If the proteins physically interact, the Ras-signaling pathway is activated, allow-Keywords G-protein signaling; membrane localization of Gc subunit; protein–protein interaction; yeast t
Trang 1protein–protein interactions using yeast G-protein
signaling
Nobuo Fukuda1, Jun Ishii2, Tsutomu Tanaka2, Hideki Fukuda2and Akihiko Kondo1
1 Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Japan
2 Organization of Advanced Science and Technology, Kobe University, Japan
Protein–protein interactions are essential for normal
cellular function, and numerous studies have provided
important insight into the molecular mechanisms
underlying these interactions In particular,
develop-ment and use of the yeast two-hybrid (Y2H) system
has greatly facilitated the study of protein–protein
interactions In order to exhaustively identify protein
interaction pairs, including membrane-associated
pro-teins, the SOS and Ras recruitment systems (SRS or
RRS) using the Ras-signaling pathway in yeast cells as the readout have proven to be successful [1,2] Mem-brane-associated proteins, which constitute approxi-mately 40% of the total cellular proteins, include many important drug receptors, channels and enzymes [3] In the SRS and RRS systems, temperature-sensi-tive mutant strains are required for detection of pro-tein–protein interactions If the proteins physically interact, the Ras-signaling pathway is activated,
allow-Keywords
G-protein signaling; membrane localization
of Gc subunit; protein–protein interaction;
yeast two-hybrid system
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 ⁄ Tel: +81 78 803 6196
E-mail: akondo@kobe-u.ac.jp
(Received 18 December 2008, revised 18
February 2009, accepted 3 March 2009)
doi:10.1111/j.1742-4658.2009.06991.x
In the current study, we report the construction of a novel system for the detection of protein–protein interactions using yeast G-protein signaling It
is well established that the G-protein c subunit (Gc) is anchored to the inner leaflet of the plasma membrane via lipid modification in the C-termi-nus, and that this localization of Gc is required for signal transduction In our system, mutated Gc (Gccyto) lacking membrane localization ability was genetically prepared by deletion of the lipid modification site Complete disappearance of G-protein signal was observed when Gccyto was expressed
in the cytoplasm of yeast cells from which the endogenous Gc gene had been deleted In order to demonstrate the potential use of our system, we utilized the Staphylococcus aureus ZZ domain and the Fc portion of human immunoglobulin G (IgG) as a model interaction pair To design our detec-tion system for protein–protein interacdetec-tion, the ZZ domain was altered so that it associates with the inner leaflet of the plasma membrane, and the Fc part was then fused to Gccyto The Fc–Gccyto fusion protein migrated towards the membrane via the ZZ–Fc interaction, and signal transduction was therefore restored This signal was successfully detected by assessing growth inhibition and transcription in response to G-protein signaling Finally, several Z variants displaying affinity constants ranging from 8.0· 103to 6.8· 108m)1were prepared, and it was demonstrated that our system was able to discriminate subtle differences in affinity In conclusion, our system appears to be a reliable and versatile technique for detection of protein–protein interactions, and may prove useful in future protein inter-action studies
Abbreviations
EGFP, enhanced green fluorescent protein; RRS, Ras recruitment system; SRS, Sos recruitment system; ZI31A, single-site mutant of the Z domain by altering isoleucine at position 31 to alanine; Z K35A , single-site mutant of the Z domain by altering the lysine at position 35 to alanine; ZWT, wild-type Z domain derived from the B domain of Staphylococcus aureus protein A.
Trang 2ing yeast cells to grow at 37C Although this system
is advantageous for analysis of membrane-associated
proteins, the yeast growth rate using this system is
slow given that the optimal temperature for yeast
repli-cation is 30C
To establish a system that allows rapid identification
of protein–protein interactions, we focused on the
yeast G-protein signaling pathway The yeast
G-pro-tein signaling pathway is a well characterized pathway
that is activated via pheromone stimulation
Phero-mone stimulation leads to activation of heterotrimeric
G-proteins comprising Gpa1 (Ga), Ste4 (Gb) and
Ste18 (Gc) through the G-protein-coupled receptor
(Fig 1A) The activated G-protein subsequently
disso-ciates into Ga and Gbc complex subunits, and the
Gbc complex induces activation of the
mitogen-acti-vated protein kinase cascade The amplified signal
results in various cellular responses, including global
changes in transcription, growth arrest in the G1
phase, and polarized morphogenesis for mating One
significant advantage of using G-protein signaling for
the detection of protein–protein interactions is that the
assays are undertaken at 30C and thus yeast cells are
able to rapidly grow at their preferred temperature
Several methods of detection of protein–protein interactions have been developed using G-protein sig-naling Medici et al established the Gpa1–Gas chime-ric system in which a receptor is fused to protein X and Gpa1–Gas is fused to protein Y, restoring G-pro-tein signaling in response to the proG-pro-tein X–proG-pro-tein Y interaction [4] Subsequently, Ehrhard et al reported the use of a Gbc interfering system, in which the inter-action between protein X fused to Gc with the integral membrane protein Y disturbed contact of Gb with its effectors and thus inhibited G-protein signaling [3] These assays can be undertaken at 30C and are therefore suitable for rapid yeast growth, unlike the SRS and RRS methods, which required temperature-sensitive mutant strains Furthermore, these systems can be applied to the study of biologically important membrane-bound proteins Unfortunately, however, previously reported systems using G-protein signaling resulted in high background signals, making it difficult
to distinguish between subtle differences in affinity, and have therefore been considered unfavorable for extensive screening processes
In the current study, we established a technique for the successful identification of protein–protein interac-tions using yeast G-protein signaling We previously utilized the G-protein-coupled receptor assay system, which involves growth inhibition following G1 arrest and transcription of the enhanced green fluorescent protein (EGFP) reporter gene to detect protein–protein interactions [5] Signal transduction defects resulting from dissociation of the Gc subunit from the mem-brane require localization of the Gbc complex to the plasma membrane through the lipidated Gc subunit [6], and our method to detect protein–protein interac-tions is based on this finding The sequence encoding target protein ‘binder X’ is genetically fused to a Gc gene from which the lipidation sites (Gccyto) have been deleted The gene encoding the binder X–Gccytofusion protein replaces the STE18 gene, which encodes intact
Gc Then the lipidation motif is genetically introduced
to ‘binder Y’ and co-expressed with the binder X–Gc-cyto protein Binder X–Gccyto protein is expressed in the cytosol and the lipidated binder Y protein is local-ized to the plasma membrane As a result, signal trans-duction did not occur When binder X and binder Y interact with each other, the binder X–Gccyto fusion protein becomes localized to the plasma membrane and thus activates G-protein signaling (Fig 1C) In this study, we selected the Fc portion of human IgG and the ZZ domain derived from Staphylococ-cus aureus protein A as the model interaction pair (Fig 2), and demonstrated protein–protein interactions using growth inhibition and transcription assays Use
C
Pheromone
A
Receptor
Signal transduction Signal transduction
Growth arrest
(growth inhibition assay) (transcription assay)
Fig 1 Schematic outline of the experimental design (A) The
wild-type Gc subunit induces pheromone-stimulated signaling (B)
Engi-neered Gc lacking membrane-localization ability (Gccyto) leads to a
significant defect in G-protein signaling As a result, the Gb and
Gccyto (Gbccyto) complex is released into the cytosol following
dissociation from Ga due to ablation of plasma membrane
associa-tions (C) Protein–protein interaction re-establishes
pheromone-stimulated signaling Interaction between protein X fused to Gc cyto
and protein Y anchored to the plasma membrane results in
migra-tion of Gbc cyto to the inner leaflet of the plasma membrane In our
system, a transcription assay using the EGFP reporter gene fused
to the pheromone-inducible FIG1 gene allows positive selection A
growth inhibition assay based on cell-cycle arrest permits negative
selection The conditions used for this system are suitable for yeast
cell growth (30 C).
Trang 3of this assay system also resulted in very low
back-ground signal
Results and Discussion
General strategy
The aim of this study was to establish a rapid and
reli-able method for the detection of protein–protein
inter-actions using yeast G-protein signaling In our system,
protein–protein interactions were detected utilizing the
knowledge that the Gc subunit localizes to the inner
leaflet of the plasma membrane and that this
localiza-tion is required for G-protein signaling (Fig 1)
For-mation of Gc mutants by deletion of their lipidation
sites completely interrupts G-protein signaling [6], thus
we expected that our system would permit a more
accurate determination of protein–protein interactions
We chose the Fc portion of human IgG and the ZZ
domain derived from protein A [7–9], and an STE18
gene encoding the yeast Gc subunit in which the
lipi-dation sites had been mutated (Gccyto), as the key
components of our system The Fc was genetically
fused to the C-terminus of Gccyto (Gccyto–Fc) and the
lipidation motif was genetically added to the
C-termi-nus of the ZZ domain (ZZmem) Gccyto–Fc and ZZmem
were then co-expressed a yeast strain lacking
endoge-nous STE18 Interaction between Fc and ZZmemwould
then result in localization of Gccyto to the plasma
membrane, and signal transduction in response to
pheromone stimulation will occur (Fig 1C)
Construction of a yeast strain lacking
endogenous Gc
In order to prepare a host strain that would accept the
mutated Gc (Gccyto), which would in turn result in its
altered localization to the membrane and therefore
cause a strong defective signal (Fig 1B), we
con-structed an endogenous Gc-defective strain termed
BWG2118 by deletion of the STE18 gene BWG2118 was derived from the MC-F1 yeast strain that induces expression of the EGFP reporter gene in response to G-protein signaling (Table 1) To confirm STE18 gene deletion in BWG2118, pheromone-dependent growth inhibition (halo) and transcription assays were carried out For growth inhibition assays, cells were plated and then exposed to synthetic pheromone spotted onto filter disks MC-F1, in which endogenous Gc is intact, produced a clear halo in response to G-protein signal-ing, but BWG2118, in which endogenous Gc is defec-tive, did not exhibit a clear halo due to loss of signaling ability (Fig 3A) For transcription assays, expression of the EGFP reporter gene under the con-trol of the pheromone-inducible FIG1 promoter was analyzed by flow cytometry MC-F1 exhibited high flu-orescence as a result of signaling, but BWG2118 did not show EGFP reporter fluorescence even after the addition of pheromone (Fig 3B) The fluorescence intensity for BWG2118 appeared similar to that for strain BY4741, which was the original BWG2118 strain and does not encode the EGFP reporter gene (data not shown) These results demonstrate that G-protein signaling was interrupted due to the absence
of the STE18 gene, and that a yeast strain BWG2118, lacking endogenous Gc, had been successfully constructed
Co-expression of ZZmemand Gccyto–Fc proteins in
an endogenous Gc-defective yeast strain
To demonstrate detection of protein–protein interac-tions using mutated Gc, we used the ZZ domain and the Fc portion as the model pair in this system The lipidation-defective Gc mutant (Gccyto) was con-structed by deleting five amino acids from the C-termi-nus, and then fusing Gccyto with Fc (Fig 2A) Alternatively, the ZZ domain, which demonstrates high specific affinity for Fc, was genetically fused
to the lipidation motif sequence of yeast Gc at the
Table 1 Yeast strains used in this study.
BZFG2118 MC-F1 ste18D::kanMX4-PPGK-ZZmemhis3D::URA3-Pste18-Gccyto-Fc Present study
BFG2Z18-WT MC-F1 ste18D::kanMX4-PPGK-ZWT, memhis3D::URA3-Pste18-Gccyto-Fc Present study
BFG2Z18-K35A MC-F1 ste18D::kanMX4-P PGK -Z K35A, mem his3D::URA3-P ste18 -Gc cyto -Fc Present study
BFG2Z18-I31A MC-F1 ste18D::kanMX4-PPGK-ZI31A, memhis3D::URA3-Pste18-Gccyto-Fc Present study
Trang 4C-terminus (ZZmem; ZZ-SNSVCCTLM-COOH;
Fig 2A) [6] The strains in which ZZmem and the
Gccyto–Fc fusion genes were integrated into BWG2118
were termed BZG2118 (ZZmem), BFG2118 (Gccyto–Fc)
and BZFG2118 (ZZmem⁄ Gccyto–Fc) (Table 1)
Expres-sion of ZZmem proteins in BZG2118 (lane 2) and
BZFG2118 (lane 4), or of Gccyto–Fc fusion proteins in
BFG2118 (lane 3) and BZFG2118 (lane 4) was
con-firmed by Western blot analysis using anti-protein A
or anti-human IgG (Fig 4A)
Migration of mutated Gc to the plasma
membrane by protein–protein interaction
restores signal transduction in an endogenous
Gc-defective yeast strain
To test our hypothesis that the mutated Gc (Gccyto)
migrates to the plasma membrane and restores signal
transduction via protein–protein interaction, we
inves-tigated whether the endogenous Gc-defective yeast
strain expressing the ZZmem protein or the Gccyto–Fc
fusion protein induced signal transduction in growth
inhibition assays In order to achieve this, cells were
plated and then exposed to synthetic pheromone
spot-ted onto filter disks (Fig 4B) The endogenous
Gc-defective yeast strain (BWG2118) and the cells
expressing ZZmem or Gccyto–Fc (BZG2118 or
BFG2118) did not show halo formation even after pheromone stimulation (Fig 4B, panels 1–3) How-ever, the yeast strain BZFG2118, which expresses both ZZmem and Gccyto–Fc, did show a clear halo in response to pheromone induction, demonstrating that co-expression of ZZmem and Gccyto–Fc was able to restore signal transduction (Fig 4B, panel 4) We also prepared a yeast strain expressing ZZ without the lipidation motif in place of ZZmem (termed BFG2118⁄ ZZ), which co-expressed Gccyto–Fc and ZZ without the lipidation motif, as a negative control strain As ZZ and Fc are known to interact in the cytosol [9], the BFG2118⁄ ZZ strain did not exhibit cell-cycle arrest in the halo assay (data not shown)
In addition, as a positive control, yeast cells express-ing the lipidation motif attached to Gccyto–Fc (Gccyto–Fcmem) formed a clear halo in response to pheromone induction, as expected (data not shown) These results demonstrate that the mutated Gc (Gccyto) strain utilized in this study had completely lost its membrane associations; however, recruitment
of Gccyto to the membrane following interaction with
ZZ and Fc recovered G-protein signaling These results suggest that interactions between membrane proteins or cytoplasmic proteins modified to contain membrane lipidation motifs and cytoplasmic proteins may be detected using our system As the growth inhibition assay based on cell-cycle arrest allowed for negative selection, our system may also be success-fully used in high-throughput screening of signal-defective mutants to determine the specific amino acids required for protein–protein interactions
Evaluation of the affinity constant via transcription assays using the EGFP fluorescence reporter gene
To corroborate the results of the growth inhibition assay, we performed reporter transcription assays As shown in Fig 4C, co-expression of ZZmemand Gccyto–
Fc (BZFG2118) resulted in remarkably high fluores-cence following transcriptional activation of the EGFP reporter gene The fluorescence intensity was equiva-lent to that of the MC-F1 positive control strain shown in Fig 3B In contrast, BFG2118, which expressed Gccyto–Fc without ZZmem, did not show reporter expression, and the fluorescence intensity was equivalent to that of the negative control strain BWG2118 (Fig 3B) These results demonstrate that our system resulted in very low background signal and therefore confers a significantly high signal-to-noise (S⁄ N) ratio in the detection of protein–protein inter-actions Detection of interactions in the absence of
A
Fig 2 Schematic outline of gene construction (A) Structural
fea-tures of the yeast endogenous Gc gene (STE18), and design of the
Gc–Fc fusion gene excluding the lipidation motif (Gc cyto –Fc) and the
lipidation motif attached to the ZZ gene (ZZ mem ) (B) Plasmid map
for integration of the ZZmemgene into the yeast chromosome (C)
Plasmid map for integration of the Gccyto–Fc gene into the yeast
chromosome.
Trang 5background signal generation was also shown for the
growth inhibition assay (Fig 4B) Transcription assays
using the EGFP reporter allow the quantitative
assess-ment of changes in G-protein signaling and
high-throughput selection of positive interaction pairs by
flow cytometric screening [5]
Numerous previous studies have reported detection
of protein–protein interactions; however, few methods
have allowed evaluation of affinity constant To assess
the correlation between the affinity constant and the
fluorescence intensity, we prepared several partners for
Fc (ZWT, 5.9· 107m)1; ZK35A, 4.6· 106m)1; ZI31A,
8.0· 103m)1) instead of ZZ (6.8· 108m)1) [10]
(where ZWT is the wild-type Z domain derived from
the B domain of Staphylococcus aureus protein A,
ZK35Ais a single-site mutant of the Z domain by
alter-ing the lysine at position 35 to alanine, and ZI31Ais a
single-site mutant of the Z domain by altering
isoleu-cine at position 31 to alanine), and introduced them
into yeast chromosomes (WT,
BFG2Z18-K35A and BFG2Z18-I31A, as shown in Table 1)
Expression of ZZmem and the ZZmem variants was
confirmed by Western blot analysis (Fig 5A), and reporter transcription assays were performed for each strain (Fig 5B) The fluorescence intensities of the strains were obviously altered according to the affinity constants of the Fc partners It was notable that the relatively faint interaction between Fc and ZI31A, whose affinity constant was 8.0· 103m)1, could be successfully detected Furthermore, we identified a log-arithmic proportional relationship between fluores-cence intensity and affinity constant (Fig 5C) Such accurate quantitative capability may be helpful for discrimination of doubtful interaction candidates using our system
(kDa)
14
1
A
B
C
a
b
c
14.3 36.9
(ZZmem) (G γ cyto -Fc) 42.0 (β-actin)
3 4
1 2
3 4
Fig 4 Restoration of signal transduction following interaction between ZZmemand Gccyto–Fc (A) Western blot analyses were per-formed using the following primary antibodies: (a) anti-protein A for the ZZ domain, (b) anti-IgG for Fc, and (c) anti-b-actin as the loading control (B) Halo bioassays were performed with 10 ng of synthetic a-factor pheromone spotted onto filter disks (C) Transcription assays were performed using flow cytometric EGFP fluorescence analysis The histogram plots show the analytical data for 10 000 cells ‘1’ indicates BWG2118 (negative control strain), ‘2’ indicates BZG2118 (the constructed strain expressing ZZ mem ), ‘3’ indicates BFG2118 (the constructed strain expressing Gccyto–Fc), and ‘4’ indi-cates BZFG2118 (the constructed strain expressing both ZZ mem and Gc cyto –Fc).
A
B
Fig 3 Confirmation of signal response in the endogenous
Gc-defective yeast strain (A) Halo bioassays were performed with
10 ng of synthetic a-factor pheromone spotted onto filter disks (B)
Transcription assays were performed by flow cytometric EGFP
fluo-rescence analysis The histogram plots show the analytical data for
10 000 cells ‘1’ indicates BWG2118 (the constructed ste18D
strain), and ‘2’ indicates MC-F1 (the STE18-intact strain).
Trang 6In conclusion, we have established a novel detection
system based on G-protein signaling for detection of
protein–protein interactions, using a mutated Gc that
lacks membrane-localization ability Our assay can be
performed under conditions suitable for maximal yeast
cell growth, and the effects can be assessed in terms of
transcription (positive selection) and growth inhibition
assays (negative selection) In addition, our system is a reliable, quantitative technique that largely avoids background signals As a result, we were able to evalu-ate a wide range of affinity constants from 8.0 · 103to 6.8· 108m)1 We suggest that our system can be uti-lized as a reliable and versatile system for detection of protein–protein interactions using G-protein signaling
Experimental procedures Strains and media
Details of Saccharomyces cerevisiae BY4741 [11], MC-F1 (J Ishii, M Moriguchi, S Matsumura, K Tatematsu,
S Kuroda, T Tanaka, T Fujiwara, H Fukuda &
A Kondo, unpublished results) and other constructed strains used in this study and their genotypes are given in Table 1 MC-F1 derived from BY4741 was engineered to express the EGFP fusion gene in response to a-factor pher-omone induction using the pherpher-omone-inducible FIG1 gene The yeast strains were grown in YPD medium containing 1% w⁄ v yeast extract, 2% peptone and 2% glucose, or in
SD medium without uracil (SD-Ura) containing 0.67% yeast nitrogen base without amino acids (Becton Dickinson and Company, Franklin Lakes, NJ, USA), 2% glucose,
20 mgÆL)1 histidine, 30 mgÆL)1 leucine and 30 mgÆL)1 methionine Agar (2% w⁄ v) was added to the media described above to produce YPD and SD-Ura agar
Construction of plasmids for yeast chromosome substitution
Plasmids used for deletion of STE18 gene by substitution
of the kanMX4 gene (G418 resistance gene) on the yeast chromosome were constructed by amplifying the fragment encoding the upstream region of STE18 (STE18p, STE18 promoter region) from MC-F1 genomic DNA using prim-ers 1 and 2 (Table 2) This fragment was then inserted into the XhoI site of pGK426 (J Ishii, K Izawa, S Matsumura,
K Wakamura, T Tanino, T Tanaka, C Ogino,
H Fukuda & A Kondo, unpublished results), yielding plasmid pGK426-GP The fragment encoding the down-stream region of STE18 (STE18t, STE18 terminator region) was amplified from MC-F1 genomic DNA using primers 3 and 4 (Table 2), and inserted into the BamHI– EcoRI sites of pGK426-GP yielding plasmid pGK426-GPT The fragment containing kanMX4 was amplified from pUG6 (EUROSCARF, Frankfurt, Germany) [12] using primers 5 and 6 (Table 2), and inserted into the XhoI–SalI site of pGK426-GPT yielding plasmid pGK426-GPTK The plasmid used for integration of the ZZ domain fused
to the lipidation motif gene (ZZmem) at the STE18 locus of the yeast chromosome was constructed by amplifying the fragment encoding the ZZ domain from pMWIZ1 [13]
1
2 3 4 5
1
10
100
1000
Affinity constant [ M–1 ]
2
3 4
5
(kDa) 14.3 8.1 36.9 42.0
(ZZ mem )
( β-actin)
(Z mem ) (Gγ cyto -Fc)
1
A
B
C
a
b
c
2 3 4 5
Fig 5 Quantitative analysis of the signal responses and interaction
strength (A) Western blot analyses were performed using the
fol-lowing primary antibodies: (a) anti-protein A for the ZZ domain, (b)
anti-IgG for Fc, and (c) anti-b-actin as the loading control (B) Flow
cytometric EGFP fluorescence analysis (C) Logarithmic plots of
flu-orescence intensity against the affinity constants ‘1’ indicates
BFG2118 (negative control strain), ‘2’ indicates BFG2Z18-I31A (the
constructed strain expressing Z I31A ), ‘3’ indicates BFG2Z18-K35A
(the constructed strain expressing ZK35A), ‘4’ indicates
BFG2Z18-WT (the constructed strain expressing ZWT), and ‘5’ indicates
BZFG2118 (the strain expressing ZZ mem ) Standard errors of three
independent experiments are presented.
Trang 7using primers 7 and 8 (Table 2), and inserting it into the
SalI–BamHI site of pGK426, yielding plasmid pUMZZ
The fragment encoding the PGK1 promoter (PGK5¢), the
ZZmemgene and the PGK1 terminator (PGK3¢) was
ampli-fied from pUMZZ using primers 9 and 10 (Table 2), and
inserted into the XhoI site of pGK426-GPTK, yielding
plas-mid pUMGPT-ZZK (Fig 2)
The plasmid used for integration of the Gccyto–Fc gene
at the HIS3 locus of the yeast chromosome was constructed
by amplifying the fragment encoding STE18p and Gc
delet-ing the lipidation sites (Gccyto) from MC-F1 genomic DNA
using primers 11 and 12 (Table 2), and inserted into the
XhoI–BamHI sites of pGK426, yielding plasmid
pUMGP-GcM The fragment encoding Fc was amplified from
pUF318-Fc [14] using primers 13 and 14 (Table 2), and inserted into the BamHI–EcoRI site of pUMGP-GcM, yielding plasmid pUMGP-GcMFc A fragment encoding the HIS3 terminator region (HIS3t) was amplified from MC-F1 genomic DNA using primers 15 and 16 (Table 2), and inserted into the NotI–SacI sites of pUMGP-GcMFc, yielding plasmid pUMGP-GcMFcH (Fig 2)
The plasmid used for integration of the Zmemgene at the STE18 locus of the yeast chromosome was constructed by amplifying the fragment encoding the Z domain from pMWIZ1 using primers 17 and 18 (Table 2), and inserted into the SalI–BamHI sites of pGK426 yielding plasmid pUMZ-WT To prepare single amino acid-substituted Z variants, the following plasmids were constructed from pUMZ-WT using the Quick-Change method (Stratagene,
La Jolla, CA, USA) For ZK35A and ZI31A, plasmids pUMZ-K35A and pUMZ-I31A were constructed using primers 19 and 20 and primers 21 and 22, respectively The fragment encoding PGK5¢, the Zmem genes (wild-type, K35A and I31A) and PGK3¢ were amplified from
pUMZ-WT, pUMZ-K35A and pUMZ-I31A, respectively, using primers 23 and 24 (Table 2), and inserted into the XhoI site
of pGK426-GPTK, yielding plasmids pUMGPT-ZK-WT, pUMGPT-ZK-K35A and pUMGPT-ZK-I31A
Construction of yeast strains The strains used in this study are described in Table 1 The genes were introduced into yeast cells using the lithium acetate method [15]
Substitution of the STE18 gene by kanMX4 in the yeast chromosome was achieved by amplifying the DNA frag-ment containing STE18p–kanMX4–STE18t from pGK426-GPTK using primers 25 and 26 (Table 2) The amplified DNA fragment was then used to transform MC-F1, and the transformant was selected on YPD solid medium containing 500 ngÆmL)1 G418 (geneteccin; Nacalai Tesque Inc., Kyoto, Japan) to yield the BWG2118 strain
Integration of the ZZmem gene was achieved by amplifying a DNA fragment containing STE18p–PGK5¢– ZZmem–PGK3¢–kanMX4–STE18t from pUMGPT-ZZK using primers 25 and 26 (Table 2) The amplified DNA fragment was used to transform MC-F1, and the transfor-mant was selected on YPD solid medium containing
500 ngÆmL)1G418 to yield the BZG2118 strain
Integration of the GcM–Fc gene was achieved by amplifying a DNA fragment containing URA3–STE18p– GcM–Fc–PGK3¢–HIS3t from pUMGP-GcMFcH using primers with 50-nucleotide 5¢ segments that were homolo-gous to the region directly upstream of the HIS3 gene (primers 27 and 28; Table 2) The amplified DNA frag-ment was then used to transform BWG2118 and BZG2118, and the transformants were selected on SD-Ura solid medium, yielding the BFG2118 and BZFG2118 strains, respectively
Table 2 Primers used for construction of plasmids and yeast
strains.
Primer number Sequence (5¢- to 3¢)
CGTAGACAAC
CACTATTTGATTTCGGCGCCTGAGCATCA TTTAGCTTTTT
AGTCAACACTATTTGAGTTTGACATTTGGC
CTTCCCCC
CGTAGACAAC
CACTATTTGATTTCGGCGCCTGAGCATCA TTTAGCTTTTT
CAAGATAAACGAAGGCAAAGTTCAATTCA TCATTTTTTTTTTATTCTTTT
Trang 8Construction of yeast strains containing Zmem genes
rather than the ZZmemgene were achieved using a process
similar to construction of the BZG2118 strain, with the
exception that the plasmids ZK-WT,
pUMGPT-ZK-K35A or pUMGPT-ZK-I31A were used instead of
pUMGPT-ZZK In addition, integration of the Gccyto–Fc
gene into these transformants was achieved as shown in
Fig 2C, yielding BFG2Z18-WT, BFG2Z18-K35A and
BFG2Z18-I31A strains
Halo bioassay to test growth arrest via the
pheromone response
An agar diffusion bioassay (halo assay) was undertaken to
measure the response to and recovery from
pheromone-induced cell-cycle arrest as described previously [16] The
yeast strains were grown in YPD medium at 30C
over-night Sterilized paper filter disks (6 mm in diameter) were
placed on the dishes, and 10 ng of a-factor pheromone was
spotted onto the disks The cells were then inoculated into
fresh YPD medium containing 2% w⁄ v agar (20 mL,
main-tained at 60C), grown until they reached an absorbance
at 600 nm (A600) of 10)3, and the suspension was
immedi-ately poured into a dish The plates were then incubated at
30C for 24 h
Flow cytometric EGFP fluorescence analysis
The fluorescence intensity of EGFP fusion proteins in yeast
cells stimulated with 5 lm of a-factor in YPD medium for
6 h was measured using a FACSCalibur flow cytometer
equipped with a 488 nm air-cooled argon laser (Becton
Dickinson and Company), and the data were analyzed
using cellquest software (Becton Dickinson and
Com-pany) Parameters were as follows: the amplifiers were set
in linear mode for forward scattering and in logarithmic
mode for the green fluorescence detector (FL1, 530⁄ 30 nm
bandpass filter) and the orange fluorescence detector (FL2,
585⁄ 21 nm bandpass filter) The amplifier gain was set at
1.00 for forward scattering; the detector voltage was set to
E00 for forward scattering and 600 V for FL1, and the
threshold for forward scattering was set at 52 The EGFP
fluorescence signal was collected using a 530⁄ 30 nm
band-pass filter (FL1), and the fluorescence intensity of 10 000
cells was defined as the FL1-height (FL1-H) geometric
mean
Western blot analysis
Yeast cells were cultured in YPD medium overnight The
cells were then harvested, washed in NaCl⁄ Pi to remove
culture media and resuspended in sample buffer for
SDS⁄ PAGE at an A600of 20 Fractionated cell lysates were
prepared by glass bead vortex homogenization for 15 min
Protein extracts were separated by 15% SDS⁄ PAGE, and Western blot analysis was performed using the primary antibodies goat anti-protein A (Rockland, Gilbertsville, PA, USA) for the ZZ or Z domain, and goat anti-human IgG (Fc) (Kirkegaard Perry Laboratories, Gaithersburg, MD, USA) for the Fc portion Alkaline phosphatase-conjugated anti-goat IgG (Vector Laboratories, Burlingame, CA, USA) was used as the secondary antibody, and colorimetric detec-tion of alkaline phosphatase activity was performed using 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetra-zolium (Promega Co., Madison, WI, USA)
Acknowledgements This work was supported by a Research Fellowship for Young Scientists from the Japanese Society for the Promotion of Science, and in part by the Global COE Program ‘Global Center for Education and Research
in Integrative Membrane Biology’ from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan
References
1 Aronheim A, Engelberg D, Li N, ai-Alawi N, Schles-singer J & Karin M (1994) Membrane targeting of the nucleotide exchange factor Sos is sufficient for activat-ing the Ras signalactivat-ing pathway Cell 78, 949–961
2 Broder YC, Katz S & Aronheim A (1998) The ras recruitment system, a novel approach to the study of protein–protein interactions Curr Biol 8, 1121–1124
3 Ehrhard KN, Jacoby JJ, Fu XY, Jahn R & Dohlman
HG (2000) Use of G-protein fusions to monitor integral membrane protein–protein interactions in yeast Nat Biotechnol 18, 1075–1079
4 Medici R, Bianchi E, DiSegni G & Tocchini-Valentini
GP (1997) Efficient signal transduction by a chimeric yeast-mammalian G protein a subunit Gpa1-Gsa cova-lently fused to the yeast receptor Ste2 EMBO J 24, 7241–7249
5 Ishii J, Tanaka T, Matsumura S, Tatematsu K, Kuroda
S, Ogino C, Fukuda H & Kondo A (2008) Yeast-based fluorescence reporter assay of G protein-coupled recep-tor signalling for flow cytometric screening: FAR1-dis-ruption recovers loss of episomal plasmid caused by signalling in yeast J Biochem 143, 667–674
6 Manahan CL, Patnana M, Blumer KJ & Linder ME (2000) Dual lipid modification motifs in Ga and Gc subunits are required for full activity of the pheromone response pathway in Saccharomyces cerevisiae Mol Biol Cell 18, 957–968
7 Kronvall G & Williams RC Jr (1969) Differences in anti-protein A activity among IgG subgroups J Immu-nol 103, 828–833
Trang 98 Nilsson B, Moks T, Fansson B, Abrahmsen L, Elmbrad
A, Holmgren E, Henrichson C, Jones TA & Uhlen MA
(1987) Synthetic IgG-binding domain based on
staphy-lococcal protein A Protein Eng 1, 107–113
9 Shibasaki S, Kuroda K, Nguyen HD, Mori T, Zou W
& Ueda M (2006) Detection of protein–protein
interac-tions by a combination of a novel cytoplasmic
mem-brane targeting system of recombinant proteins and
fluorescence resonance energy transfer Appl Microbiol
Biotechnol 70, 451–457
10 Jendeberg L, Persson B, Anderssson R, Karlsson R,
Uhlen M & Nilsson B (1995) Kinetic analysis of the
interaction between protein A domain variants and
human Fc using plasmon resonance detection J Mol
Recognit 8, 270–278
11 Brachann CB, Davies A, Cost GJ, Caputo E, Li J,
Hieter P & Boeke JD (1998) Designer deletion strains
derived from Saccharomyces cerevisiae S288C: a useful
set of strains and plasmids for PCR-mediated gene
disruption and other applications Yeast 14, 115–132
12 Gu¨ldener U, Heck S, Fielder T, Beinhauer J &
Hege-mann JH (1996) A new efficient gene disruption cassette
for repeated use in budding yeast Nucleic Acids Res 24, 2519–2524
13 Nakamura Y, Shibasaki S, Ueda M, Tanaka A,
Fuku-da H & Kondo A (2001) Development of novel whole-cell immunoadsorbents by yeast surface display of the IgG-binding domain Appl Microbiol Biotechnol 57, 500–505
14 Fukuda N, Ishii J, Shibasaki S, Ueda M, Fukuda H & Kondo A (2007) High-efficiency recovery of target cells using improved yeast display system for detection of protein–protein interactions Appl Microbiol Biotechnol
76, 151–158
15 Gietz D, St Jean A, Woods RA & Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells Nucleic Acids Res
20, 1425
16 Ishii J, Matsumura S, Kimura S, Tatematsu K, Kuroda
S, Fukuda H & Kondo A (2006) Quantitative and dynamic analyses of G protein-coupled receptor signal-ing in yeast ussignal-ing Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein Biotechnol Prog 22, 954–960