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

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-

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

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]

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

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-

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-

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

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

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

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

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–

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

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

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