RESEARCH ARTICLE
Evidence for functional links between the Rgd1-Rho3 RhoGAPGTPase module and Tos2, a protein involved in polarized growth in
Saccharomyces cerevisiae
Sandra Claret1,2, Olivier Roumanie1,2, Valérie Prouzet-Mauleon1,2, Fabien Lefebvre1,2, Didier
Thoraval1,2, Marc Crouzet1,2 & François Doignon1,2
1
RDPR, Institute of Cellular Biochemistry and Genetics, University of Bordeaux 2, Bordeaux, France; and 2CNRS, UMR 5095, Bordeaux, France
Correspondence: François Doignon, RDPR,
Institute of Cellular Biochemistry and
Genetics, University of Bordeaux 2, Box 64,
146 rue Léo Saignat, 33076 Bordeaux Cedex,
France. Tel.: 133 5 57 57 47 18; fax: 133 5
57 57 47 19; e-mail: francois.doignon@
u-bordeaux2.fr
Received 23 July 2010; revised 20 October
2010; accepted 5 November 2010.
Final version published online 8 December
2010.
DOI:10.1111/j.1567-1364.2010.00704.x
Editor: Ian Dawes
Abstract
The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity
of Rho3p and Rho4p, which are involved in bud growth and cytokinesis,
respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening
identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region.
Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle
and associated with detergent-resistant membranes. We observed that TOS2
overexpression suppressed rgd1D sensitivity to a low pH. In the tos2D strain, the
amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on
Rgd1p activity in vivo. We also showed a functional interaction between the TOS2
and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect
of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein
involved in polarized growth and most probably associated with the plasma
membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.
Keywords
Saccharomyces cerevisiae; polarized growth;
Rho GTPases; Rgd1p; Tos2p.
YEAST RESEARCH
Introduction
The budding yeast Saccharomyces cerevisiae exhibits polarized growth at several stages of its life cycle (Drubin &
Nelson, 1996; Pruyne & Bretscher, 2000a). Polarized growth
requires coordination between the synthesis of cell growth
material and the asymmetrical delivery of cellular components between mother and daughter cells (Pruyne &
Bretscher, 2000a, b; Brennwald & Rossi, 2007). Rho GTPases
play a role in establishing and maintaining polarized growth
during bud and septum formation (Johnson, 1999; Gladfelter et al., 2002; Bretscher, 2003). In the yeast S. cerevisiae, six
Rho proteins, Cdc42p and Rho1p-Rho5p, were identified
(Garcia-Ranea & Valencia, 1998; Roumanie et al., 2001). These
small GTPases cycle between the GTP-bound active and the
GDP-bound inactive conformations. The active form interacts
with its specific targets and signals to different pathways
involved in actin cytoskeleton organization and exocytosis
(Tanaka & Takai, 1998; Adamo et al., 1999, 2001). This
GDP–GTP cycle is tightly regulated spatiotemporally in cells
FEMS Yeast Res 11 (2011) 179–191
by the positive regulators, guanine nucleotide-exchange factors (GEFs), and by the negative regulators, the GTPaseactivating proteins (GAPs). GEFs activate Rho GTPases by
promoting the exchange of GDP for GTP (Cerione & Zheng,
1996) and GAPs enhance the intrinsic GTPase activity, leading
to the inactive state of the GTPase (Tcherkezian & LamarcheVane, 2007). The dynamic properties of GDP–GTP cycling are
important as shown for the Cdc42p GTPase in septin ring
assembly and in cell fusion during mating (Gladfelter et al.,
2002; Barale et al., 2006).
The Rho3p and Rho4p GTPases are thought to be required
for the maintenance, but not for the initiation, of polarized
cell growth. (Matsui & Toh-e, 1992a, b). Indeed, the combined
depletion of Rho3p and Rho4p causes cell death at 30 1C and
results in large, round cells with a small bud and a disorganized actin cytoskeleton. The deletion of RHO3 alone also
causes severe defects in cell growth. The overexpression of
RHO4 suppresses the growth defects of rho3D, suggesting that
certain functions of Rho3p and Rho4p may be overlapping
(Matsui & Toh-e, 1992b). However, Rho3p and Rho4p
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180
S. Claret et al.
GTPases appear to play distinct roles in bud extension and
bud neck formation, respectively. Indeed, the production of
GTP-blocked Rho3p in wild-type yeast results in abnormally
elongated cells, whereas yeast cells producing GTP-blocked
Rho4p are larger and rounder, with poorly defined bud necks
(Fernandes et al., 2006).
We showed previously that the Rgd1p protein displays
RhoGAP activity for Rho3p and Rho4p GTPases (Doignon
et al., 1999). Rgd1p has a molecular organization similar to
several members of the F-BAR family (Dawson et al., 2006;
Itoh & De Camilli, 2006); indeed, in addition to the
C-terminal RhoGAP domain (aa 486–666), Rgd1p possesses
an F-BAR domain (aa 1–300) at its N-terminus (ProuzetMauleon et al., 2008). The F-BAR domains, which include a
FER/CIP4-homology (FCH) domain, followed by a coiledcoil sequence, are involved in membrane binding and
tubulation (Itoh et al., 2005; Tsujita et al., 2006; Shimada
et al., 2007). We showed that the F-BAR region of Rgd1p
binds phosphoinositides in vitro and also plays a key role in
the localization of the RhoGAP to the bud tip and neck
during the cell cycle (Prouzet-Mauleon et al., 2008).
Increasing evidence indicated that RhoGAPs are regulated by distinct mechanisms and play a role in a number of
Rho-mediated signalling pathways (Bos et al., 2007; Tcherkezian & Lamarche-Vane, 2007). Therefore, we searched for
partners of the Rgd1 RhoGAP to investigate for potential
upstream regulators of the Rho3 and Rho4 GTPases and
consequently of polarized growth. Using a two-hybrid
screen, we identified a truncated form of Tos2p interacting
with Rgd1p. The TOS2 gene belongs to a family of five genes
called TOS (Horak & Snyder, 2002) for Target Of SBF (Swi4Swi6 cell cycle box Binding Factor) and it displays a strong
cell cycle-dependent expression that peaks in G1 (Spellman
et al., 1998). The Tos2 protein localizes to the bud tip and
bud neck and is a component of a cell polarity protein
interaction map (Drees et al., 2001; Sundin et al., 2004).
Additionally, the Tos2 protein, which may act as a membrane anchor, is involved in the localization of Cdc24p, the
GEF for Cdc42p, to the bud growth site (Toenjes et al.,
2004). More recently, the TOS2 gene was found to be a highcopy suppressor of a temperature-sensitive strain carrying
mutations in GIC1 and GIC2, which encode two effectors of
the Cdc42p GTPase (Gandhi et al., 2006). In this study, we
addressed the functional links between the Rgd1p RhoGAP
and its binding partner Tos2p. Our analysis revealed a
functional relationship between the protein Tos2 and the
module Rgd1-Rho3.
Materials and methods
Yeast strains and growth conditions
All basic and genetic yeast manipulations were carried out
according to Guthrie & Fink (1991). The genotypes of yeast
strains used are listed in Table 1. Yeast strains were grown at
30 1C in standard yeast extract peptone dextrose medium or
synthetic dextrose (SD) minimal medium supplemented
with appropriate amino acids. Acidic shock was performed
by adding hydrochloric acid until the pH of the liquid
medium was 2.8 (Gatti et al., 2005). Cell death was determined by methylene blue staining (de Bettignies et al.,
2001). Yeast transformations were performed as described
previously (Gietz et al., 1995).
Plasmids
The TOS2 gene was inserted into the low-copy vectors
pUG35 with the URA3 marker and pUG23 with the HIS3
marker (Niedenthal et al., 1996); the TOS2 coding sequence
Table 1. Saccharomyces cerevisiae strains
Name
Genotype
Source
FY1679a
BY4742
rgd1D
tos2D
rgd1D
tos2D
tos2D rgd1D
rho3-1 rho4D
rho3D
rho4D
rho3-V51
BY647
BY646
HY (HF7c Y187)
MATa, ura3-52, trp1D63, leu2D1
MATa, his3D1, leu2D0, lys2D0, ura3D0
BY4742; rgd1<kanMX4
BY4742; tos2<kanMX4
MATa, ura-52, his3 11,15 rgd1<HIS3
MATa, ura3-52, trp1D63, his3D200, tos2<kanMX4
MATa, ura3-52, trp1D63, his3 11,15 rgd1<HIS3, tos2<kanMX4
MATa, ura3-52, leu2-3,112, trp1, his3, rho3-1<TRP1, rho4<HIS3
MATa, ura3-52, trp1D63, rho3<kanMX4
MATa, ura3-52, leu2D1, trp1D63, rho4<kanMX4
MATa, his3D200, rho3<LEU2, RHO3-V51
MATa, CDC42, ura3-52
MATa, cdc42-6, ura3-52
MATa/MATa, ura3-52/ura3-52, his3-D200/his3-D200, ade2-101/ade2-101, trp1-901/trp1-901,
leu2-3,112/leu2-3,112, gal4-542/gal4-542, gal80-538/gal80-538, lys2<GAL1-HIS3/LYS2,
URA3<(GAL4 17-mers)3-CYC1-lacZ/URA3<GAL1-lacZ
EUROFAN
EUROSCARF
EUROSCARF
EUROSCARF
Our lab
D. Gallwitz
Our lab
Y. Matsui
Our lab
Our lab
P. Brennwald
P. Brennwald
P. Brennwald
Louvet et al. (1997)
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FEMS Yeast Res 11 (2011) 179–191
181
Tos2p a functional partner of the Rgd1-Rho3 module
C-terminus was fused to the yEGFP. The TOS2–GFP fusion
gene in pUG35-TOS2 and pUG23-TOS2 plasmids was under
the control of the MET25 promoter. TOS2 expression from
the MET25 promoter was repressed by adding 2 mM methionine in the medium. Plasmids pUHA35 and pUHA36 were
constructed by replacing the green fluorescent protein (GFP)
sequence of pUG35 and pUG36 (Niedenthal et al., 1996) by a
sequence encoding the 3HA epitope. The pUHA35 and
pUHA36 vectors allow 3HA tagging at the C-terminal and
N-terminal ends of the protein, respectively. The TOS2
coding sequence was inserted into the pUHA35 to yield
pUHA35-TOS2. Similarly, the wild-type and constitutively
activated (Q74L) forms of Rho3p (Roumanie et al., 2000)
were tagged with 3HA at the N-terminus by inserting the
coding sequences into the pUHA36 vector. The Rgd1p
localization was followed by the use of the RGD1-3xGFP
construct (Prouzet-Mauleon et al., 2008) integrated into the
RGD1 locus of appropriate strains.
reported previously (Doignon et al., 1999). The pODB80
plasmids encoding the DNA-binding domain fused to the
inactivated (GDP-bound) of Rho3p (T30N) and Rho4p
(T86N) forms or to wild-type forms were constructed after
PCR amplification corresponding to RHO3 and RHO4
sequences cloned in the pCM185 plasmid (Roumanie et al.,
2000). The two-hybrid interactions were analysed both by
monitoring growth on selective medium and b-galactosidase activity. Yeast strains containing plasmid combinations
that expressed only the different forms of Rgd1p or Tos2p
fused with the different domains of Gal4p did not grow on
selective medium or express b-galactosidase activity. c-Fos
and c-Jun, two proteins known to interact strongly, were
used as a standard positive control in the yeast two-hybrid
protein–protein interaction assay by introducing the plasmids pPC76 (Fos-DB) and pPC79 (Jun-AD) (Chevray &
Nathans, 1992) into the HY strain.
In vitro protein-binding assays
Yeast two-hybrid assays
The RGD1 coding sequence (aa 1–666) fused to the Gal4p
DNA-binding domain of pODB80 (TRP1 marker) was used
as a bait and introduced into the yeast HY diploid strain
(Louvet et al., 1997). The resulting strain was transformed
with a yeast DNA library fused to the Gal4 activation domain
(Clontech). The library was estimated to contain 6 106
independent clones with an insert size of 1.1 kb. Transformed
HY cells were plated onto synthetic medium without leucine,
tryptophan and histidine to allow the detection of clones that
trigger the activation of reporter genes. A stringent response
was elicited by adding 3 mM 3-amino 1,2,3-triazole; His1
clones were scored after replating on selective medium.
Further confirmation of the interaction was obtained by
measuring the expression of the second reporter gene, lacZ,
in yeast strains (Louvet et al., 1997).
Interactions between different forms of the RGD1 and
TOS2 genes were tested using the pODB80 and the pACT2
(LEU2 marker) plasmids carrying the Gal4p activation
domain (Clontech). The entire ORF of TOS2 (aa 1–622)
and sequences corresponding to the TOS2D TM sequence
(aa 62–622) and C-TOS2 (aa 410–622) identified in the twohybrid screen were amplified by PCR from genomic DNA
and inserted in-frame into the activation domain (AD) of
pACT2. Likewise, the RGD1 sequences encoding truncated
forms containing the FCH and coiled coil (aa 1–183), the
coiled coil and the GAP (aa 141–666) and the GAP (aa
483–666) regions were PCR amplified and fused to the DB
domain in pODB80. DNA encoding the entire coding
sequence (aa 1–734) and the C-ter (aa 522–734) of SKG6
was also PCR-amplified and inserted into both pODB80 and
pACT2 plasmids. The production of activated forms of
Rho3 (Q74L) and Rho4 (Q131L) using pODB80 has been
FEMS Yeast Res 11 (2011) 179–191
The GST-Rgd1 and GST proteins used for in vitro binding
reactions were produced in yeast S. cerevisiae using a
galactose-inducible expression system and proteins were
purified with glutathione-Sepharose beads essentially as
described previously (Roumanie et al., 2001). The yield of
GST-Rgd1 and GST proteins was estimated by Coomassie
blue staining after separation by sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) and by
Western blotting with anti-Rgd1 antibodies. The GSTRgd1p was estimated to 4 90% of the total protein amount
in the pull-down extract. The protein concentration was
determined by comparing the intensities of bands with
calibrated molecular weight standards (GE healthcare).
The region encoding amino acids 410–622 of Tos2p was
PCR-amplified from yeast genomic DNA and inserted into
the pIVEX2.3-MCS vector (Roche). The resulting plasmid
was used in coupled in vitro transcription–translation reactions using the RTS 100 E. coli HY kit (Roche) in the
presence of [35S] methionine, following the manufacturer’s
instructions. For the binding assay, 5 mL of the [35S]
methionine-labelled reaction mixture containing the radioactive C-Tos2p diluted in 85 mL binding buffer (10 mM
HEPES pH 7.4, 140 mM KCl, 2 mM MgCl2, 1% Triton
X-100) was preincubated with 30 mL glutathione-Sepharose
beads for 30 min on ice, followed by centrifugation at
10 000 g for 2 min. The supernatant was then used in a
100 mL binding reaction containing 1 mM of GST or GSTRgd1 proteins immobilized on glutathione–Sepharose beads
(15 mL). The reaction was incubated for 2 h at 4 1C with
slight agitation and centrifuged at 10 000 g for 2 min. The
supernatant (80 mL) was boiled in sample buffer; the pellet
was washed five times with 400 mL binding buffer to remove
nonspecific interacting proteins before boiling in sample
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182
buffer. Equal volumes of samples were subjected to SDSPAGE, stained with coomassie blue, dried and exposed to an
imaging screen on an FLA-5100 (Fujifilm).
Fractionation and membrane extraction of
proteins
Experiments were carried out using the protocols as
described previously (Lodder et al., 1999; Toenjes et al.,
2004). The BY4742 strain, the BY4742 strain transformed
with pUHA-TOS2 and expressing Tos2-3HA, and the
FY1679 strain transformed with YEp352-WSC13HA (Rajavel
et al., 1999) and expressing Wsc1-3HA (Fernandes et al.,
2006), were grown in minimal medium to the mid-log phase
(0.5 OD600 nm). Cells (25 U OD600 nm) were harvested,
washed once with cold water and lysed in lysis buffer
[0.8 M sorbitol, 50 mM Tris HCl pH 7.6, 1 mM EDTA,
1 mM phenylmethylsulphonyl fluoride supplemented with
a protease inhibitor cocktail (Sigma)] with glass beads using
a Mini-beadbeater (Biospec Products) at 4 1C. Cell lysates
were clarified by spinning at 500 g for 5 min at 4 1C. The
supernatants were centrifuged at 13 000 g for 10 min at 4 1C
to yield supernatant (S13) and pellet (P13) fractions. P13
was resuspended in a volume equal to that of the supernatant. Equal volumes of supernatant and pellet fractions
were separated on 8% SDS-PAGE and immunoblotted with
anti-HA (12CA5) or anti-Rgd1 antibodies.
For membrane extraction experiments, the lysate obtained
after clearing at 500 g was divided into several aliquots.
Aliquots were incubated at 4 1C for 15 min with 1 M NaCl,
1% Triton X-100, 1 M urea or a mixture of 1% Triton X-100
and 1 M urea in a final volume of 500 mL of lysis buffer. As a
control, one aliquot was incubated without adding any
extracting reagent. Lysates were then spun at 13 000 g for
10 min at 4 1C. P13 was resuspended in lysis buffer and S13
and P13 were separated on polyacrylamide gels and immunoblotted. The intensity of bands on a Western blot was
evaluated by densitometry analysis.
Pull-down assay for GTP-bound Rho3p
The amount of GTP-bound Rho3p was analysed in vivo
using the Rho pull-down assay (Ren et al., 1999) and
developed in the yeast Schizosaccharomyces pombe by
Calonge et al. (2003). It is based on the ability of the Rhobinding domain (RBD) from the effector protein Rhotekin
to bind human Rho proteins (Ren et al., 1999). The wildtype, rgd1D and tos2D strains were transformed with the
plasmid pUHA36-RHO3 expressing the wild-type 3HARho3p; the wild-type strain was also transformed with the
plasmid pUHA36-GTP-RHO3 expressing constitutively
activated Rho3p. The TOS2-3HA construct from the appropriate plasmid was also expressed in the wild-type strain
carrying the pUHA36-RHO3. For this purpose, the URA3
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S. Claret et al.
marker of pUHA35-TOS2 was replaced by the HIS3 marker
from pUG34 (Niedenthal et al., 1996). Transformants were
selected on SD medium for uracil and/or histidine prototrophy. Cells were grown in the absence of methionine for
the production of different tagged proteins and collected at
0.6 OD600 nm. Lysates were obtained by breaking cells with
glass beads at 4 1C. The protein concentration of lysates was
determined using the Bradford assay, the samples were
adjusted to the same concentration and for each sample a
total of 500 mg protein was used in the pull-down assay.
GTP-bound Rho3p was pulled down from the cell extract by
binding to GST-C21RBD (rhotekin RBD), previously
obtained and purified from Escherichia coli (Reid et al.,
1996). One fifth of Rho3 retained on GST-RBD beads was
loaded on a polyacrylamide gel. The total amount of Rho3p in
different strains was determined by loading equal quantities of
protein (2.5 mg per lane) from yeast lysates on the gel. AntiHA 12CA5 monoclonal antibodies were used for Western blot
analysis. Determination of the amounts of pulled-down GTPbound and total Rho3p levels in each condition were based on
band intensity quantification. Densitometric analysis was
performed from nonsaturated films.
Results
Identification of Tos2p as an Rgd1p physical
partner
Using a two-hybrid approach, a yeast genomic DNA library
for proteins interacting with full-length Rgd1p was screened.
We identified one clone that displayed a specific interaction.
Using a positive control in a two-hybrid interaction based
on c-Fos and c-Jun, two proteins known to interact strongly,
this interaction was estimated at third of the control.
Sequence analysis revealed that it encoded the carboxyterminal 213 amino acids of the protein Tos2. In silico
analysis of full-length Tos2p revealed a single transmembrane domain in the amino-terminal part of the protein (aa
37–62) and a potential PEST sequence (aa 295–328; score
6.05 with ePESTfind program – http://www.expasy.org/
tools/; threshold of 5.0) (Fig. 1a). To determine whether
these sequences affected the binding efficiency of Tos2p to
Rgd1p, we performed two-hybrid analysis using full-length
and truncated forms of Tos2p (Fig. 1b). We observed that
the strength of interaction was not modified in the presence
of the transmembrane domain or of the PEST motif. The
region between amino acids 410 and 622 was sufficient for
interaction with Rgd1p. We reported (Roumanie et al.,
2000) that the Rgd1 protein comprises an N-terminal FCH
domain (aa 33–144), followed by a coiled-coil motif (aa
139–172), both belonging to an F-BAR domain (ProuzetMauleon et al., 2008) and a RhoGAP domain at the carboxyterminal end (aa 486–666). We then determined the Rgd1p
FEMS Yeast Res 11 (2011) 179–191
183
Tos2p a functional partner of the Rgd1-Rho3 module
Fig. 1. The C-terminal region of the Tos2 protein interacts with the Rgd1p RhoGAP. (a) Schematic representation of Tos2 protein domains.
Transmembrane (TM) and PEST motifs were identified using the TMpred and ePESTfind algorithms. Numbers indicate amino acid positions. (b) Twohybrid interactions between Rgd1p and Tos2p. Yeast two-hybrid assays were carried out as described in Materials and methods. HY diploid cells containing
various combinations of the Gal4 DNA-binding domain and Gal4-activating domain fusions were tested for the transcriptional activation of the HIS3
reporter gene onto SD minus tryptophan, leucine and histidine (SD–TLH) containing 3-amino-1,2,3-triazole (3 mM). Plates were shown after 2 days of
incubation at 30 1C. b-Galactosidase activity was indicated as nanomoles of cleaved O-nitrophenyl-b-D-galactopyranoside per minute and per milligram
of yeast total proteins. , no interaction; 1, significant interaction. (c) Interaction of in vitro translated carboxy-terminal region of Tos2p (C-Tos2) with
Rgd1p. C-Tos2p was produced and radiolabelled by coupled in vitro transcription–translation. Reaction products were incubated with 1 mM of
immobilized GST or GST-Rgd1 fusion protein and washed. Bound and unbound C-Tos2p was separated by SDS-PAGE and analysed by autoradiography.
The band corresponding to the C-Tos2 translation product is indicated by an arrow and the percentage of protein product bound to GST-Rgd1 is
determined by comparison with the total C-Tos2 amount. The lower band indicated by an asterisk presumably represents a breakdown product.
domain able to bind Tos2p in a two-hybrid assay testing
different regions of Rgd1p with C-Tos2p, the carboxyterminal region of Tos2p encompassing residues 410–622.
We found that the FCH and coiled-coil domains were not
necessary for interaction (Fig. 1c). In fact, a truncated form
FEMS Yeast Res 11 (2011) 179–191
of Rgd1p (aa 483–666) containing only the GAP domain
was found to be sufficient for binding C-Tos2p (Fig. 1c).
Tos2p is similar to Skg6p (Tomishige et al., 2005), with an
overall 35% identity. C-Tos2p, in particular, is closely related
to the Skg6p carboxy-terminal region (aa 522–734), with a
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184
higher identity score of 52%. We tested the ability of the
Rgd1p RhoGAP domain to interact with the Skg6 fulllength protein or its C-terminal region (aa 522–734) using
two-hybrid assays. No interaction was detected with either
of the two forms of Skg6p tested. Tos2p interacts with the
GAP domain of Rgd1p, the GAP for the Rho3p and Rho4p
GTPases; thus, we tested whether Tos2p also interacted with
Rho3p or Rho4p. We did not observe any interaction
whatever the GTPase form used, i.e. wild-type, activated or
inactivated forms. Thus, the two-hybrid interaction of
Tos2p is restricted to the Rgd1 RhoGAP domain.
To support our results, we produced C-Tos2p (aa 410–622)
– identified above as sufficient to interact with Rgd1p – in
vitro. We tested the ability of this construct to bind the
GST-Rgd1p fusion protein produced in yeast and immobilized on beads. We found that C-Tos2p specifically associated
with GST-Rgd1p and not with GST alone (Fig. 1d). Altogether, these results show that the Tos2 protein through its
carboxy-terminal region is a physical partner of Rgd1p and
two-hybrid assays provide evidence that this interaction might
be mediated by the RhoGAP domain of Rgd1p.
Both Tos2p and Rgd1p fractionate in detergentresistant membranes (DRMs)
Toenjes et al. (2004) reported that Tos2p may help anchor
Cdc24p, the GEF for Cdc42p, to sites of polarized growth.
Thus, we examined whether Tos2p was associated with
Rgd1p in vivo and influenced the Rgd1p localization.
In silico analysis of Tos2p predicted one transmembrane
domain. We confirmed that Tos2p was an intrinsic membrane protein. The Tos2p protein tagged with 3HA at its
carboxy-terminus was produced from the low-copy plasmid
pUHA35. Its fractionation was analysed after a centrifugation at 13 000 g for 10 min. Tos2p was recovered in the pellet
(P13) and a large smear (Fig. 2) ranging from 85 to
4 130 kDa was observed instead of a band corresponding
to the predicted molecular mass (73 288 Da), suggesting the
existence of multiple post-translational modifications. This
indicated that Tos2-3HA was associated with large membrane structures. The solubility of Tos2-3HA was examined
by incubating cell extracts with various chemicals (Fig. 2).
The tagged protein was resistant to solubilization after
treatment with 1 M NaCl, 1% Triton X-100, 1 M urea and
with a mixture of Triton X-100 and urea, followed by
spinning at 13 000 g. Control experiments using the single
transmembrane Wsc1p-3HA protein (Fig. 2) showed partial
solubilization with triton X-100 and total solubilization with
the mixture of triton X-100 and urea (Fig. 2), as reported
previously (Lodder et al., 1999). Our results suggest that
Tos2p is associated with DRMs (Bagnat et al., 2000; Wachtler & Balasubramanian, 2006). As Rgd1p possesses an
F-BAR domain at its N-terminus, a domain described as
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S. Claret et al.
Fig. 2. Tos2p and Rgd1p are distributed in DRM fractions. Cell lysates
were prepared from reference cells (BY4742) and from cells expressing
Tos2-3HA or Wsc1-3HA grown at 30 1C at OD600 nm of 0.5 in minimal
medium. The supernatants obtained after a 5-min centrifugation at
500 g were centrifuged at 13 000 g for 10 min. The supernatant (S) and
pellet (P) were separated on an 8% polyacrylamide gel and immunoblotted with anti-HA or with anti-Rgd1p antibodies. The supernatant
collected after the 500 g centrifugation was incubated for 15 min at 4 1C
with the indicated compounds before centrifugation at 13 000 g.
involved in membrane binding and tubulation (Itoh et al.,
2005; Tsujita et al., 2006; Shimada et al., 2007), we also
examined the fractionation of Rgd1p in the supernatant and
the pellet after centrifugation. The RhoGAP sedimented at
13 000 g, in agreement with the presence of an F-BAR;
treatment with the same chemicals used for Tos2p did not
modify the fractionation of the RhoGAP (Fig. 2). Although
surprising for a protein without a transmembrane domain,
these findings suggest that Rgd1p was also associated with
DRMs like its partner Tos2p and that both proteins are
located in the same cellular compartment. These fractionation studies are consistent with previous studies showing
that both Tos2p and Rgd1p localize to sites of polarized
growth (Drees et al., 2001; Prouzet-Mauleon et al., 2008).
As shown previously for Cdc24p (Toenjes et al., 2004), we
hypothesized that Tos2p may affect Rgd1p anchoring at
membrane structures. Thus, we examined the fractionation
of the RhoGAP in tos2D and in wild-type cells overexpressing TOS2. We observed in both genetic backgrounds that
Rgd1p was recovered in P13 similar to what happens in the
wild type (data not shown). To further address the role of
Tos2p in the interaction with the RhoGAP Rgd1p, we
investigated the effect of TOS2 inactivation and overexpression on the subcellular distribution of Rgd1p. Rgd1-3GFP
was localized to polarized growth sites in tos2D mutant as in
wild-type cells overexpressing TOS2; the distribution of
Rgd1p at the bud and the neck and other localization in
both strains (Supporting Information, Fig. S1) were similar
to that already reported (Prouzet-Mauleon et al., 2008).
However, when TOS2 was overexpressed, nearly half the cells
presented an elongated bud. Nevertheless, the distribution
of Rgd1p between the bud tip and the bud neck in cells
FEMS Yeast Res 11 (2011) 179–191
185
Tos2p a functional partner of the Rgd1-Rho3 module
showing an elongated bud was not significantly different
from wild-type cells. In the cells with an elongated bud, the
fluorescence associated with Rgd1p-3GFP appeared to be
restricted to the tip of the growing bud (Fig. S1). Although
Tos2p and Rgd1p could locate in the same cellular compartment, the latter results indicated that the Rgd1p/Tos2p
interaction was not necessary for the Rgd1p localization
and distribution in yeast.
The level of activated Rho3p is regulated by
Tos2p
The two-hybrid interaction of a Tos2p region with the
RhoGAP domain of Rgd1p suggested that Tos2p acts
through Rgd1p to activate GTP hydrolysis by the Rho3p
and Rho4p GTPases. Thus, we tested Rgd1p RhoGAP
activity in the presence and absence of Tos2p. The addition
of full-length Tos2p or C-Tos2p (aa 410–622) to an in vitro
RhoGAP activity assay for Rgd1p (Doignon et al., 1999) did
not affect the quantity of GTP hydrolysed by Rho3p or
Rho4p (data not shown). To examine the effect of Tos2p in
vivo, we analysed the amount of activated GTPases in S.
cerevisiae tos2D mutant and in wild-type cells overexpressing
TOS2. We used the Rho-GTP pull-down assay developed by
Ren et al. (1999) and previously applied to fission yeast S.
pombe (Calonge et al., 2003). The RBD from human
Rhotekin fused to a GST tag was used for affinity precipitation of cellular GTP-Rho3 and GTP-Rho4 proteins. However, under our experimental conditions, this approach was
only successful with Rho3p. The Rho3p GTPase was tagged
at its N-terminus with the 3HA epitope. The amount of
Rho3 protein from the extracts was determined by Western
blotting using the anti-HA 12CA5 monoclonal antibody.
GTP-bound Rho3 proteins were pulled down from the
extracts with GST-C21RBD, carrying the rhotekin-binding
domain (Fig. 3a). A wild-type strain expressing the constitutively GTP-bound or the wild-type 3HA-Rho3 form was
used to validate this GST-pull-down assay. GST-RBD beads
retained substantial amounts of the GTP-bound Rho3p
from lysates of cells expressing the constitutively GTPbound 3HA-Rho3 form, but only a small fraction of the
total wild-type 3HA-tagged Rho3p (Fig. 3a); Rho3 proteins
were not precipitated with the GST domain alone. We
repeated this experiment using the rgd1D strain producing
wild-type tagged Rho3p. The proportion of Rho3p obtained
from rgd1D lysates was higher than that obtained from wildtype lysates, consistent with Rgd1p GAP activity in these
cells (Fig. 3a). These findings demonstrated that the GSTRBD bait could be used to evaluate the proportion of active
GTP-bound Rho3p in S. cerevisiae.
We then determined the effect of TOS2 inactivation and
overexpression on the amount of active Rho3p in vivo. The
amount of GTP-bound Rho3p detected in the TOS2-overFEMS Yeast Res 11 (2011) 179–191
Fig. 3. Inactivation of TOS2 increases the amount of active Rho3p. (a)
WT cells (BY4742) overexpressing Tos2-3HA or not and the rgd1D and
tos2D mutant cells in the BY background were transformed by the vector
pUHA36-RHO3 or pUHA36-GTP-RHO3. The plasmids pUHA36-RHO3
and pUHA36-GTP-RHO3- allow the production of either the wild-type
(HA-Rho3) or constitutively GTP-bound Rho3 (HA-GTP-Rho3) tagged
with 3HA. GTP-bound Rho3p was pulled down from cell extracts with
GST-C21RBD and detected using the 12CA5 antibodies, as described in
Materials and methods. GTP-bound Rho3p (lanes 1) and total HA-Rho3p
(lanes 2) were revealed by a Western blot. The presence (1) or absence
( ) of vectors expressing the proteins is indicated on the left-hand side.
(b) Quantitative analysis of GTP-bound Rho3p in the different strains
after the pull-down assays. Bands were scanned and the corresponding
protein levels were evaluated. The amount of pulled-down GTP-Rho3p
relative to the total Rho3p levels was determined for each strain and the
ratios were expressed as folds vs. the control from wild type (WT). Data
are mean SD from three independent experiments.
expressing strain was similar to that observed in wild-type
cells. By contrast, TOS2 inactivation led to increased
amounts of GTP-bound Rho3p, with the signal intensity
higher than that observed in the reference strain (Fig. 3a).
The amounts of pulled-down GTP-bound relative to the
total Rho3p levels under each condition were determined on
band intensity quantification from three independent experiments and the ratios were expressed as folds vs. the WT
control (Fig. 3b). This analysis highlighted the effect of
Tos2p absence on the abundance of the active Rho3p in yeast
cells. When TOS2 was overexpressed, the quantification was
equivalent to that observed for the wild type. Altogether,
these data show that Tos2p participates in the in vivo
modulation of Rho3p activation.
Functional relationship between TOS2 and RGD1
To investigate the functional significance of the physical
interaction between Rgd1p and Tos2p and to know whether
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186
S. Claret et al.
vector lowered the rgd1 sensitivity to low pH (Fig. 4). The
same effect was observed with pRS426-TOS2, a high-copy
vector in which the TOS2 gene was not modified and under
the control of its own promoter (data not shown). This
response indicated that the TOS2 gene partially suppressed
the RGD1 inactivation. This suppression, observed in the
absence of Rgd1p, demonstrated that TOS2 must act on
alternative pathways to suppress the rgd1D sensitivity to low
pH through mechanisms that do not involve the interaction
between Tos2p and the RhoGAP.
Functional relationship between TOS2 and RHO3
Fig. 4. Cell death in wild-type and rgd1D cells overexpressing TOS2
after a shift to a low pH. Wild-type (FY1679a) and rgd1D cells transformed with the vector pUG35 with or without the TOS2 gene were
grown at 30 1C to an OD600 nm of 0.5 in minimal medium. pH was
decreased from 3.6 to 2.8. Cell lethality was determined 6 h after the
acid shock. Results were obtained from three independent experiments;
the bar indicates the SDs.
these proteins act in common pathways, we first examined
the effects of TOS2 deletion or overexpression in wild-type
cells and compared these phenotypes with the rgd1D mutant
phenotype. RGD1 inactivation leads to sensitivity to low
pH; mortality of exponentially growing rgd1D cells reaches
50% 6 h after an acidic shock (Gatti et al., 2005). Cells
inactivated for TOS2 had a normal size and morphology, as
reported previously (Gandhi et al., 2006), and their viability
was not affected by growing the mutant cells at a low pH.
Concerning wild-type cells overexpressing TOS2, they
exhibit normal growth and viability under standard growth
conditions; however, as mentioned above for cells containing the pUHA35-TOS2 vector, overexpression from the
pUG35-TOS2 vector also resulted in cell morphology alterations. Induction of TOS2 expression from the MET25 promoter in a low-copy plasmid led to elongated buds as
described previously (Gandhi et al., 2006). Our findings
showed the involvement of TOS2 in polarized growth and in
controlling the timing of apical and isotropic growth as
proposed by Gandhi et al. (2006). Cells overexpressing TOS2
from the MET25 promoter were slightly sensitive to a low pH;
a shift to acidic pH induced cell death in about 18% of
growing cells (Fig. 4).
To carry on the study on the links between Rgd1p and
Tos2p, the effects of TOS2 overexpression or inactivation
were also analysed in the rgd1D genetic background. The
deletion of TOS2 in the rgd1D strain did not change the rgd1
mutant sensitivity to low-pH shock (data not shown). On
the contrary, the overexpression of TOS2 from the pUG35
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Pull-down experiments revealed the effect of Tos2p absence on
the abundance of the active Rho3p in yeast cells. Therefore, we
also analysed the functional relationships between TOS2 and
the genes encoding the Rho3 and Rho4 GTPases regulated by
Rgd1. Initially, we overexpressed the TOS2 gene in the
temperature-sensitive rho3-1 rho4D strain; rho3-1 rho4D cells
did not grow at 37 1C (Imai et al., 1996). The TOS2 overexpressing cells as well as the control grew normally at 25 1C,
but did not grow at 37 1C. However, we observed that
overexpression of TOS2 reproducibly enhanced the Ts growth
phenotype with the absence of growth at 35 1C (Fig. 5),
indicating a negative effect of TOS2 overexpression in this
strain. This effect may be linked to a lack of function of Rho3p,
Rho4p or both GTPases. Thus, the effect of TOS2 overexpression was examined in the single mutants (Fig. 5). We used the
strains deleted for RHO3 and RHO4; the rho3D has a nearly
normal vegetative growth in a medium without an osmotic
stabilizer. The rho3D and rho4D strains were transformed with
the pUG35-TOS2. We observed that the overexpression of
TOS2 was detrimental to the growth of rho3D. This effect was
eliminated by repressing the MET25 promoter controlling
TOS2 expression by adding methionine in the medium. No
growth difference was detected in the rho4D background when
TOS2 expression was induced. These results suggest that the
negative effect observed in the rho3-1 rho4D double mutant
was linked to Rho3 function.
Rho3 acts as a regulator of cell polarity and exocytosis,
coordinating several distinct events for the delivery of
proteins to specific sites on the cell surface. The rho3-V51
allele exhibited a well-polarized actin cytoskeleton, but
showed a severe defect in secretion after a shift to the
restrictive temperature (Adamo et al., 1999). rho3-V51 cells
grown at 14 1C show a cold-sensitive defect in the delivery of
vesicles from mother to bud. Whereas the rho3-V51 strain
transformed with the control vector did not grow at 14 1C,
the same strain overexpressing TOS2 had a growth partially
restored (Fig. 5). These strains presented a similar growth at
the permissive temperature (25 1C). Thus, the overexpression of TOS2 can suppress the growth defect due to the
rho3-V51 mutation. The cdc42-6 allele also revealed a role
FEMS Yeast Res 11 (2011) 179–191
187
Tos2p a functional partner of the Rgd1-Rho3 module
for Cdc42 in docking and fusion of secretory vesicles that
was independent of its major functions in cell polarity
(Adamo et al., 2001; Roumanie et al., 2005). Similar to what
was observed for the rho3-V51 mutant, cdc42-6 cells have a
severe exocytic defect, but show normal localization of
markers for polarized exocytosis (Roumanie et al., 2005).
In addition, the thermosensitive rho3-V51 and cdc42-6
mutations are suppressed by a same set of genes (Adamo
et al., 2001). Therefore, we also tested TOS2 overexpression
in the cdc42-6 strain (Fig. 5). cdc42-6 cells did not grow at 33
and 37 1C (Roumanie et al., 2005). TOS2 overexpression did
not restore cdc42-6 growth at the restrictive temperature,
but we observed a strongly reduced growth at 25 1C. This
negative effect was not observed in the CDC42 background
and was dosage dependent (data not shown). Thus, the
TOS2 gene is not a suppressor of the cdc42-6 mutation and
the suppression effect is restricted to the rho3-51 mutation.
TOS2 might compensate a specific function associated with
the Rho3 GTPase.
Discussion
Fig. 5. Effect of TOS2 overexpression in RHO3, RHO4 and CDC42
mutated backgrounds. The rho3-1 rho4D double mutant and the rho3D
and rho4D single mutants were transformed with the control vector
pUG35 ( ) and with pUG35-TOS2 (1TOS2). Serial dilutions were
spotted onto SD medium minus uracil. rho3-1 rho4D cells were incubated at the permissive temperature (25 1C), at the nonpermissive
temperature (37 1C) and at 35 1C; rho3D and rho4D cells were incubated
at 25 1C under the inducing ( methionine) or the repressing (1methionine) conditions for TOS2 expression. rho3-1 rho4D and rho4D strains
were grown for 2 days and rho3D for 4 days. rho3-V51 cells were
transformed with the control vector pUG23 ( ) and with pUG23-TOS2
(1TOS2) and spotted onto SD medium minus histidine. rho3-V51 cells
were grown at 25 1C for 3 days and at the restrictive temperature (14 1C)
for 5 days. The cdc42-6 mutant and the CDC42 control strains were
transformed with pUG35 ( ) and pUG35-TOS2 (1TOS2). Transformed
cells were spotted onto SD medium minus uracil and grown for 2 days at
25 and 33 1C (restrictive temperature for the cdc42-6 mutation).
FEMS Yeast Res 11 (2011) 179–191
We reported previously that Rgd1p is functionally linked to
Las17p/Vrp1p, the yeast homologues of human WASP and
WIP (Roumanie et al., 2000) as well as components of the
protein kinase C (PKC) pathway, which control cell wall
integrity in S. cerevisiae (de Bettignies et al., 1999, 2001). To
gain an insight into Rgd1p function, we screened for
physical partners in a two-hybrid screening assay. Tos2p
was identified as a candidate Rgd1p-binding protein.
Further analyses confirmed that Tos2p directly binds to
Rgd1p through its C-terminal region and two-hybrid studies allowed us to speculate that the RhoGAP domain of
Rgd1p is involved in this interaction. Consistent with this
interaction, the Tos2p protein localizes to sites of cell growth
including the nascent bud site, bud tip and bud neck (Drees
et al., 2001; Sundin et al., 2004), overlapping with the
localization of Rgd1p (Prouzet-Mauleon et al., 2008).
To confirm the interaction between Tos2p and Rgd1p, we
explored the characteristics of these proteins as well as their
functions in a yeast cell. Based on our fractionation results,
Tos2p is an integral membrane protein and, given its cellular
localization and effect on the GEF Cdc24p (Toenjes et al.,
2004), Tos2p must be inserted into the yeast plasma
membrane. Following the treatment of cell membranes with
Triton X-100, Tos2p was recovered after centrifugation in
the nonsolubilized residue, indicating that it is associated
with DRMs. Surprisingly, for a protein without transmembrane domain, the fractionation and solubilization of Rgd1p
RhoGAP yielded results similar to those obtained for Tos2p,
indicating that Rgd1 is also associated with DRMs. A large
majority of yeast plasma membrane proteins studied for
their potential association with rafts have been found in
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188
DRMs (Malinska et al., 2004); however, DRMs do not
simply equate to lipid rafts in their native state (Wachtler &
Balasubramanian, 2006). For instance, it was reported that
the TOR kinases, TORC1 and TORC2 in yeast fractionate
with a novel form of detergent-resistant-associated membranes (Aronova et al., 2007). A significant number of
proteins identified within TOR-DRM correspond to proteins implicated in processes related to actin polymerization
and/or endocytosis. Preliminary experiments using an iodixanol gradient indicate that Rgd1p associated with DRMs
that are distinct from Pma1p-containing plasma membrane
rafts. These fractionation properties are due to the F-BAR
domain. Recently, in membrane flotation experiments in an
Optitrep gradient, the F-BAR protein Cdc15 from S. pombe
was also described to be located between the bottom of the
gradient and the top fraction containing Pma1p (RobertsGalbraith et al., 2010). The F-BAR domain that was involved
in interaction with lipid bilayers in vitro and in membrane
binding (Itoh et al., 2005; Tsujita et al., 2006; Shimada et al.,
2007) could have particular characteristics with regard to the
membrane solubilization. Thus, Tos2p and Rgd1p are associated with membranes structures, and their similar behaviour
following chemical treatment and centrifugation could be
indicative of a localization of these proteins in a same cellular
compartment consistent with an in vivo interaction.
We observed that Tos2p did not affect the RhoGAP activity
of Rgd1p in vitro, suggesting that Tos2p does not act directly
on the ability of Rgd1p to activate GTP hydrolysis by Rho3p
and Rho4p. However, using a pull-down assay, we showed that
the depletion of Tos2p results in sustained levels of activated
Rho3p in yeast. To explain this variation, it is tempting to
propose an in vivo regulation of the GAP activity of Rgd1p by
Tos2p. Tos2p might influence the RhoGAP activity through
protein interaction with Rgd1p in a particular conformation
or within a protein complex. Nevertheless, considering that
the Rgd1p GAP activity was not directly measured from the
TOS2-inactivated strain, other mechanisms cannot entirely be
ruled out, for example an action of Tos2p on the GEF of
Rho3p. Tos2p might also influence the active Rho3p abundance in other ways. Indeed, Tos2p interacts with Cdc24p, the
guanidine nucleotide exchange factor required for the activation of Cdc42, and with the protein kinase C Pkc1p, which
functions downstream of the Rho1 GTPase (Drees et al.,
2001). Tos2p may provide a regulatory connection between
Rho1- and Cdc42-regulated pathways and the Rgd1p/Rho3p
module. In this case, an increase in active Rho3p abundance in
the tos2D strain could result from multiple effects including
the lack of Tos2p and those related to the Tos2 partners.
However, whatever the mechanism for explaining the variation of active Rho3p, the data support the functional links
between Tos2p and the GTPase.
The suppressor effect of TOS2 overexpression on rgd1D
sensitivity to low pH supports the existence of a pathway
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S. Claret et al.
compensating the absence of the Rgd1p protein. The
suppression of rgd1D sensitivity may be mediated by interactions of Tos2p with other proteins such as Pkc1p or
Cdc24p (Drees et al., 2001; Toenjes et al., 2004). The Pkc1p
kinase could be implicated in this suppression phenomenon. We demonstrated that components of the PKC pathway
are required for tolerance to low pH and that low-pH stress
activates the PKC signalling pathway through phosphorylation of Slt2p, the last kinase in the MAP kinase cascade
(Claret et al., 2005). Moreover, we reported that the overexpression of several components of the PKC pathway
restores the viability of rgd1 cells at the stationary phase (de
Bettignies et al., 2001). Cell death is caused by acidification
of the medium during growth (Gatti et al., 2005); thus,
activation of the PKC pathway can prevent cell death of the
rgd1D mutant in an acidic medium. Using PST1-lacZ as a
reporter gene for PKC pathway activation (Claret et al.,
2005), we observed that TOS2 overexpression in wild-type
cells increases the basal and activated levels of this pathway
after an acidic shock (Fig. S2). Thus, the suppressor effect of
TOS2 overexpression on rgd1D sensitivity to low pH could
be explained by increased PKC-pathway activation. Altogether, these data, with those already reported (de Bettignies
et al., 2001; Claret et al., 2005), suggest that Tos2p, Pkc1p
and Rgd1p might belong to the same functional network.
Finally, we showed functional interactions between TOS2
and mutated RHO3 alleles. During this analysis, it is
interesting to note the suppressive effect of TOS2 overexpression on the rho3-V51 growth defect. This effect seems
to be associated with a Rho3p-specific function, strengthening the functional relationship between Tos2p and the
GTPase Rho3p. At the restrictive temperature, the rho3-V51
mutant has a defect in the transport of vesicles from the
mother cell into the bud and shows a large accumulation of
vesicles in mother cells. Thus, it would be particularly
interesting to examine the vesicles distribution in the rho3V51 mutant overexpressing TOS2.
We demonstrated in this study that Tos2p is a physical
partner of the Rgd1p RhoGAP and acts on active Rho3
abundance, suggesting an overall effect of Tos2p on the
Rgd1p/Rho3p module. Taking into account our current
results, we can imagine that the protein Tos2p could
participate in the establishment of a cellular environment
modulating in vivo the GAP activity of Rgd1. In addition, we
provided further evidence for the functional links between
Tos2p and Pkc1p (Fig. S2). Toenjes et al. (2004) previously
reported functional links between Tos2p and Cdc24p, the
GEF for Cdc42p. Hence, through interactions with these
partners, Tos2p is involved in several pathways controlling
polarized growth in S. cerevisiae and could fine-tune signalling between the different pathways to optimize yeast
growth. Phosphorylation of Tos2p by Cdc28p, a cell cycle
cyclin-dependent kinase (Ubersax et al., 2003), together
FEMS Yeast Res 11 (2011) 179–191
189
Tos2p a functional partner of the Rgd1-Rho3 module
with the localization of Tos2p at the bud tip and bud neck,
might contribute to this cellular role during growth by
interacting with Cdc24p, Pkc1p and Rgd1p at particular
times and locations. The lack of an obvious secondary
structure for Tos2p could explain its ability to interact
physically with different proteins, with Tos2p adapting its
conformation according to the protein partner. Further
studies will be necessary to understand the intriguing role
of Tos2p in signalling between these different pathways all
involved in polarized growth.
Acknowledgements
We thank D. Gallwitz for providing the tos2D in an FY
background. We also thank P. Brennwald for the gift of rho3V51 and cdc42-6 strains and Y. Matsui for the rho3-1 rho4D
strain. We are grateful to C. Barthe for the isolation of the
TOS2 clone in the initial two-hybrid screening. This work was
supported by grants from University Bordeaux 2 and CNRS.
Authors’contribution
S.C. and O.R. contributed equally to this work.
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Tos2p a functional partner of the Rgd1-Rho3 module
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Rgd1-3GFP was observed by fluorescence microscopy in tos2D and wild-type strains as well as in wild-type
strains overexpressing TOS2 from pUHA35 or not.
FEMS Yeast Res 11 (2011) 179–191
Fig. S2. Effect of TOS2 overexpression on the expression of
the PST1-lacZ reporter gene after low-pH shock.
Please note: Wiley-Blackwell is not responsible for the
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material) should be directed to the corresponding author
for the article.
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