Báo cáo khoa học: Protein–protein interactions and selection: yeast-based approaches that exploit guanine nucleotide-binding protein signaling doc

MINIREVIEW
Protein–protein interactions and selection: yeast-based
approaches that exploit guanine nucleotide-binding
protein signaling
Jun Ishii
1,
*, Nobuo Fukuda
2,
*, Tsutomu Tanaka
1
, Chiaki Ogino
2
and Akihiko Kondo
2
1 Organization of Advanced Science and Technology, Kobe University, Japan
2 Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Japan
Introduction
Protein–protein interactions have fundamental roles in
a variety of biological functions, and are of central
importance for virtually every process in a living cell.
Hence, many methodologies for elucidating protein
interactions have been developed during the past cou-
ple of decades. To investigate interactions inside cells
under physiological conditions, especially, yeast would
be a most typical organism, and various in vivo selec-
tion approaches are now available.
The budding yeast Saccharomyces cerevisiae is one
of the simplest unicellular eukaryotes, and is often
used as a eukaryotic model organism for cellular and
molecular biology [1–5]. Yeast has several benefits,
including the possession of eukaryotic secretory

machinery, post-translational modifications, rapid cell
growth, and well-established and versatile genetic tech-
niques. Thus, it is also used to establish technologies
with which to survey interactions of eukaryotic
Keywords
guanine nucleotide-binding protein;
protein-protein interaction; screening;
signaling; yeast; yeast two-hybrid
Correspondence
A. Kondo, Department of Chemical Science
and Engineering, Graduate School of
Engineering, Kobe University, 1-1
Rokkodaicho, Nada-ku, Kobe 657-8501,
Japan
Fax: +81 78 803 6196
Tel: +81 78 803 6196
E-mail:
*These authors contributed equally to this
work
(Received 29 October 2009, revised 5
February 2010, accepted 24 February 2010)
doi:10.1111/j.1742-4658.2010.07625.x
For elucidating protein–protein interactions, many methodologies have
been developed during the past two decades. For investigation of interac-
tions inside cells under physiological conditions, yeast is an attractive
organism 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 guanine
nucleotide-binding protein (G-protein) signaling pathway in yeast have
been established to investigate the interactions of proteins for biological

study and pharmaceutical research. G-proteins involved in various cellular
processes are mainly divided into two groups: small monomeric G-proteins,
and heterotrimeric G-proteins. In this minireview, we summarize the basic
principles and applications of yeast-based screening systems, using these
two types of G-protein, which are typically used for elucidating biological
protein interactions but are differentiated from traditional yeast two-hybrid
systems.
Abbreviations
GAP, GTPase-activating proteins; GEF, guanine nucleotide exchange factor; GPCR, guanine nucleotide-binding protein-coupled receptor;
G-protein, guanine nucleotide-binding protein; Gc
cyto
, mutated yeast Gc lacking membrane localization ability; MAPK, mitogen-activated
protein kinase; M3R, M
3
muscarinic acetylcholine receptor; mRas, mammalian Ras; RRS, Ras recruitment system; SRS, Sos recruitment
system; Y2H, yeast two-hybrid; yRas, yeast Ras.
1982 FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS
proteins. The yeast two-hybrid (Y2H) system, which
was originally designed to detect protein–protein inter-
actions in vivo by separation of a transcription factor
into a DNA-binding domain and a transcription acti-
vation domain, is a typical representative of a yeast-
based genetic approach [6], and numerous improved
Y2H systems have been developed to overcome its
potential problems [7–14]. The utility of Y2H systems
has been demonstrated to varying degrees, involving
analyses 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 an
attractive organism for analyzing the interactions of
eukaryotic proteins.
Guanine nucleotide-binding proteins (G-proteins)
are signaling molecules that are highly conserved
among various eukaryotes, and that engage in a wide
variety of cellular processes [3,29]. They switch from
an inactive to an active state by exchanging a GDP
molecule for GTP, and they return to the inactive state
by hydrolyzing GTP to GDP. They are divided into
two main groups: small monomeric G-proteins and
heterotrimeric G-proteins [29]. Because eukaryotic
yeast cells have both types of G-protein, but are not as
complicated as higher eukaryotes, yeast has been used
as the model organism for the study of G-protein
machinery [30–32]. Much knowledge of G-protein
signaling in yeast has been accumulated and used to
study cellular processes, including protein interactions.
In this minireview series highlighting the methodolo-
gies for elucidating protein–protein interactions, the
other two minireviews by K. Tomizaki et al. [33] and
M. Umetsu et al. [34] deal with array based-technolo-
gies for detecting protein interactions in vitro, and con-
structive approaches to the generation of novel
binding proteins on the basis of tertiary structural
information, respectively. In this first minireview, we
focus on and summarize the unique technologies used
to exploit yeast G-protein signaling, which are com-
monly used for the exploration of biological protein
interactions under physiological in vivo conditions but

are distinguishable from conventional Y2H systems
from a scientific and engineering perspective.
Ras signaling-based screening systems
for protein–protein interactions
Small monomeric G-protein signaling in yeast
Small monomeric G-proteins, such as Ras and Ras-like
proteins, are found mainly at the inner surface of the
plasma membrane as monomers. They function as GTP-
ases on their own, and are involved in controlling cell
proliferation, differentiation, and apoptosis [29]. The
Ras 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 metabolism
in 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 cytosolic
cAMP levels activates the cAMP-dependent protein
kinase, which has an essential role in the progression
from the G
1
phase to the S phase of the cell cycle.
Owing to their intrinsically slow GTPase and GTP–
GDP exchange activities, Ras proteins are strictly
controlled by two classes of regulatory proteins:
GTPase-activating proteins (GAPs), and guanine nucle-
otide exchange factors (GEFs) [35]. RasGAPs, which
act as negative regulators of Ras–cAMP signaling by
accelerating hydrolysis of GTP to GDP on Ras pro-
teins, can stimulate the GTPase activity of Ras proteins

to 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–cAMP
signaling pathway. Whereas Cdc25p is essential in most
genetic backgrounds, Sdc25p is dispensable and is
normally expressed only during nutrient depletion or in
nonfermentative situations. Through its role in regulat-
ing cAMP levels, Cdc25p is involved in fermentative
growth, nonfermentative growth, cell cycling, sporula-
tion, and cell size regulation. Thus, the main positive
regulator of yeast Ras proteins is Cdc25p.
Characteristic aspects of Ras signaling-based
screening systems
Ras signaling-based yeast screening systems for the
exploration of protein interaction partners allow for
positive selection of interactions between soluble cyto-
solic proteins or between a soluble protein and a hydro-
phobic membrane protein through the restoration of
Ras signaling [36–38]. These systems employ the cdc25
yeast strain, which is deficient in Ras signaling and
regains 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 in
yeast, can be investigated because of the restitution of
Ras signaling on the plasma membrane but the absence
of reconstitution of DNA-binding transcription factors

J. Ishii et al. Screening systems using yeast G-protein signaling
FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1983
in the nucleus. The restricted cell survival with Ras
signaling-based selection is suitable for screening large
libraries (Table 1), although the method has compara-
tive difficulty in accurately assessing relative interaction
strengths.
Sos recruitment system
The Sos recruitment system (SRS) was initially
reported as a Ras signaling-based screening system,
and it takes advantage of the fact that the human
RasGEF protein, hSos, can substitute for the GEF of
yeast endogenous Ras (yRas) protein, Cdc25p, to
allow cell survival and proliferation (Fig. 1A) [36]. In
the SRS, a yeast variant strain that has the tempera-
ture-sensitive cdc25-2 allele is required. The cdc25-2
strain cannot survive at a restrictive temperature
(36 °C), owing to a lack of function of Cdc25p to
activate Ras signaling, whereas it can grow at a
lower temperature (25 °C). One protein should be
Table 1. Protein–protein interaction pairs identified or applied in G-protein signaling-based systems.
Interaction pair Reference
Sos recruitment system
c-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]
GABA
A
receptor 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 system
c-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-Fos
a
[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 system
Snf1 (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 system
ZZ domain or Z variants (Z domain: B domain mutant derived from protein A)–Fc part (of human IgG) [80]
Competitor-introduced Gc recruitment system
b
ZZ domain or Z variants–Fc part [102]
a
This system is to be used for monitoring receptor tyrosine kinase activity.
b
This 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 FEBS
membrane-associated or be attached to an inner mem-
brane translocating signal involved in myristoylation
and palmitoylation, and the other protein should be
soluble and be fused to hSos to prevent false autoacti-
vation by membrane localization of hSos. Only when
the membrane-localized protein interacts with the
hSos fusion protein will hSos be recruited to the
plasma membrane and yeast Ras signaling be rescued.
As a consequence, the temperature-sensitive mutant
that expresses interacting protein pairs can grow at
36 °C.
Using the SRS, a novel repressor that interacts with
the c-Jun subunits of AP-1 and represses its activity was
isolated [36] (Table 1). AP-1 is a transcription factor

that binds to DNA through a leucine zipper motif.
Thus, the ability of the SRS to identify transcriptional
regulators has been reasonably well established, owing
to the membrane-localized interaction, unlike conven-
tional Y2H systems based on the reconstitution of
DNA-binding transcription factors in the nucleus.
Ras recruitment system
The Ras recruitment system (RRS), using mammalian
Ras (mRas), was later developed as an improved ver-
sion of the SRS [38]. The RRS has the advantages of
the SRS without some of its limitations. For example,
the RRS permits more strict selection, owing to the
stringent requirement for membrane localization of
mRas, can eliminate the isolation of predictable
Ras false positives, owing to the introduction of
mRasGAP, and can more broadly detect interactions,
owing to the relatively small size of Ras as compared
with hSos [37,38]. The RRS is based on the absolute
requirement that Ras be localized to the plasma mem-
brane for its function (Fig. 1B). In the RRS, mRas
lacking its CAAX motif for localization to the plasma
membrane, but possessing a constitutively active muta-
tion, is used as a substitute for hSos, and mRasGAP is
additionally expressed. The membrane localization of
mRas through protein–protein interactions in a cdc25-2
yeast strain results in the activation of its downstream
effector, adenylyl cyclase, and restores its growth abil-
ity. In an initial report, the usefulness of the RRS was
confirmed by practical screening of a cDNA library
of 500 000 independent transformants [38] (Table 1).

Later, the RRS was applied to detect the activity and
inhibition of a dimerization-dependent receptor tyrosine
kinase and to identify an interacting pair of human glu-
cocorticoid receptors from a HeLa cell cDNA library
[39,40] (Table 1).
Pheromone signaling-based screening
systems
Heterotrimeric G-protein signaling in yeast
As peripheral membrane proteins, heterotrimeric
G-proteins associate with the inner side of the plasma
membrane. Heterotrimeric G-proteins consisting of
three subunits, Ga,Gb, and G c, exist in various sub-
families and are widely conserved among eukaryotic
species. They transduce messages from ubiquitous
receptors, which control important functions such as
taste, smell, vision, heart rate, blood pressure, neuro-
transmission, and cell growth [29]. Yeast has only two
types of heterotrimeric G-protein: pheromone signaling-
related and nutrient signaling-related [30–32]. Nutrient
signaling is profoundly and intricately linked to Ras
signaling [30,31], whereas the pheromone signaling
pathway is connected to mating processes [32].
The yeast pheromone signaling-related G-protein
comprises three subunits, Gpa1p, Ste4p, and Ste18p,
which structurally correspond to mammalian Ga,Gb,
(b)(a)
A
B
(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 Ras
signaling 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. The
active form of GTP-bound yRas allows cell survival. (B) The RRS
system using a constitutively active mutant of mammalian Ras lack-
ing the lipid modification motif (mRas). (a) Noninteracting protein
pairs are unable to activate the yeast Ras signaling pathway, and
are also unable to drive cell growth. (b) Interacting protein pairs
bring mRas to plasma membrane, where it can activate the yeast
Ras signaling pathway. Ras signaling allows cell survival. X and Y
represent test proteins for interaction analysis.
J. Ishii et al. Screening systems using yeast G-protein signaling
FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1985
and Gc, respectively [32]. The heterotrimeric G-protein
is divided into two key components from the perspec-
tive of structure and function. Ga (Gpa1p) is associ-
ated with the intracellular plasma membrane through
dual lipid modifications of myristoylation and palmi-
toylation in the N-terminus [41], whereas the Gbc
dimer (the Ste4p–Ste18p complex) is also localized to
the inner leaflet of the plasma membrane through dual
lipid modifications of farnesylation and myristoylation
in the C-terminus of Ste18p, and the formation of a
complex between Ste4p and lipidated Ste18p [41,42].
They form part of the signaling cascade activated by
G-protein-coupled receptors (GPCRs), and mediate
cellular processes in mating in response to the presence
of pheromone (Fig. 2A).

The yeast haploid a-cell has a sole pheromone recep-
tor, Ste2p, which is classified as a GPCR, and the
tridecapeptide a-factor functions as a pheromone and
binds to the Ste2p receptor on the cell surface [32].
The heterotrimeric G-proteins are closely associated
(a) (b)
B
A
Fig. 2. Yeast pheromone signaling pathway and its utilization for a GPCR biosensor. (A) Schematic illustration of the pheromone signaling
pathway. (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 from
Gpa1p 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 stimulates
hydrolysis of GTP to GDP on Gpa1p, and helps to inactivate pheromone signaling. (B) Schematic illustration of typical genetic modifications
enabling 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. Transcription
machineries 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 ligand
sensitivity, 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 FEBS
with the intracellular domain of the Ste2p receptor,
and the pheromone-bound receptor is conformational-
ly changed and activates the G-protein [43]. Gpa1p
is thereby changed from an inactive GDP-bound state
to an active GTP-bound state and dissociates the
Ste4p–Ste18p complex. Subsequently, the dissociated
Ste4p–Ste18p complex binds to effectors through
Ste4p, and then activates the mitogen-activated protein
kinase (MAPK) cascade [44,45]. The Ste5 scaffold pro-

tein binds to the kinases of the MAPK cascade and
brings them to the plasma membrane. The concentra-
tion of the bound kinases on the membrane possibly
promotes amplification of the signal [46,47]. As a con-
sequence, the activated pheromone signaling leads to
the phosphorylation of Far1p and the transcription
factor Ste12p. These phosphorylated proteins trigger
cell cycle arrest in G
1
[48–50] and global changes in
transcription [51,52]. FUS1 gene expression is repre-
sentative of the drastic changes in transcription in
response to pheromone signaling [53,54]. As a princi-
pal negative regulator, the Gpa1-specific GAP Sst2p, a
member of the regulator of G-protein signaling family,
is also involved in the pathway [55,56].
Pheromone signaling-based screening
systems – ligand–GPCR or
GPCR–G-protein interactions
Background of pheromone signaling-based
screening systems
GPCRs constitute the largest family of integral mem-
brane proteins, and have a variety of biological func-
tions. They are the most frequently addressed drug
targets, and modulators of GPCRs form a key area
for the pharmaceutical industry, representing nearly
30% of all Food and Drug Administration-approved
drugs [57,58]. Yeast permits the functional expression
of various heterologous GPCRs and other signaling
molecules such as G-proteins. Yeast also facilitates

versatile genetic techniques for screening and quantifi-
cation. Therefore, it offers opportunities to establish
fundamental technologies for drug discovery or basic
medicinal study [59,60]. Yeast-based screening systems
exploiting pheromone GPCR signaling enable the
analysis of several interactions, including not only
protein–protein but also ligand–receptor and receptor–
protein interactions. These systems can recognize the
on–off switching of a signal, such as the binding of an
agonist ⁄ antagonist to a receptor, and critical mutations
involved in ligand-dependent or constitutive acti-
vation ⁄ inactivation of signaling molecules. In addi-
tion, assays can be performed at the yeast optimum
temperature of 30 °C, unlike with Ras signaling-based
systems, which require the incubation of yeast cells
at suboptimal temperatures (25 and 36 °C), and the
monitoring or discrimination of the signaling changes
through quantitative and survival readouts. Hence,
they have been applied in various experiments, includ-
ing target identification, ligand screening, and receptor
mutagenesis.
Pheromone signaling as a biosensor for
understanding GPCRs
GPCRs have a common tertiary structure, composed of
seven hydrophobic integral membrane domains, and the
mechanism of signaling that is mediated by heterotri-
meric G-proteins is also conserved between yeast and
mammalian cells. This has led to the construction of
ingenious systems that provide for the mutual exchange
of signals between heterologous GPCRs and yeast

G-proteins in yeast without generating dysfunctions.
With versatile screening techniques, yeast can be used
as a sensor to detect the initiation of GPCR-associated
signaling [59,60]. Briefly, in wild-type yeast a-cells,
Ste2p receptor or mammalian receptors can activate the
yeast pheromone signaling pathway via intracellular
heterotrimeric G-proteins, including the native form or
an engineered form of Gpa1p, in response to ligand
binding. The activated pheromone signals cause cell
cycle arrest and transcription activation, which are
exploited as signaling readouts (Fig. 2A,B). These
biosensing techniques have been established in yeast
with engineered pheromone signaling, and numerous
characteristics of pheromone signaling molecules
have been successfully elucidated [43–45, 47–50, 53–55].
Moreover, pheromone signaling-related molecules, such
as Ste2p receptor, G-proteins, and peptidic a-factor
pheromone, have been extensively mapped with muta-
genesis techniques, demonstrating their usefulness for
screening huge libraries and for identification of impor-
tant domains or amino acids [61–66].
Bioassay and transcriptional assay for signaling
detection
The arrest of the cell cycle completely prevents cell
growth during signaling. Monitoring of cell densities in
liquid media with or without pheromones can distin-
guish signaling on the basis of delay of entry into the
logarithmic growth phase. The agar diffusion bioassay
(halo assay), in which cells are mixed with unsolidified
fresh agar medium in which pheromone-spotted paper

filter 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 signaling
FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1987
forming halos, owing to the robust inhibition of cell
growth (the halos may look blacked out on a mono-
chromatic figure) [55,62,63,66,67].
On the other hand, the use of transcriptional
changes that are closely regulated by the signaling
makes possible versatile procedures for detection. The
FUS1 gene, which is engaged in drastic augmentation
of the transcription level responding to the signal, is
commonly taken as a reflector of signaling and is fused
with various reporter genes associated with growth
and photometry. Auxotrophic or drug-resistant repor-
ter genes, such as HIS3 or hph, are generally used for
selection, and are suitable for screening large-scale
libraries [66–68]. Colorimetric, luminescent and fluores-
cent reporters, such as lacZ, luc,orGFP, are usually
used for numerical conversion and are appropriate for
relative and quantitative assessment of signaling levels
[61–64,66–68].
Gene disruption for system modification
The 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, this
makes growth reporter genes for positive selection,
such as HIS3, useless for the detection of signaling,
owing to stagnation of cell growth [66,67], whereas the
synchronization of the cell cycle in G

1
arrest provides
uniform levels of expression of reporter genes such as
GFP for each cell [69]. For that reason, FAR1 is usu-
ally disrupted in positive selection screens using growth
selection (Fig. 2B). Because the far1D strain never
induces 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 expression
of the HIS3 reporter gene on histidine-defective plates
[66,67]. At the same time, it has been reported that the
arrest of the cell cycle causes the drastic dropout of
episomal plasmids, resulting in a serious problem when
the 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]. SST2
gene encodes the Gpa1-specific GAP that stimulates
hydrolysis of GTP to GDP on Gpa1p and helps in the
inactivation of pheromone signaling. Removal of Sst2p
function causes a considerable decrease in GTPase
activity for Gpa1p, and makes the conversion of GTP
to GDP difficult, owing to a lack of competence of
GTPase activity (Fig. 2B). The loss of SST2 could pro-
vide supersensitivity, even to a 250–10 000-fold lower
concentration of a -factor [67]. However, a relatively

high background signal of the sst2D strain, especially
when grown in rich medium such as YPD, has been
confirmed in the absence of a-factor pheromone by a
transcription assay using the FUS1–GFP reporter gene
[69]. Although the SST2-deficient strategy is a powerful
technology for experiments requiring high sensitivity, it
does not necessarily produce the best signal-to-noise
ratio. Accordingly, choosing the correct situation for
using 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 GPCRs
Many heterologous GPCRs containing adrenergic,
muscarinic, serotonin, neurotensin, somatostatin, olfac-
tory and many other receptors have been successfully
expressed in yeast, and the feasibility of yeast-based
GPCR screening systems has been demonstrated
[59,60,68,70–75]. Yeast Gpa1p, which is equivalent to
Ga, shares high homology, in part, with human Ga
i
classes, and a number of GPCRs of human and other
species are able to interact with Gpa1p and activate
pheromone signaling in yeast [73–75]. Many other
human GPCRs can also function as yeast signaling
modulators as a result of various genetic modifications,
including one in which chimeric Gpa1p systems
(so-called ‘transplants’) have only five amino acids in
the C-terminus of Gpa1p substituted for those of
human Ga subunits, including the Ga
i ⁄ o

,Ga
s
and
Ga
q
families (Fig. 2B) [71]. Indeed, these transplants
have allowed functional coupling of serotonin, mus-
carinic, purinergic and many other receptors to the
yeast pheromone pathway [71–73,76].
The rat M
3
muscarinic acetylcholine receptor has
been used for rapid identification of functionally criti-
cal amino acids, with random mutagenesis of the entire
sequence [72]. In this system, the CAN1 reporter gene
coding for arginine–canavanine permease was inte-
grated into the locus of a pheromone response gene in
yeast cells whose endogenous CAN1 gene was deleted,
and the recombinant strain expressed Can1p in
response to ligand-dependent signaling. Owing to the
cytotoxicity of canavanine caused by Can1p expres-
sion, recombinant strains with inactivating mutations
in the receptor can survive on agar media containing
canavanine and receptor-specific agonists. The recov-
ered mutant M
3
muscarinic acetylcholine receptors in
this system also show substantial functional impair-
ments in transfected mammalian cells, and the utility
Screening 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 FEBS
of the yeast-based procedure for GPCR mutagenesis
has been proven.
Human formyl peptide receptor like-1, which was
originally identified as an orphan GPCR, has been
used to isolate agonists for GPCRs of unknown func-
tion [77]. Histidine prototrophic selection by the
FUS1–HIS3 reporter gene was performed with secreted
random tridecapeptides as a library and a mamma-
lian ⁄ yeast hybrid Ga subunit which allows functional
coupling with the receptor. As a result, surrogate
agonists as peptidic candidates have been successfully
screened, and the promoted activation of formyl
peptide receptor like-1 expressed in human cells has
been validated with synthetic versions of the peptides.
Pheromone signaling-based screening
systems – protein–protein interactions
Yeast–mammal chimeric Ga system
Medici et al. [78] constructed an intelligent system for
analysis of protein–protein interactions by managing
heterotrimeric G-protein signaling in yeast (Fig. 3A).
They initially found that a fusion protein between the
yeast Ste2p receptor lacking the last 62 amino acids of
the cytoplasmic tail and the full-length Gpa1p trans-
duced the signal in response to the binding of a-factor
in cells devoid of both endogenous STE2 and endoge-
nous GPA1. Subsequently, a yeast–mammal chimeric
Ga composed of the N-terminal 362 amino acids of
Gpa1p and the C-terminal 128 amino acids of rat Ga
s

was prepared. The chimeric Ga is able to interact with
the yeast Gbc complex, but is not able to interact with
the yeast Ste2p receptor, and it was fused to the trun-
cated Ste2p receptor. Although a gpa1D yeast strain
harboring the yeast–rat chimeric Ga does not respond
to pheromone, a ste2D gpa1D yeast strain expressing
the Ste2p–Gpa1p–Ga
s
fusion protein that is covalently
linked to Ste2p and the chimeric Ga displayed a strong
pheromone response in the presence of a-factor. These
results suggest that the specific interaction of the recep-
tor with the C-terminus of Ga is necessary to bring
the two proteins into close proximity. This hypothesis
was applied to the analysis of protein–protein inter-
actions. It was demonstrated that the interaction of
Gpa1p–Ga
s
fused to protein X and Ste2p receptor
fused to protein Y permitted pheromone response
signaling through the contact between Ste2p and
Gpa1p–Ga
s
, using the interaction between Snf1 and
Snf4, which form a kinase complex regulating transcrip-
tional activation in glucose derepression, or between
Raf and the constitutively active form of Ras (Table 1).
In this system, a gpa1D haploid strain harboring the
plasmid, which complements Gpa1p function to capture
Ste4p/Ste18p subunits, or a GPA1 ⁄ gpa1 D diploid yeast

strain was used to avoid lethality by spontaneous signal-
ing from the liberated Ste4p ⁄ Ste18p subunits.
Gc interfering system
The Gc interfering system (it was called a G-protein
fusion system in the original literature) has been devel-
oped to monitor integral membrane protein–protein
interactions and to screen for negative mutants with
loss of the interaction capacity (Fig. 3B) [79]. The
yeast Gc -subunit Ste18p was genetically fused to the
C-terminus of cytoplasmic protein X, and the pro-
tein X–Gc fusion protein and integral membrane
protein Y in its native form were coexpressed in a
ste18D strain. The interaction between protein X–Gc
and protein Y inhibits pheromone signaling through
the Gbc complex, in spite of the presence of a-factor,
whereas a lack of interaction between protein X and
protein Y normally leads to signaling. This event might
be attributed to the fact that restrictive localization or
structural interruption by trapping of the Gbc complex
at the position of protein Y on the membrane disturbs
the 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 nSec1
mutants that lost the ability to bind to syntaxin 1a were
successfully identified by taking advantage of growth
arrest induced through the protein–protein interaction
[79] (Table 1).
Gc recruitment system
The above-described systems for analysis of protein–

protein interactions using pheromone signaling are
proven techniques for selecting target proteins
involved with membrane proteins. However, they
might generate relatively high background signals,
making them unfavorable for screening candidates by
growth selection, because the machinery for distin-
guishing interactions does not always ensure com-
plete inactivation of signaling in the presence of
pheromone.
The Gc recruitment system has recently been devel-
oped using the pheromone signaling pathway, and is a
dependable system that completely eliminates back-
ground signals for noninteracting protein pairs in the
presence of pheromone (Fig. 3C) [80]. This system can
be used to investigate cytosolic–cytosolic or cytosolic–
membrane protein interactions. A yeast strain with a
mutated Gc lacking membrane localization ability
J. Ishii et al. Screening systems using yeast G-protein signaling
FEBS Journal 277 (2010) 1982–1995 ª 2010 The Authors Journal compilation ª 2010 FEBS 1989
(Gc
cyto
) should be prepared by deletion of the dual
lipid modification sites in the C-terminus of Ste18p,
because yeast pheromone signaling strictly requires
the localization of the Gbc complex to the plasma
membrane [41,42]. The release of Ste18p into the
cytosol eliminates the signaling ability mediated by
the Ste4p–Ste18p complex [41], and this technique
therefore leads to absolute interruption of background
A

B
C
(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 Ste2p
receptor. Chimeric Gpa1p is fused to protein X, and yeast Ste2p receptor is fused to protein Y. (a) Noninteracting protein pairs are
unable 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 screen
for negative mutants that do not interact. Ste18p genetically fused to the C-terminus of cytoplasmic protein X and integral membrane
protein 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 the
Gbc 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 are
coexpressed in a ste18D strain. (a) Noninteracting protein pairs completely lack pheromone signaling, owing to the release of the Ste4p–Ste18p
complex into the cytosol. (b) Interacting protein pairs restore signaling, owing to the recruitment of the Gbc complex onto the plasma
membrane.
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 FEBS
signals. One test protein must be soluble and fused
with Gc
cyto
to be expressed in the cytosol but not the
membrane, whereas the other may be soluble but
should have an added lipid modification site to allow
association with the inner leaflet of plasma membrane,
or it may be an intrinsically hydrophobic integral
membrane protein or lipidated element of a mem-
brane-associated protein. Consequently, when the

cytosolic protein X–Gc
cyto
fusion protein and the
membrane-associated protein Y are expressed in a
ste18D haploid strain in the presence of a-factor phero-
mone, the interaction between protein X and protein Y
restores signaling, owing to the recruitment of the Gbc
complex onto the plasma membrane, which can be
monitored, but a lack of interaction between protein X
and protein Y results in no background signaling.
In an original report, the ZZ domain derived from
protein A of Staphylococcus aureus and the Fc portion
of human IgG, which are both soluble proteins, were
used as a model interaction pair (Table 1). The ZZ
domain is a tandemly repeated Z domain that binds to
human Fc protein and displays higher affinity than a
Z domain monomer [81]. The interaction between the
ZZ domain with an attached dual lipidation motif in
its C-terminus and Fc fused to the C-terminus of
Gc
cyto
was easily detected with a transcriptional assay
using the pheromone response FIG1 promoter and a
GFP reporter gene or a halo bioassay by growth
arrest, whereas background signals from noninteract-
ing pairs were never observed, owing to the loss of
localization of the yeast Gbc complex at the plasma
membrane.
The wild-type and two variants of the Z domain
that each possess a single mutation and exhibit differ-

ent affinity constants were expressed as additional
interaction pairs for the Fc fusion protein [82]. All
variants with a wide range of affinity constants, from
8.0 · 10
3
to 6.8 · 10
8
m
)1
[83], were clearly detectable,
and moreover, the relatively faint interaction with an
affinity constant of 8.0 · 10
3
m
)1
was successfully
detected because of the complete elimination of back-
ground signal for noninteracting pairs (Table 1). Sur-
prisingly, a logarithmic proportional relationship
between affinity constants and fluorescence intensities
measured by the transcriptional assay was observed,
suggesting that this approach may facilitate the rapid
assessment of affinity constants.
Finally, the Gc recruitment system has more
recently been improved by the expression of a third
cytosolic protein that competes with the candidate pro-
tein [102]. The competitor-introduced Gc recruitment
system could specifically isolate only affinity-enhanced
variants from libraries containing a large majority of
original proteins, clearly indicating the applicability of

this new approach to directed evolution.
Concluding remarks
Yeast-based approaches with the G-protein signaling
machineries presented here are remarkably useful for
the detection and screening of interactions of proteins
involved in various biological processes. These systems
are essentially comparable to the Y2H systems that
have been predominantly used to screen protein–pro-
tein interaction partners from large-scale libraries and
to estimate the relative strengths of interactions, but
are additionally able to detect activation or inactiva-
tion associated with the switching machinery of signal-
ing molecules, such as major pharmaceutical targets of
GPCRs. Yeast-based and signaling-mediated screening
systems are obviously powerful and practical tools
with which to quickly screen for possible candidates.
In the future, we can be sure that they will be
improved, with more powerful and user-friendly
advanced modifications, and will be widely applied to
various fields, such as protein engineering.
Acknowledgements
This work was supported in part by a Research
Fellowship for Young Scientists from the Japan Society
for 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 Bioproduction
Kobe), from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT), Japan.
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