure. Rajagopala et al. [43] therefore curated the literature andcompiled a list of 33 published PPIs among lambda proteins (notethat this list includes PPIs among proteins that are not in the par-ticle). This set of 33 known interactions is considered as a ‘‘gold-standard’’ set here. These authors then tested all possible proteinpairs encoded by the lambda genome for interactions using a ma-trix-based Y2H screen that employed 6 different Y2H vectors(Fig. 4). Several lessons can be learned from this analysis. First,the screen detected more than half of all previously known interac-tions (including 4 interactions among regulatory proteins not pres-ent in the virion). Rajagopala et al. speculate that the remaininginteractions were not detected because of the lack of chaperones,assembly factors, post-translational modifications, or other effects.Second, each vector pair detected a certain fraction of the known‘‘gold-standard’’ set of PPIs; which interaction was detected de-pended primarily on the vector pair used (Fig. 4C). For instance,pGBKT7/pGADC produced the largest absolute number of ‘‘gold-standard’’ interactions although the pDEST vectors produced thelargest fraction of gold-standard interactions. 11 out of 16 interac-tions were detected only by one vector pair, namely pGBKT7g/pGADCg (5 PPIs), pDEST (4), and pGBKCg/pGADCg (2). The other5 PPIs were detected with multiple vectors each. Interestingly, only4 of the gold-standard PPIs were detected exclusively with N-ter-minal fusions, the system with which the vast majority of allY2H screens are carried out. Two gold-standard PPIs were detectedexclusively with C-terminal fusions. The majority was detectedwith either NC fusions or multiple vectors.

3.6. The spliceosome

The spliceosome is probably the largest protein complexknown, at least in terms of complexity, containing more than200 different proteins as well as multiple RNAs [44]. It is thusmuch bigger than the ribosome. Although spliceosomal proteinsassemble into smaller complexes, such as the U1, U2, U4/6 andU5 snRNPs, they transiently associate with each other during theprocess of splicing, which makes them a ‘‘functional’’ complex. Gi-ven its complexity, the spliceosome has not been crystallized andthere are only certain subcomplexes whose structure is reasonablywell-known, such as the U1 snRNP [45]. Hegele et al. [46] recentlypresented a systematic analysis of protein–protein interactionsamong 244 spliceosomal proteins, of which 141 proteins are clas-sified as ‘‘core proteins’’, based on their high abundance. Initially,Hegele et al. cloned 244 proteins known to be associated withthe human spliceosome into yeast two-hybrid bait and prey vec-tors and tested them in a pairwise fashion for interactions. Thisstudy found a total of 632 interactions among 196 of the 244 pro-teins. Notably, 390 interactions were among non-core and 242interactions were found among core proteins. Importantly, thisstudy also curated 311 binary interactions previously publishedin 201 papers. Of these 311 PPIs, 72 were reproduced by Hegele’sY2H analysis. While this number corresponds to only 23% of thepublished PPIs, the fraction of reproduced PPIs among the core pro-teins was 41% (43 PPIs). Reproducing only a quarter of all pub-lished interactions certainly have the same reasons as discussedabove: first, only one vector pair was used in the Y2H analysis. Sec-ond, splicesomal interactions may depend on both RNA support aswell as tertiary interactions. Third, many interactions are dynamicand thus rather weak, and may require additional factors such asassembly proteins or other catalysts [47]. The Hegele paper unfor-tunately did not attempt to integrate their data into structuralmodels of the spliceosomal subunits, so it remains unclear to whatextent these interactions facilitate a structural understanding of

Fig. 4. Bacteriophage lambda. (A) Phage particle and its protein components. (B) Interaction map of the proteins shown in (A). Proteins inside box are virion proteins, proteinsoutside the box are assembly factors that are not part of the assembled virion. It remains unclear if gpM (‘‘M’’) and gpL (‘‘L’’) are part of the virion. (C) Contribution of eachvector pair to the known lambda interactome [43]. The left panel shows the number of interactions generated by each vector pair, including previously known interactions(shown in blue). The right panel illustrates the fraction of previously known interactions relative to the total number of PPIs generated by each vector. The pDEST vectorsproduce a high fraction of such ‘‘gold-standard’’ PPIs although their total number of known PPIs is still smaller than those generated by the pGBKT7/pGADC pair. Note thatthese numbers include 4 PPIs among regulatory proteins not in the virion. (A) and (B) after [43].

398 S.V. Rajagopala et al. / Methods 58 (2012) 392–399

the spliceosome. However, many interactions suggested local sub-complexes that can be subjected to crystallography and otheranalyses.

A good example how Y2H screens can dissect the interactionswith a large complex is an analysis of the U1 snRNP. This subcom-plex of the spliceosome consists of 10 U1 proteins and a ring of se-ven Sm proteins in yeast. Several groups have solved the overallstructure of the complex by cryo-EM analysis and crystallography[48,49]. However, their model of the human U1 snRNP containsonly three U1 proteins and the Sm ring (7 proteins). It remains un-clear where the remaining proteins are that are clearly parts of theyeast U1 snRNP. Ester & Uetz [50] showed that a subcomplex of 3proteins (Snu71, Prp40, and Luc7) can be detected within the yeastU1 snRNP and these authors mapped their interaction domains. Itis hoped that such subcomplexes are stable enough for crystalliza-tion, so that their atomic structure can be resolved eventually.

4. Conclusions

From the data discussed here, it becomes clear that the Y2H sys-tem can contribute significantly to the understanding of proteincomplexes. However, there is also much room for improvements.First, multiple vectors need to be used routinely to achieve maxi-mum coverage. In addition, the existing vectors can be certainlyfurther improved. Notably, vector variations as mentioned herehave not even been used for alternative Y2H systems such as thesplit-ubiquitin system or protein fragment complementation as-

says. Second, and similar to crystallography, it is likely that com-parative studies will shed much light on protein complexes. Forinstance, while crystallization of E. coli and eukaryotic ribosomeswas unsuccessful for many decades, the ribosomes (or ribosomalsubunits) of other species could be crystallized more easily. Simi-larly, it is quite likely that many interactions may be easier to de-tect with proteins from non-model organisms although this issuehas not been systematically studied on a larger (experimental)scale [51]. That said, it will be interesting to see how complexesand their interactions look in different, distantly related species.There is certainly much to be discovered by comparative analysisusing improved methodology.

Acknowledgements

SVR and PU were supported by NIH Grant R01GM079710; ATwas supported by NSF Grant 1048199.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ymeth.2012.07.015.

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