1.Introduction and Background

There is a great need for general methods to characterize the proteins that contemporary biology makes available.The list of such proteins needing further characterization is growing and includes proteins already known to be important for specific cellular functions,mutant proteins identified in vivo or made in vitro,and very large numbers of protein being identified by genome projects.Here we describe the extension of two-hybrid approaches so that they can bear on this problem.

The recent success of two-hybrid systems is due to the fact that many cellular functions are carried out by proteins that touch one another.For example,the complex process of transcription initiation requires the ordered assembly of numerous interacting transcription factors with RNA polymerase and ancillary proteins,into a protein machine that initiates transcription (Guarente,1996; Tjian and Maniatis,1994).This machine can be viewed as a network of interacting proteins,as can the machines that control other processes,such as DNA replication,protein translation,and the cell cycle.A full understanding of these processes will require knowledge of,not only the proteins (parts)that make up each machine,but also of the topological relationships (connections)that individual parts make with one another.

Likewise,a full understanding of the function of any new protein will require knowledge of the interactions it makes with previously identified proteins.Currently,most new proteins are being identified by large scale sequencing projects.For many of these new proteins the sequence alone sheds little or no light on their function.

Two-hybrid systems have been used to probe the function of new proteins ever since they were developed (Chien et al.,1991; Fields and Song,1989).The first application of two-hybrid methods to probe protein function was to examine the interactions between proteins isolated by two hybrid methods and relatively small numbers of test proteins (see for example,Durfee et al.,1993; Gyuris et al.,1993; Harper et al.,1993; Zervos et al.,1993),but their use quickly spread to the analysis of many other proteins (Choi et al.,1994; Kranz et al.,1994; Marcus et al.,1994; Printen and Sprague,1994; Van Aelst et al.,1993; Yuan et al.,1993).In anticipation of the utility of applying these methods to larger sets,we and others began devising ways to do so.

Larger scale two hybrid approaches typically rely on interaction mating.In this method the protein fused to the DNA-binding domain (the bait)and the protein fused to the activation domain (here called the prey)are expressed in two different haploid yeast strains of opposite mating type (MATa and MATa),and the strains are mated to determine if the two proteins interact.Mating occurs when haploid yeast strains of opposite mating type come into contact,and results in fusion of the two haploids to form a diploid yeast strain.Thus,an interaction can be determined by measuring activation of a two-hybrid reporter gene in the diploid strain.

As described below,interaction mating has been used to examine interactions between small sets of tens of proteins (Finley and Brent,1994; Finley and Brent,1995; Reymond and Brent,1995),larger sets of hundreds of proteins (R.L.F.and R.B.,unpublished),to screen libraries (Bendixen et al.,1994),and to attempt to comprehensively map connections between proteins encoded by a small genome (Bartel et al.,1996).The primary advantage of this technique is that it reduces the number of yeast transformations needed to test individual interactions.For example,to test for interactions between a set of 10 bait proteins and 5 prey proteins without interaction mating would require 50 transformations to create 50 strains that carry the pair-wise combinations of baits and preys.With mating however,only 15 transformations would be needed; 10 for the different bait plasmids,and 5 for the different prey plasmids; and the resulting two sets of transformants would be mated to create the 50 combinations.The microbiology of the mating procedure (which is extremely simple)is detailed in Section 2.

Interaction mating techniques have facilitated a number of two-hybrid studies of protein protein interaction.Among its first uses was to determine the specificity of interactors isolated in library screens or interactor hunts (Harper et al.,1993).As described in the previous chapters,in the first steps of an interactor hunt,one isolates genes that encode proteins that interact with a particular bait.Before the interacting proteins are further characterized,it is necessary to determine if their interaction with the bait is specific by showing that they do not interact with other unrelated baits or with the DNA-binding domain portion of the bait.When mating is used to test specificity,the strain that contains the activation domain fused protein (prey)is mated with different yeast strains which express either the original bait protein or other,preferably unrelated baits,and the investigator verifies that the reporters are only active in diploids that contain the original bait (Finley and Brent,1994; Finley and Brent,1995; Harper et al.,1993).

For example,Harper,Elledge and colleagues used a mating assay to test the specificity of newly isolated interactors (Harper et al.,1993).The methods of these investigators also circumvented the need to isolate the prey plasmid.In their experiments,they performed two-hybrid hunts with a bait plasmid that contains a dominant marker,CYH2,that can be selected against by plating the yeast on medium containing cycloheximide,which is toxic to yeast that carry CYH2.Yeast isolated in an interactor hunt were plated on cycloheximide plates to select those that had lost the original bait plasmid but retained the library plasmid.The resulting strain was then mated with a collection of bait strains,including ones that expressed the original bait,to determine the specificity of the library-encoded prey.A mating scheme has also been used directly in an interactor hunt by mating a strain expressing a bait with a strain transformed with the library DNA; here,mating promises to bypass the need to perform separate transformations with library DNA for each new hunt (Bendixen et al.,1994).

In addition to its use in interactor hunts,mating can be used to characterize small sets of proteins as described in Section 2.1 and Protocol 1.In one example of this approach,we used interaction mating to characterize a set of seven Drosophila Cyclin-dependent kinases (Cdk)interactors,or Cdis (Finley and Brent,1994).Strains expressing versions of the Cdis fused to an activation domain were mated with 74 different strains expressing different bait proteins,including Cdks from other species and four of the Cdis themselves.The results from this study illustrate the types of information that can be derived from such a characterization.First,the experiments showed that some of the Cdis interacted with different subgroups of seven highly related Cdk baits,suggesting that the Cdis recognize structural features shared by these Cdks but absent in the non-interacting Cdks; inspection of an alignment of the Cdk protein sequences suggested residues that may be important for specific interactions with certain Cdis.Second,Cdi3,Drosophila Cyclin D,interacted much more strongly with human Cdk4 than with any of the other Cdks in the panel including the Drosophila Cdks,suggesting that there may be an as yet unidentified Drosophila Cdk4 homolog which is the true partner for Cyclin D.Third,two of the Cdis interacted with two other Cdis,indicating in each instance that each Cdi has surfaces for binding to the Cdk and to another Cdi,and suggesting that these proteins form ternary or higher order complexes.Finally,the demonstration that tw℃dis with no sequence similarity to previously identified proteins interact with each other as well as with the Cdk,but not with a panel of over 60 other proteins,provided an additional clue to their functions,strongly supporting the idea that they function along with the Cdk in the network of proteins that regulates the cell cycle.These results demonstrate that examination of the interactions between even small numbers of proteins can provide a number of functional insights.Much larger sets of proteins can be characterized by scaling up these procedures as described in Section 2.2 and discussed in Sections 6 and 7.

2.Interaction mating

In this section we present methods for performing interaction mating assays on small or large sets of proteins using the interaction trap,and in Section 3 we discuss use of interaction mating with other two-hybrid systems.The interaction trap (see Chapter 4 and references therein)uses the E.coli protein LexA as the DNA-binding domain and a protein encoded by random E.coli sequences,the B42 "acid blob",as the transcription activation domain.Both proteins are expressed from multicopy (2µ)plasmids; the LexA fusion,or bait,is expressed from a plasmid containing the HIS3 marker,and the activation domain fused protein,or prey,is expressed from a plasmid containing the TRP1 marker.In the most commonly used bait plasmid,pEG202,the bait is expressed from the constitutive yeast ADH1 promoter.Related bait plasmids are available which express the bait fused to a nuclear localization signal (pNLex,see Chapter 4),or which express the bait conditionally from the GAL1 promoter (pGILDA,D.Shaywitz and C.Kaiser,personal communication).The most commonly used prey plasmid,pJG4-5,expresses proteins fused to the B42 activation domain,the SV40 nuclear localization signal,and an epitope tag derived from hemagglutinin,all driven by the yeast GAL1 promoter which is active only in yeast grown on galactose (Gyuris et al.,1993).Use of the GAL1 promoter to express the prey allows toxic proteins to be expressed transiently and helps eliminate many false positives in interactor hunts (Chapter 4).The interaction trap uses two reporter genes that carry upstreamlexA binding sites (operators): LEU2 and lacZ.The LEU2 reporters are integrated into the yeast genome and the lacZ reporters typically reside on 2µ plasmids bearing the URA3 marker,though integrated versions are also available (R.L.F.,R.B.,S.Hanes,unpublished).Several versions of the LEU2 and lacZ reporters have been made that have a range of sensitivities based on the number of upstreamlexA operators.In general the LEU2 reporters are more sensitive to a given interacting pair of proteins than the lacZ reporters (Estojak et al.,1995); however,recently highly sensitive lacZ reporters have been used that contain several LexA operators and transcription terminator sequences downstream of the lacZ gene (S.Hanes,personal communication).

Several different combinations of strains,plasmids,and reporters can be used for mating (Section 3).In one common version (Finley and Brent,1994),the strain expressing the bait (bait strain)is RFY206 (MATa ura3-52 his3Æ200 leu2-3 lys2Æ201 trp1::hisG)transformed with the HIS3 bait plasmid and a URA3 lacZ reporter plasmid like pSH₁8-34.The strain expressing the activation domain-tagged protein (prey strain)is EGY48 (MATa ura3 his3 leu2::3LexAop-LEU2 trp1 LYS2)transformed with the TRP1 prey plasmid.Patches of these two strains on agar plates are brought into contact by replica plating (see below)and grown on a rich medium overnight.During this time cells in the patches mate and fuse to form diploids.The cells are then transferred by replica plating to plates on which only diploids can grow: these plates lack uracil,histidine,and tryptophan so that neither parental haploid can grow on them.To avoid an additional step,the diploid selection plates are also indicator plates,which allows an interaction to be scored by testing for expression of the reporter genes.In the protocols presented here the lacZ reporter is measured,using diploid selection indicator plates containing X-Gal,a chromogenic substrate for the lacZ gene product.However,it is worth mentioning that expression of the LEU2 reporter can also be easily scored by putting the diploids on plates that lack leucine,and that the future will likely bring other reporters.Furthermore,because both reporter genes exhibit a reduced sensitivity in diploid strains compared to haploid strains,the most sensitive versions of the lacZ or LEU2 reporters are recommended for interaction mating assays.

Variants of this simple procedure are sometimes useful.In particular,because some baits activate transcription by themselves,it is often useful to conditionally express the prey protein so that one scores patches that show an increase in reporter gene expression in the presence of the prey.To do this,the diploids are placed on two different X-Gal plates,one that contains galactose,which results in expression of the prey,and one that contains glucose which represses expression of the prey.Here,an interaction between the bait and prey is detected when the diploid yeast containing them turn more blue on the galactose X-Gal plate than on the glucose X-Gal plate.

2.1 Interaction mating^- small scale

It is often informative to look for interactions between small sets of proteins,or between a given protein and a test set of ten to a hundred proteins.The test set,for example,might contain different allelic forms of the original bait,sets of structurally related proteins,sets of proteins known or suspected to be involved in some process,and unrelated proteins used to demonstrate the specificity of an interaction.Protocol 1 describes a convenient method to test small sets of proteins for interactions.

The collections of bait and prey strains used here can be maintained on yeast plates stored at 4℃ for two to three months,or stored frozen for several years (see Protocol 2).For mating,the two strains are first streaked to the appropriate selection plates: the bait strains (RFY206 containing the URA3 lacZ reporter plasmid and HIS3 bait plasmid)are streaked to plates lacking uracil and histidine -u-h Glu)to maintain selection for the two plasmids; the prey strains (EGY48 containing the TRP1 prey plasmid)are streaked to plates lacking tryptophan (-w Glu)to maintain selection for the prey plasmid.The haploid strains are then brought into contact by placing both plates sequentially on the same replica velvet and lifting the double imprint with a YPD plate (see Protocol 1).If the bait strains are streaked in parallel horizontal stripes and the prey strains are streaked in vertical stripes,physical contact between the strains will occur at the intersections of the stripes on the YPD plate.After a brief period of growth to allow diploids to form,the yeast are transferred to diploid selection indicator plates by replica plating.Diploid colonies that contain a pair of interacting bait and prey proteins are more blue on the galactose X-Gal plate than the glucose X-Gal plate.

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Protocol 1.Mating assay^- small scale for tens of different bait or prey proteins.

Materials

_ Bait strains: S.cerevisiae strain RFY206 (MATa ura3-52 his3Æ200 leu2-3 lys2Æ201 trp1::hisG)transformed with a URA3 plasmid containing a lacZ reporter,such as pSH₁8-34,and various HIS3 bait plasmids,such as derivatives of pEG202 that produce different LexA fusions.Each bait strain will contain a different bait plasmid.

Prey strains: S.cerevisiae strain EGY48 (MATa ura3 his3 leu2::3LexAop-LEU2 trp1 LYS2)transformed with TRP1 prey plasmids,such as derivatives of pJG4-5 that produce different activation domain-tagged proteins or preys

Sterile wooden applicator sticks (e.g.FisherBrand 01-340)

Minimal glucose yeast plates lacking uracil and histidine (-u-h Glu),see Chapter 4

Minimal glucose plates lacking tryptophan (-w Glu),see Chapter 4

YPD plates,see Chapter 4

Minimal X-Gal glucose plates lacking uracil,histidine,and tryptophan (-u-h-w Glu X-Gal),see Chapter 4.

Minimal X-Gal galactose/raffinose plates lacking uracil,histidine,and tryptophan (-u-h-w Gal/Raf X-Gal),see Chapter 4

Replica plater and sterile replica velvets

Optional

Minimal glucose plates lacking uracil,histidine,tryptophan,and leucine (-u-h-w-l Glu),see Chapter 4

Minimal galactose/raffinose plates lacking uracil,histidine,tryptophan,and leucine (-u-h-w^- Gal/Raf),see Chapter 4

Method

1.Streak different bait strains in horizontal parallel stripes on a -u-h Glu plate.Streaks should be at least 3 mm wide and at least 5 mm apart,with the first streak starting about 15 mm from the edge of the plate.A 100 mm plate (which for some reason is typically 90 mm in diameter)will hold 8 different bait strains.Create a duplicate plate of bait strains for each different plate of prey strains to be used.

2.Likewise,streak different prey strains in vertical parallel stripes on a -w Glu plate.As a control for baits that may activate transcription,include a prey strain that contains the prey vector pJG4-5 not encoding a fusion protein (i.e.encoding only the activation domain).Create a duplicate plate of prey strains for each plate of bait strains to be used.

3.Incubate plates at 30℃ until there is heavy growth on the streaks.When taken from reasonably fresh cultures,for example plates that have been stored at 4℃ for less than a month,streaked RFY206-derived bait strains take about 48 hours to grow and EGY48-derived prey strains take about 24 hours.

4.Press a plate of prey strains to a replica velvet,evenly and firmly so that yeast from all along each streak are left on the velvet.This plate may be reused if necessary.Press a plate of bait strains to the same replica velvet.This plate of bait strains cannot be reused as it is now contaminated with prey strains.

5.Lift the impression of the bait and prey strains from the velvet by pressing a YPD plate on it.Incubate the YPD plate for 24 hours at 30℃.

6.Replica YPD plates to the following diploid selection,indicator plates: -u-h-w Glu X-Gal,-u-h-w Gal/Raf,and (optional: -u-h-w-l Glu,and -u-h-w-l Gal/Raf).The YPD plate should contain sufficient growth to enable a single impression on the velvet to be lifted by at least four indicator plates.

7.Patch control strains (see text)onto the indicator plates and incubate at 30℃.Examine results daily.Diploids will grow and blue color will develop within 2 days.

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2.2 Interaction mating^- large scale

With a few modifications,the procedure described above can be used to test for interactions between a single prey protein and hundreds of baits (Protocol 3,see Figure 1 below).Large panels of bait strains can be collected and stored frozen indefinitely (Protocol 2)and then screened against any number of preys.One such set of bait strains contains over 700 different LexA fusion proteins from our own work and from numerous other labs that use the interaction trap (R.L.F.,R.B.,A.Reymond,unpublished).Screening a protein against such a panel enables one to quickly test its ability to interact with a large number of known proteins,most of which have been characterized to some extent,and have been chosen for study because of their known or suspected involvement in some biological process.Thus,the finding of an interaction between a tested protein and a member of the panel can often lead to immediate clues about the biological function of both proteins (see Section 5).While the number of proteins in the existing panel is far less than the number of proteins in a good library,this approach does offer the advantage of screening the test protein against a set of proteins enriched for those of current interest to the biological community.It is worth noting that these proteins come from many different organisms in which they are expressed in different tissues and at different developmental stages.Thus it becomes possible to identify interacting partners that have not yet been isolated from the same species,or that are not expressed in tissues from which interaction libraries have been made.

For some proteins,this approach offers additional advantages over screening a library using a traditional two-hybrid scheme.Proteins that activate transcription when fused to LexA or another DNA-binding domain can be difficult to use in conventional interactor hunts.Though methods are available to reduce the sensitivity of the reporter genes (Durfee et al.,1993; Estojak et al.,1995; Chapter 2,3,4)it is not always possible to reduce the reporter sensitivity below the threshold of activation for some baits.Moreover,reduction in reporter sensitivity carries with it the risk that the reporters will not detect weakly interacting proteins.Furthermore,spontaneously occurring yeast mutations,for example those that increase the copy number of the bait plasmid,can increase the activating potential of weakly activating baits (R.L.F.,R.B.,A.Mendelsohn,unpublished data); such mutations are typically scored as positive in the early stages of an interactor hunt,and they are not readily detected in schemes where the specificity test is performed by removing the bait plasmid from the strain containing the prey and mating the strain with other bait strains.Thus,an alternative for proteins that activate transcription as baits,is to use them as preys to screen existing panels of baits,or even libraries of baits.Interaction mating approaches also have clear advantages for proteins that are somewhat toxic to yeast; the prey vector allows conditional expression of toxic proteins in the presence of a bait,and often the interaction can be observed as the reporters are activated even if the cells are inviable.An example of the use of interaction mating together with a large panel of bait strains to characterize a protein that both activates transcription and is toxic to yeast,Drosophila Cyclin E (Finley,Zavitz,Thomas,Richardson,Zipursky,and Brent,in prep),is discussed in Section 7.

Figure 1.Mating assay for interactions between a prey and 96 baits

Figure 1.

Top.The plate on the left holds 96 different yeast strains in patches (or colonies)that each express a different bait protein.The plate on the right holds 96 patches,each of the same yeast strain (prey strain)that expresses a protein fused to an activation domain (prey).The plate of bait strains and the plate of prey strains are each pressed to the same replica velvet and the impression is lifted with a plate containing YPD medium.After one day of growth on the YPD plate,during which time the two strains mate to form diploids,the YPD plate is pressed to a new replica velvet and the impression is lifted with a plate containing diploid selection medium and an indicator like X-Gal.Blue patches (dark spots)on the X-Gal plate indicate that the lacZ reporter is transcribed,suggesting that the prey interacts with the bait at that location.

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Protocol 2.Collecting bait (and prey)strains

Materials

Freezing media: 1:1 solution of minimal glucose media lacking appropriate amino acids (e.g.-u-h Glu for bait strains): sterile glycerol solution (65% (v/v)glycerol,0.1 M MgSO₄,25 mM Tris-HCl pH 7.4)

1.0 to 1.5 ml cryotubes

Yeast strains freshly streaked to minimal glucose plates

Sterile wooden applicator strips

Methods

1.Streak bait strains to -u-h Glu plates,or prey strains to -w Glu plates,and incubate at 30℃ for 24 to 48 hours.Yeast should be taken from the plates and frozen no more than 4 days after being streaked.

2.With a sterile wooden applicator stick,grab a dollop of yeast from the plates and inoculate 0.5 ml of freezing solution in a cryotube.Vortex lightly.This solution should have an OD₆₀₀ over 3.0.

3.Alternatively,inoculate 0.5 ml of -u-h Glu liquid media to an OD₆₀₀ less than 0.2,incubate at 30℃ with shaking until OD₆₀₀ = 1.5 to 2.0 (log phase),and add 0.25 ml of this culture to 0.25 ml of sterile glycerol solution in a cryotube.

4.Freeze by placing cryotubes in -80℃ freezer.Most strains can be recovered after up to at least two years by scraping the surface of the ice and streaking to minimal glucose plates.Avoid allowing entire contents of cryotube to thaw.

Protocol 3.Mating assay^- large scale for hundreds of different bait or prey strains.

Materials

Freshly streaked bait and prey strains (see Protocol 1)

One set of the following 150 x 15 mm plates for each test of interactions between an activation domain-tagged protein (in a prey strain)and 96 baits (bait strains): -u-h- Glu; -w Glu; YPD; -u-h-w Glu X-Gal; -u-h-w Gal/Raf X-Gal

Replica plater and sterile velvets for 150 mm diameter plates.(A replica devise can be fashioned from a box of 200 µl pipet tips by stretching a velvet over the top of the box)

96-prong device (e.g.DanKar MC-96)with 3 mm diameter flat ended metal prongs in a 96-well configuration.Similar devices can be used in 48-well configurations for use with 100 mm plates.

0.5 to 4.0 ml sterilized tubes arranged in a 96-well configurations (e.g.cluster tubes such as Costar #4411).Ideally these tube can be capped and frozen at -80℃.

-u-h Glu liquid media,see Chapter 4

-w Glu liquid media,see Chapter 4

Sterile glycerol solution (65% (v/v)glycerol,0.1 M MgSO₄,25 mM Tris-HCl pH 7.4)

Methods

1.It is most convenient to place large numbers of bait strains in a 96-well configuration (Figure 1).This can be done by inoculating 2 ml of -u-h Glu media in cluster tubes and growing to OD₆₀₀ = 1.5 to 2.0.After making plates from these cultures (see step 2 below)add an equal volume of sterile glycerol solution,cap and freeze at -80℃.

2.Use the 96-prong device,sterilized in ethanol and flame,to transfer bait strains from the culture to the center of a 150 mm -u-h Glu plate.Each plate can contain 96 different bait strains.Tens of identical plates can be made from one culture.Incubate the plates at 30℃ for 48 hours or until all bait strains have grown to colonies 5 mm in diameter.These plates can be stored at 4℃ for up to 2 months and used to inoculate another liquid culture when more plates are needed.Several positions on each plate should contain control strains with baits that activate various levels of transcription (see Section 4 and Table 1).

3.Inoculate 50 ml of -w Glu liquid media with a prey strain and grow at 30℃ with shaking to OD₆₀₀ = 1.5 to 3.0.Pour the culture into a sterile 150 mm plate,or into the sterile top from a box of 200 µl pipets,and use the 96-prong device,sterilized in ethanol and flame,to transfer the culture to -w Glu plates.On these plates,all 96 positions will contain the same prey strain.

4.Follow the replica plating procedure from Protocol 1 to combine the bait and prey strains to a YPD plate,and then after growth on the YPD plate at 30℃ for 24 hours,replica to X-Gal indicator,diploid selection plates (-u-h-w Glu X-Gal and -u-h-w Gal/Raf X-Gal)(see Figure 1 above).

5.Examine results after two days.

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3.Interaction mating assay with other yeast two-hybrid systems

In addition to the interaction trap,many other yeast two-hybrid systems have been developed (see Chapter 1 and Allen et al.,1995; Fields and Sternglanz,1994; Mendelsohn and Brent,1994,for reviews).All of these allow the analysis of individual protein-protein interactions,and permit interactor hunts to isolate new proteins that interact with a bait.In some instances plasmids or strains from one system can be used in another,but often the components are incompatible.Most often,the yeast selectable markers on the different components differ.In addition,systems that use Gal4 as the DNA binding domain cannot be used with yeast strains that have a wild-type GAL4 gene,and therefore,since the Gal4 protein is required to activate the GAL1 promoter,cannot be used with systems that use the GAL1 promoter to drive expression of the prey protein.Finally,use of interaction mating requires careful attention to the mating types of the strains and the selectable markers used to select the diploids.

4.Recording the results

Interaction between bait and prey results in the interaction phenotypes: growth of the .