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MINIREVIEWProtein–protein interactions and selection: yeast-basedapproaches that exploit guanine nucleotide-bindingprotein signalingJun Ishii1,*, Nobuo Fukuda2,*, Tsutomu Tanaka1, Chiaki Ogino2and Akihiko Kondo21 Organization of Advanced Science and Technology, Kobe University, Japan2 Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, JapanIntroductionProtein–protein interactions have fundamental roles ina variety of biological functions, and are of centralimportance for virtually every process in a living cell.Hence, many methodologies for elucidating proteininteractions have been developed during the past cou-ple of decades. To investigate interactions inside cellsunder physiological conditions, especially, yeast wouldbe a most typical organism, and various in vivo selec-tion approaches are now available.The budding yeast Saccharomyces cerevisiae is oneof the simplest unicellular eukaryotes, and is oftenused as a eukaryotic model organism for cellular andmolecular biology [1–5]. Yeast has several benefits,including the possession of eukaryotic secretorymachinery, post-translational modifications, rapid cellgrowth, and well-established and versatile genetic tech-niques. Thus, it is also used to establish technologieswith which to survey interactions of eukaryoticKeywordsguanine nucleotide-binding protein;protein-protein interaction; screening;signaling; yeast; yeast two-hybridCorrespondenceA. Kondo, Department of Chemical Scienceand Engineering, Graduate School ofEngineering, Kobe University, 1-1Rokkodaicho, Nada-ku, Kobe 657-8501,JapanFax: +81 78 803 6196Tel: +81 78 803 6196E-mail: akondo@kobe-u.ac.jp*These authors contributed equally to thiswork(Received 29 October 2009, revised 5February 2010, accepted 24 February 2010)doi:10.1111/j.1742-4658.2010.07625.xFor elucidating protein–protein interactions, many methodologies havebeen developed during the past two decades. For investigation of interac-tions inside cells under physiological conditions, yeast is an attractiveorganism with which to quickly screen for hopeful candidates using versa-tile genetic technologies, and various types of approaches are now avail-able. Among them, a variety of unique systems using the guaninenucleotide-binding protein (G-protein) signaling pathway in yeast havebeen established to investigate the interactions of proteins for biologicalstudy and pharmaceutical research. G-proteins involved in various cellularprocesses are mainly divided into two groups: small monomeric G-proteins,and heterotrimeric G-proteins. In this minireview, we summarize the basicprinciples and applications of yeast-based screening systems, using thesetwo types of G-protein, which are typically used for elucidating biologicalprotein interactions but are differentiated from traditional yeast two-hybridsystems.AbbreviationsGAP, GTPase-activating proteins; GEF, guanine nucleotide exchange factor; GPCR, guanine nucleotide-binding protein-coupled receptor;G-protein, guanine nucleotide-binding protein; Gccyto, mutated yeast Gc lacking membrane localization ability; MAPK, mitogen-activatedprotein kinase; M3R, M3muscarinic acetylcholine receptor; mRas, mammalian Ras; RRS, Ras recruitment system; SRS, Sos recruitmentsystem; Y2H, yeast two-hybrid; yRas, yeast Ras.1982 FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBSproteins. The yeast two-hybrid (Y2H) system, whichwas originally designed to detect protein–protein inter-actions in vivo by separation of a transcription factorinto a DNA-binding domain and a transcription acti-vation domain, is a typical representative of a yeast-based genetic approach [6], and numerous improvedY2H systems have been developed to overcome itspotential problems [7–14]. The utility of Y2H systemshas been demonstrated to varying degrees, involvinganalyses of comprehensive interactome networks[15–18], identification of novel interaction factors[19–22], investigations of homodimerization or hetero-dimerization [23–25], and the obtaining of conforma-tional information [26–28]. Thus, yeast is definitely anattractive organism for analyzing the interactions ofeukaryotic proteins.Guanine nucleotide-binding proteins (G-proteins)are signaling molecules that are highly conservedamong various eukaryotes, and that engage in a widevariety of cellular processes [3,29]. They switch froman inactive to an active state by exchanging a GDPmolecule for GTP, and they return to the inactive stateby hydrolyzing GTP to GDP. They are divided intotwo main groups: small monomeric G-proteins andheterotrimeric G-proteins [29]. Because eukaryoticyeast cells have both types of G-protein, but are not ascomplicated as higher eukaryotes, yeast has been usedas the model organism for the study of G-proteinmachinery [30–32]. Much knowledge of G-proteinsignaling in yeast has been accumulated and used tostudy cellular processes, including protein interactions.In this minireview series highlighting the methodolo-gies for elucidating protein–protein interactions, theother two minireviews by K. Tomizaki et al. [33] andM. Umetsu et al. [34] deal with array based-technolo-gies for detecting protein interactions in vitro, and con-structive approaches to the generation of novelbinding proteins on the basis of tertiary structuralinformation, respectively. In this first minireview, wefocus on and summarize the unique technologies usedto exploit yeast G-protein signaling, which are com-monly used for the exploration of biological proteininteractions under physiological in vivo conditions butare distinguishable from conventional Y2H systemsfrom a scientific and engineering perspective.Ras signaling-based screening systemsfor protein–protein interactionsSmall monomeric G-protein signaling in yeastSmall monomeric G-proteins, such as Ras and Ras-likeproteins, are found mainly at the inner surface of theplasma membrane as monomers. They function as GTP-ases on their own, and are involved in controlling cellproliferation, differentiation, and apoptosis [29]. TheRas proteins are, in addition, necessary for the comple-tion of mitosis and the regulation of filamentous growth[35]. In the yeast S. cerevisiae, growth and metabolismin response to nutrients, particularly glucose, is regu-lated to a large degree by the Ras–cAMP pathway[30,31,35]. Ras proteins activate adenylate cyclase,which synthesizes cAMP, and the increase in cytosoliccAMP levels activates the cAMP-dependent proteinkinase, which has an essential role in the progressionfrom the G1phase to the S phase of the cell cycle.Owing to their intrinsically slow GTPase and GTP–GDP exchange activities, Ras proteins are strictlycontrolled by two classes of regulatory proteins:GTPase-activating proteins (GAPs), and guanine nucle-otide exchange factors (GEFs) [35]. RasGAPs, whichact as negative regulators of Ras–cAMP signaling byaccelerating hydrolysis of GTP to GDP on Ras pro-teins, can stimulate the GTPase activity of Ras proteinsto terminate the signaling event. On the other hand,RasGEFs, which contain Cdc25p and Sdc25p in yeast,stimulate the exchange of GDP for GTP on Ras pro-teins. The stimulated RasGEFs activate the Ras–cAMPsignaling pathway. Whereas Cdc25p is essential in mostgenetic backgrounds, Sdc25p is dispensable and isnormally expressed only during nutrient depletion or innonfermentative situations. Through its role in regulat-ing cAMP levels, Cdc25p is involved in fermentativegrowth, nonfermentative growth, cell cycling, sporula-tion, and cell size regulation. Thus, the main positiveregulator of yeast Ras proteins is Cdc25p.Characteristic aspects of Ras signaling-basedscreening systemsRas signaling-based yeast screening systems for theexploration of protein interaction partners allow forpositive selection of interactions between soluble cyto-solic proteins or between a soluble protein and a hydro-phobic membrane protein through the restoration ofRas signaling [36–38]. These systems employ the cdc25yeast strain, which is deficient in Ras signaling andregains it with the presence of interacting protein pairs.The machinery of intrinsic cell survival and prolif-eration of Ras signaling is utilized for the readout.Interactions of proteins of interest, including transcrip-tional activators or repressors that might induce tran-scription of a reporter or disable vital functions inyeast, can be investigated because of the restitution ofRas signaling on the plasma membrane but the absenceof reconstitution of DNA-binding transcription factorsJ. Ishii et al. Screening systems using yeast G-protein signalingFEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1983in the nucleus. The restricted cell survival with Rassignaling-based selection is suitable for screening largelibraries (Table 1), although the method has compara-tive difficulty in accurately assessing relative interactionstrengths.Sos recruitment systemThe Sos recruitment system (SRS) was initiallyreported as a Ras signaling-based screening system,and it takes advantage of the fact that the humanRasGEF protein, hSos, can substitute for the GEF ofyeast endogenous Ras (yRas) protein, Cdc25p, toallow cell survival and proliferation (Fig. 1A) [36]. Inthe SRS, a yeast variant strain that has the tempera-ture-sensitive cdc25-2 allele is required. The cdc25-2strain cannot survive at a restrictive temperature(36 °C), owing to a lack of function of Cdc25p toactivate Ras signaling, whereas it can grow at alower temperature (25 °C). One protein should beTable 1. Protein–protein interaction pairs identified or applied in G-protein signaling-based systems.Interaction pair ReferenceSos recruitment systemc-Jun–JDP1 or c-Jun–JDP2 (Jun dimerization proteins) [36]c-Jun–Fra-2, c-Jun–FosB or c-Jun–c-Fos (Fos) [36]p110–p85 [36]BRCA1 (breast cancer susceptibility gene 1)–CtIP (CtBP-interacting protein) [84]Sox9–PKA-Ca (protein kinase A catalytic subunit a) [85]VDAC1 (voltage-dependent anion-selective channel 1)–Tctex1 (t-complex testis expressed-1) [86]VDAC1–PBP74 (peptide-binding protein 74) [86]p5–p5 [87]GABAAreceptor c2 subunit–GODZ (Golgi-specific DHHC zinc finger protein) [88]IRS-1 (insulin receptor substrate 1)–HDAC2 (histone deacetylase 2) [89]p73–PKA-Cb (protein kinase A catalytic subunit b) [90]Truncated ERb (estrogen receptor b)–truncated ERb [91]HBO1 (histone acetyltransferase binding to ORC-1)–PR (progesterone receptor) [92]CMV 1a (cucumber mosaic virus 1a)–TIP1 or CMV 1a–TIP2 (tonoplast intrinsic proteins) [93]TRAF2 (tumor necrosis factor receptor associated factor 2)–Smurf2 (SMAD-specific E3 ubiquitin protein ligase 2) [94]EF3 (elongation factor 3)–Cch1 (high-affinity calcium channel) [95]Ras recruitment systemc-Jun–c-Fos [38]p110–p85 [38]JDP2–C ⁄ EBPc (CCAAT ⁄ enhancer-binding protein) [38]Pac65 (Pac2; p21-activated kinase 2)–Rac1 mutant [38]Pac65–Grb2 (growth factor receptor-binding protein 2) [38]Sos (son of sevenless)–Grb2 (growth factor receptor-bound protein 2) [38]Truncated EGFR (epidermal growth factor receptor) fused with M-Jun–truncated EGFR fused with M-Fosa[39]Glucocorticoid receptor NR3C1–ZKSCAN4 (zinc finger with KRAB and SCAN domains 4) [40]PacR (Pac2 regulatory domain)–Chp (Cdc42Hs homologous protein) [96]b-Catenin–CBP (CREB-binding protein) [97]JNK (c-Jun N-terminal kinase)–IKAP (IjB kinase complex-associated protein) [98]ErbB (EGFR)–Grb2 [99]c-Myc–Krim-1A or c-Myc–Krim-1B (Krab box proteins interacting with Myc) [100]RalA (Ras-like protein A)–ZONAB (ZO-1-associated nucleic acid-binding protein) [101]Yeast–mammal chimeric Ga systemSnf1 (AMP-activated protein kinase)–Snf4 (regulatory subunit of Snf1 kinase complex) [78]Raf–Ras mutant [78]Gc interfering system (G-protein fusion system)Syntaxin 1a–nSec1 (neuronal Sec1) [79]FGFR3 (fibroblast-derived growth factor receptor 3)–SNT-1 (FGFR signaling adaptor) [79]Gc recruitment systemZZ domain or Z variants (Z domain: B domain mutant derived from protein A)–Fc part (of human IgG) [80]Competitor-introduced Gc recruitment systembZZ domain or Z variants–Fc part [102]aThis system is to be used for monitoring receptor tyrosine kinase activity.bThis system is to be used for selective isolation of affinity-enhanced variants.Screening systems using yeast G-protein signaling J. Ishii et al.1984 FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBSmembrane-associated or be attached to an inner mem-brane translocating signal involved in myristoylationand palmitoylation, and the other protein should besoluble and be fused to hSos to prevent false autoacti-vation by membrane localization of hSos. Only whenthe membrane-localized protein interacts with thehSos fusion protein will hSos be recruited to theplasma membrane and yeast Ras signaling be rescued.As a consequence, the temperature-sensitive mutantthat expresses interacting protein pairs can grow at36 °C.Using the SRS, a novel repressor that interacts withthe c-Jun subunits of AP-1 and represses its activity wasisolated [36] (Table 1). AP-1 is a transcription factorthat binds to DNA through a leucine zipper motif.Thus, the ability of the SRS to identify transcriptionalregulators has been reasonably well established, owingto the membrane-localized interaction, unlike conven-tional Y2H systems based on the reconstitution ofDNA-binding transcription factors in the nucleus.Ras recruitment systemThe Ras recruitment system (RRS), using mammalianRas (mRas), was later developed as an improved ver-sion of the SRS [38]. The RRS has the advantages ofthe SRS without some of its limitations. For example,the RRS permits more strict selection, owing to thestringent requirement for membrane localization ofmRas, can eliminate the isolation of predictableRas false positives, owing to the introduction ofmRasGAP, and can more broadly detect interactions,owing to the relatively small size of Ras as comparedwith hSos [37,38]. The RRS is based on the absoluterequirement that Ras be localized to the plasma mem-brane for its function (Fig. 1B). In the RRS, mRaslacking its CAAX motif for localization to the plasmamembrane, but possessing a constitutively active muta-tion, is used as a substitute for hSos, and mRasGAP isadditionally expressed. The membrane localization ofmRas through protein–protein interactions in a cdc25-2yeast strain results in the activation of its downstreameffector, adenylyl cyclase, and restores its growth abil-ity. In an initial report, the usefulness of the RRS wasconfirmed by practical screening of a cDNA libraryof 500 000 independent transformants [38] (Table 1).Later, the RRS was applied to detect the activity andinhibition of a dimerization-dependent receptor tyrosinekinase and to identify an interacting pair of human glu-cocorticoid receptors from a HeLa cell cDNA library[39,40] (Table 1).Pheromone signaling-based screeningsystemsHeterotrimeric G-protein signaling in yeastAs peripheral membrane proteins, heterotrimericG-proteins associate with the inner side of the plasmamembrane. Heterotrimeric G-proteins consisting ofthree subunits, Ga,Gb, and G c, exist in various sub-families and are widely conserved among eukaryoticspecies. They transduce messages from ubiquitousreceptors, which control important functions such astaste, smell, vision, heart rate, blood pressure, neuro-transmission, and cell growth [29]. Yeast has only twotypes of heterotrimeric G-protein: pheromone signaling-related and nutrient signaling-related [30–32]. Nutrientsignaling is profoundly and intricately linked to Rassignaling [30,31], whereas the pheromone signalingpathway is connected to mating processes [32].The yeast pheromone signaling-related G-proteincomprises three subunits, Gpa1p, Ste4p, and Ste18p,which structurally correspond to mammalian Ga,Gb,(b)(a)AB(a) (b)Fig. 1. Schematic illustration of Ras signaling-based screening sys-tems. (A) The SRS system using the human RasGEF protein, hSos.(a) Noninteracting protein pairs are unable to activate the yeast Rassignaling pathway, and are also unable to drive cell growth. (b)Interacting protein pairs bring hSos to the plasma membrane,where it can exchange GDP for GTP of yeast endogenous Ras. Theactive form of GTP-bound yRas allows cell survival. (B) The RRSsystem using a constitutively active mutant of mammalian Ras lack-ing the lipid modification motif (mRas). (a) Noninteracting proteinpairs are unable to activate the yeast Ras signaling pathway, andare also unable to drive cell growth. (b) Interacting protein pairsbring mRas to plasma membrane, where it can activate the yeastRas signaling pathway. Ras signaling allows cell survival. X and Yrepresent test proteins for interaction analysis.J. Ishii et al. Screening systems using yeast G-protein signalingFEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1985and Gc, respectively [32]. The heterotrimeric G-proteinis divided into two key components from the perspec-tive of structure and function. Ga (Gpa1p) is associ-ated with the intracellular plasma membrane throughdual lipid modifications of myristoylation and palmi-toylation in the N-terminus [41], whereas the Gbcdimer (the Ste4p–Ste18p complex) is also localized tothe inner leaflet of the plasma membrane through duallipid modifications of farnesylation and myristoylationin the C-terminus of Ste18p, and the formation of acomplex between Ste4p and lipidated Ste18p [41,42].They form part of the signaling cascade activated byG-protein-coupled receptors (GPCRs), and mediatecellular processes in mating in response to the presenceof pheromone (Fig. 2A).The yeast haploid a-cell has a sole pheromone recep-tor, Ste2p, which is classified as a GPCR, and thetridecapeptide a-factor functions as a pheromone andbinds to the Ste2p receptor on the cell surface [32].The heterotrimeric G-proteins are closely associated(a) (b)BAFig. 2. Yeast pheromone signaling pathway and its utilization for a GPCR biosensor. (A) Schematic illustration of the pheromone signalingpathway. (a) In the absence of a-factor, heterotrimeric G-protein is unable to activate the pheromone signaling pathway. (b) Binding of a-fac-tor to Ste2p receptor activates the pheromone signaling pathway through heterotrimeric G-protein. Sequestered Ste4p–Ste18p complex fromGpa1p activates effectors and subsequent kinases that constitute the MAPK cascade, resulting in phosphorylation of Far1p and Ste12p.Phosphorylation of Far1p leads to cell cycle arrest. Phosphorylation of Ste12p induces global changes in transcription. Sst2p stimulateshydrolysis of GTP to GDP on Gpa1p, and helps to inactivate pheromone signaling. (B) Schematic illustration of typical genetic modificationsenabling the pheromone signaling pathway to be used as a biosensor to represent activation of GPCRs. Intact or chimeric Gpa1p can trans-duce the signal from yeast endogenous Ste2p or heterologous GPCRs that are expressed on the yeast plasma membrane. Transcriptionmachineries that are closely regulated by the phosphorylated transcription factor, Ste12p, are used to detect activation of pheromone signal-ing with various reporter genes. FAR1, SST2 and STE2 are often disrupted (shown in light gray) to prevent growth arrest, improve ligandsensitivity, and avoid competitive expression of yeast endogenous receptor.Screening systems using yeast G-protein signaling J. Ishii et al.1986 FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBSwith the intracellular domain of the Ste2p receptor,and the pheromone-bound receptor is conformational-ly changed and activates the G-protein [43]. Gpa1pis thereby changed from an inactive GDP-bound stateto an active GTP-bound state and dissociates theSte4p–Ste18p complex. Subsequently, the dissociatedSte4p–Ste18p complex binds to effectors throughSte4p, and then activates the mitogen-activated proteinkinase (MAPK) cascade [44,45]. The Ste5 scaffold pro-tein binds to the kinases of the MAPK cascade andbrings them to the plasma membrane. The concentra-tion of the bound kinases on the membrane possiblypromotes amplification of the signal [46,47]. As a con-sequence, the activated pheromone signaling leads tothe phosphorylation of Far1p and the transcriptionfactor Ste12p. These phosphorylated proteins triggercell cycle arrest in G1[48–50] and global changes intranscription [51,52]. FUS1 gene expression is repre-sentative of the drastic changes in transcription inresponse to pheromone signaling [53,54]. As a princi-pal negative regulator, the Gpa1-specific GAP Sst2p, amember of the regulator of G-protein signaling family,is also involved in the pathway [55,56].Pheromone signaling-based screeningsystems – ligand–GPCR orGPCR–G-protein interactionsBackground of pheromone signaling-basedscreening systemsGPCRs constitute the largest family of integral mem-brane proteins, and have a variety of biological func-tions. They are the most frequently addressed drugtargets, and modulators of GPCRs form a key areafor the pharmaceutical industry, representing nearly30% of all Food and Drug Administration-approveddrugs [57,58]. Yeast permits the functional expressionof various heterologous GPCRs and other signalingmolecules such as G-proteins. Yeast also facilitatesversatile genetic techniques for screening and quantifi-cation. Therefore, it offers opportunities to establishfundamental technologies for drug discovery or basicmedicinal study [59,60]. Yeast-based screening systemsexploiting pheromone GPCR signaling enable theanalysis of several interactions, including not onlyprotein–protein but also ligand–receptor and receptor–protein interactions. These systems can recognize theon–off switching of a signal, such as the binding of anagonist ⁄ antagonist to a receptor, and critical mutationsinvolved in ligand-dependent or constitutive acti-vation ⁄ inactivation of signaling molecules. In addi-tion, assays can be performed at the yeast optimumtemperature of 30 °C, unlike with Ras signaling-basedsystems, which require the incubation of yeast cellsat suboptimal temperatures (25 and 36 °C), and themonitoring or discrimination of the signaling changesthrough quantitative and survival readouts. Hence,they have been applied in various experiments, includ-ing target identification, ligand screening, and receptormutagenesis.Pheromone signaling as a biosensor forunderstanding GPCRsGPCRs have a common tertiary structure, composed ofseven hydrophobic integral membrane domains, and themechanism of signaling that is mediated by heterotri-meric G-proteins is also conserved between yeast andmammalian cells. This has led to the construction ofingenious systems that provide for the mutual exchangeof signals between heterologous GPCRs and yeastG-proteins in yeast without generating dysfunctions.With versatile screening techniques, yeast can be usedas a sensor to detect the initiation of GPCR-associatedsignaling [59,60]. Briefly, in wild-type yeast a-cells,Ste2p receptor or mammalian receptors can activate theyeast pheromone signaling pathway via intracellularheterotrimeric G-proteins, including the native form oran engineered form of Gpa1p, in response to ligandbinding. The activated pheromone signals cause cellcycle arrest and transcription activation, which areexploited as signaling readouts (Fig. 2A,B). Thesebiosensing techniques have been established in yeastwith engineered pheromone signaling, and numerouscharacteristics of pheromone signaling moleculeshave been successfully elucidated [43–45, 47–50, 53–55].Moreover, pheromone signaling-related molecules, suchas Ste2p receptor, G-proteins, and peptidic a-factorpheromone, have been extensively mapped with muta-genesis techniques, demonstrating their usefulness forscreening huge libraries and for identification of impor-tant domains or amino acids [61–66].Bioassay and transcriptional assay for signalingdetectionThe arrest of the cell cycle completely prevents cellgrowth during signaling. Monitoring of cell densities inliquid media with or without pheromones can distin-guish signaling on the basis of delay of entry into thelogarithmic growth phase. The agar diffusion bioassay(halo assay), in which cells are mixed with unsolidifiedfresh agar medium in which pheromone-spotted paperfilter disks are placed, can also discriminate signal-ing by showing cleared-out areas around the disks,J. Ishii et al. Screening systems using yeast G-protein signalingFEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1987forming halos, owing to the robust inhibition of cellgrowth (the halos may look blacked out on a mono-chromatic figure) [55,62,63,66,67].On the other hand, the use of transcriptionalchanges that are closely regulated by the signalingmakes possible versatile procedures for detection. TheFUS1 gene, which is engaged in drastic augmentationof the transcription level responding to the signal, iscommonly taken as a reflector of signaling and is fusedwith various reporter genes associated with growthand photometry. Auxotrophic or drug-resistant repor-ter genes, such as HIS3 or hph, are generally used forselection, and are suitable for screening large-scalelibraries [66–68]. Colorimetric, luminescent and fluores-cent reporters, such as lacZ, luc,orGFP, are usuallyused for numerical conversion and are appropriate forrelative and quantitative assessment of signaling levels[61–64,66–68].Gene disruption for system modificationThe arrest of the cell cycle caused through phosphory-lation of Far1p allows for the examination of phero-mone signaling [55,59,60,62,63,66,67]. However, thismakes growth reporter genes for positive selection,such as HIS3, useless for the detection of signaling,owing to stagnation of cell growth [66,67], whereas thesynchronization of the cell cycle in G1arrest providesuniform levels of expression of reporter genes such asGFP for each cell [69]. For that reason, FAR1 is usu-ally disrupted in positive selection screens using growthselection (Fig. 2B). Because the far1D strain neverinduces cell cycle arrest, it can be used in growth selec-tion to screen for positive clones in response to phero-mone signaling, which is represented by the expressionof the HIS3 reporter gene on histidine-defective plates[66,67]. At the same time, it has been reported that thearrest of the cell cycle causes the drastic dropout ofepisomal plasmids, resulting in a serious problem whenthe library is screened and the target plasmids are col-lected, and hence the disruption of FAR1 could signifi-cantly improve plasmid retention rates [69].Accordingly, disruption of FAR1 is required for posi-tive growth screening.The SST2-deficient strategy is widely used in utiliz-ing pheromone signaling as a sensor, owing to hyper-sensitivity for ligand binding [59,60,63,67,69]. SST2gene encodes the Gpa1-specific GAP that stimulateshydrolysis of GTP to GDP on Gpa1p and helps in theinactivation of pheromone signaling. Removal of Sst2pfunction causes a considerable decrease in GTPaseactivity for Gpa1p, and makes the conversion of GTPto GDP difficult, owing to a lack of competence ofGTPase activity (Fig. 2B). The loss of SST2 could pro-vide supersensitivity, even to a 250–10 000-fold lowerconcentration of a -factor [67]. However, a relativelyhigh background signal of the sst2D strain, especiallywhen grown in rich medium such as YPD, has beenconfirmed in the absence of a-factor pheromone by atranscription assay using the FUS1–GFP reporter gene[69]. Although the SST2-deficient strategy is a powerfultechnology for experiments requiring high sensitivity, itdoes not necessarily produce the best signal-to-noiseratio. Accordingly, choosing the correct situation forusing Sst2p is required for each experiment. In addition,STE2 is often disrupted, to avoid competitive expres-sion of yeast endogenous receptor [59–64,66,69].Expression of heterologous GPCRsMany heterologous GPCRs containing adrenergic,muscarinic, serotonin, neurotensin, somatostatin, olfac-tory and many other receptors have been successfullyexpressed in yeast, and the feasibility of yeast-basedGPCR screening systems has been demonstrated[59,60,68,70–75]. Yeast Gpa1p, which is equivalent toGa, shares high homology, in part, with human Gaiclasses, and a number of GPCRs of human and otherspecies are able to interact with Gpa1p and activatepheromone signaling in yeast [73–75]. Many otherhuman GPCRs can also function as yeast signalingmodulators as a result of various genetic modifications,including one in which chimeric Gpa1p systems(so-called ‘transplants’) have only five amino acids inthe C-terminus of Gpa1p substituted for those ofhuman Ga subunits, including the Gai ⁄ o,GasandGaqfamilies (Fig. 2B) [71]. Indeed, these transplantshave allowed functional coupling of serotonin, mus-carinic, purinergic and many other receptors to theyeast pheromone pathway [71–73,76].The rat M3muscarinic acetylcholine receptor hasbeen used for rapid identification of functionally criti-cal amino acids, with random mutagenesis of the entiresequence [72]. In this system, the CAN1 reporter genecoding for arginine–canavanine permease was inte-grated into the locus of a pheromone response gene inyeast cells whose endogenous CAN1 gene was deleted,and the recombinant strain expressed Can1p inresponse to ligand-dependent signaling. Owing to thecytotoxicity of canavanine caused by Can1p expres-sion, recombinant strains with inactivating mutationsin the receptor can survive on agar media containingcanavanine and receptor-specific agonists. The recov-ered mutant M3muscarinic acetylcholine receptors inthis system also show substantial functional impair-ments in transfected mammalian cells, and the utilityScreening systems using yeast G-protein signaling J. Ishii et al.1988 FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBSof the yeast-based procedure for GPCR mutagenesishas been proven.Human formyl peptide receptor like-1, which wasoriginally identified as an orphan GPCR, has beenused to isolate agonists for GPCRs of unknown func-tion [77]. Histidine prototrophic selection by theFUS1–HIS3 reporter gene was performed with secretedrandom tridecapeptides as a library and a mamma-lian ⁄ yeast hybrid Ga subunit which allows functionalcoupling with the receptor. As a result, surrogateagonists as peptidic candidates have been successfullyscreened, and the promoted activation of formylpeptide receptor like-1 expressed in human cells hasbeen validated with synthetic versions of the peptides.Pheromone signaling-based screeningsystems – protein–protein interactionsYeast–mammal chimeric Ga systemMedici et al. [78] constructed an intelligent system foranalysis of protein–protein interactions by managingheterotrimeric G-protein signaling in yeast (Fig. 3A).They initially found that a fusion protein between theyeast Ste2p receptor lacking the last 62 amino acids ofthe cytoplasmic tail and the full-length Gpa1p trans-duced the signal in response to the binding of a-factorin cells devoid of both endogenous STE2 and endoge-nous GPA1. Subsequently, a yeast–mammal chimericGa composed of the N-terminal 362 amino acids ofGpa1p and the C-terminal 128 amino acids of rat Gaswas prepared. The chimeric Ga is able to interact withthe yeast Gbc complex, but is not able to interact withthe yeast Ste2p receptor, and it was fused to the trun-cated Ste2p receptor. Although a gpa1D yeast strainharboring the yeast–rat chimeric Ga does not respondto pheromone, a ste2D gpa1D yeast strain expressingthe Ste2p–Gpa1p–Gasfusion protein that is covalentlylinked to Ste2p and the chimeric Ga displayed a strongpheromone response in the presence of a-factor. Theseresults suggest that the specific interaction of the recep-tor with the C-terminus of Ga is necessary to bringthe two proteins into close proximity. This hypothesiswas applied to the analysis of protein–protein inter-actions. It was demonstrated that the interaction ofGpa1p–Gasfused to protein X and Ste2p receptorfused to protein Y permitted pheromone responsesignaling through the contact between Ste2p andGpa1p–Gas, using the interaction between Snf1 andSnf4, which form a kinase complex regulating transcrip-tional activation in glucose derepression, or betweenRaf and the constitutively active form of Ras (Table 1).In this system, a gpa1D haploid strain harboring theplasmid, which complements Gpa1p function to captureSte4p/Ste18p subunits, or a GPA1 ⁄ gpa1 D diploid yeaststrain was used to avoid lethality by spontaneous signal-ing from the liberated Ste4p ⁄ Ste18p subunits.Gc interfering systemThe Gc interfering system (it was called a G-proteinfusion system in the original literature) has been devel-oped to monitor integral membrane protein–proteininteractions and to screen for negative mutants withloss of the interaction capacity (Fig. 3B) [79]. Theyeast Gc -subunit Ste18p was genetically fused to theC-terminus of cytoplasmic protein X, and the pro-tein X–Gc fusion protein and integral membraneprotein Y in its native form were coexpressed in aste18D strain. The interaction between protein X–Gcand protein Y inhibits pheromone signaling throughthe Gbc complex, in spite of the presence of a-factor,whereas a lack of interaction between protein X andprotein Y normally leads to signaling. This event mightbe attributed to the fact that restrictive localization orstructural interruption by trapping of the Gbc complexat the position of protein Y on the membrane disturbsthe contact with its subsequent effector. In one exam-ple, interactions of attractive drug target candidates,syntaxin 1a and nSec1 or fibroblast-derived growth fac-tor receptor 3 and SNT-1, were monitored, and nSec1mutants that lost the ability to bind to syntaxin 1a weresuccessfully identified by taking advantage of growtharrest induced through the protein–protein interaction[79] (Table 1).Gc recruitment systemThe above-described systems for analysis of protein–protein interactions using pheromone signaling areproven techniques for selecting target proteinsinvolved with membrane proteins. However, theymight generate relatively high background signals,making them unfavorable for screening candidates bygrowth selection, because the machinery for distin-guishing interactions does not always ensure com-plete inactivation of signaling in the presence ofpheromone.The Gc recruitment system has recently been devel-oped using the pheromone signaling pathway, and is adependable system that completely eliminates back-ground signals for noninteracting protein pairs in thepresence of pheromone (Fig. 3C) [80]. This system canbe used to investigate cytosolic–cytosolic or cytosolic–membrane protein interactions. A yeast strain with amutated Gc lacking membrane localization abilityJ. Ishii et al. Screening systems using yeast G-protein signalingFEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1989(Gccyto) should be prepared by deletion of the duallipid modification sites in the C-terminus of Ste18p,because yeast pheromone signaling strictly requiresthe localization of the Gbc complex to the plasmamembrane [41,42]. The release of Ste18p into thecytosol eliminates the signaling ability mediated bythe Ste4p–Ste18p complex [41], and this techniquetherefore leads to absolute interruption of backgroundABC(a) (b)(a) (b)(a) (b)Fig. 3. Schematic illustration of pheromone signaling-based screening systems for protein–protein interaction analysis. (A) The yeast–mammal chimeric Ga system uses chimeric Gpa1p, which is able to interact with the yeast Gbc complex, but not with the yeast Ste2preceptor. Chimeric Gpa1p is fused to protein X, and yeast Ste2p receptor is fused to protein Y. (a) Noninteracting protein pairs areunable to activate the pheromone signaling pathway. (b) Interacting protein pairs bring Ste2p and chimeric Gpa1p into close proximity,and permit physical contact between the two, resulting in activation of pheromone signaling. (B) The Gc interfering system can screenfor negative mutants that do not interact. Ste18p genetically fused to the C-terminus of cytoplasmic protein X and integral membraneprotein Y are coexpressed in a ste18D strain. (a) Noninteracting protein pairs are able to activate the pheromone signaling pathway.(b) Interacting protein pairs are unable to activate the pheromone signaling pathway, owing to the interruption of contacts between theGbc complex and its effector. (C) The Gc recruitment system can completely eliminate background signals for noninteracting pro-tein pairs. Mutated Ste18p lacking membrane localization fused to cytoplasmic protein X and membrane-associated protein Y arecoexpressed in a ste18D strain. (a) Noninteracting protein pairs completely lack pheromone signaling, owing to the release of the Ste4p–Ste18pcomplex into the cytosol. (b) Interacting protein pairs restore signaling, owing to the recruitment of the Gbc complex onto the plasmamembrane.Screening systems using yeast G-protein signaling J. Ishii et al.1990 FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBSsignals. One test protein must be soluble and fusedwith Gccytoto be expressed in the cytosol but not themembrane, whereas the other may be soluble butshould have an added lipid modification site to allowassociation with the inner leaflet of plasma membrane,or it may be an intrinsically hydrophobic integralmembrane protein or lipidated element of a mem-brane-associated protein. Consequently, when thecytosolic protein X–Gccytofusion protein and themembrane-associated protein Y are expressed in aste18D haploid strain in the presence of a-factor phero-mone, the interaction between protein X and protein Yrestores signaling, owing to the recruitment of the Gbccomplex onto the plasma membrane, which can bemonitored, but a lack of interaction between protein Xand protein Y results in no background signaling.In an original report, the ZZ domain derived fromprotein A of Staphylococcus aureus and the Fc portionof human IgG, which are both soluble proteins, wereused as a model interaction pair (Table 1). The ZZdomain is a tandemly repeated Z domain that binds tohuman Fc protein and displays higher affinity than aZ domain monomer [81]. The interaction between theZZ domain with an attached dual lipidation motif inits C-terminus and Fc fused to the C-terminus ofGccytowas easily detected with a transcriptional assayusing the pheromone response FIG1 promoter and aGFP reporter gene or a halo bioassay by growtharrest, whereas background signals from noninteract-ing pairs were never observed, owing to the loss oflocalization of the yeast Gbc complex at the plasmamembrane.The wild-type and two variants of the Z domainthat each possess a single mutation and exhibit differ-ent affinity constants were expressed as additionalinteraction pairs for the Fc fusion protein [82]. Allvariants with a wide range of affinity constants, from8.0 · 103to 6.8 · 108m)1[83], were clearly detectable,and moreover, the relatively faint interaction with anaffinity constant of 8.0 · 103m)1was successfullydetected because of the complete elimination of back-ground signal for noninteracting pairs (Table 1). Sur-prisingly, a logarithmic proportional relationshipbetween affinity constants and fluorescence intensitiesmeasured by the transcriptional assay was observed,suggesting that this approach may facilitate the rapidassessment of affinity constants.Finally, the Gc recruitment system has morerecently been improved by the expression of a thirdcytosolic protein that competes with the candidate pro-tein [102]. The competitor-introduced Gc recruitmentsystem could specifically isolate only affinity-enhancedvariants from libraries containing a large majority oforiginal proteins, clearly indicating the applicability ofthis new approach to directed evolution.Concluding remarksYeast-based approaches with the G-protein signalingmachineries presented here are remarkably useful forthe detection and screening of interactions of proteinsinvolved in various biological processes. These systemsare essentially comparable to the Y2H systems thathave been predominantly used to screen protein–pro-tein interaction partners from large-scale libraries andto estimate the relative strengths of interactions, butare additionally able to detect activation or inactiva-tion associated with the switching machinery of signal-ing molecules, such as major pharmaceutical targets ofGPCRs. Yeast-based and signaling-mediated screeningsystems are obviously powerful and practical toolswith which to quickly screen for possible candidates.In the future, we can be sure that they will beimproved, with more powerful and user-friendlyadvanced modifications, and will be widely applied tovarious fields, such as protein engineering.AcknowledgementsThis work was supported in part by a ResearchFellowship for Young Scientists from the Japan Societyfor the Promotion of Science and a Special Coordi-nation Fund for Promoting Science and Technology,Creation of Innovation Centers for Advanced Inter-disciplinary Research Areas (Innovative BioproductionKobe), from the Ministry of Education, Culture, Sports,Science and Technology (MEXT), Japan.References1 Sychrova´H (2004) Yeast as a model organism to studytransport and homeostasis of alkali metal cations.Physiol Res 53, S91–S98.2 Lushchak VI (2006) Budding yeast Saccharomycescerevisiae as a model to study oxidative modificationof proteins in eukaryotes. 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