Institut für Mikrobiologie, Heinrich-Heine-Universität
Düsseldorf, Universitätsstr. 1, Geb. 26. 12, D-40225
Düsseldorf, Germany
* Present address: Universität Hohenheim, Institut für
Lebensmitteltechnologie, Fachgebiet Gärungstechnologie (150f), Garbenstr.
25, D-70599 Stuttgart, Germany
Author for correspondence (e-mail:
heinisch{at}uni-hohenheim.de
)
Accepted 15 May 2002
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Summary |
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Key words: Saccharomyces cerevisiae, MAP kinase, RHO5, Signal transduction, GTPase
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Introduction |
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Similar to many other small GTPases, Rho1p has a set of different target
proteins. In its GTP-bound state it binds to and thereby activates the
ß-1,3-glucan synthase complex
(Drgonova et al., 1996). It is
also involved in regulation of the actin cytoskeleton by interacting with
Bni1p (Fujiwara et al., 1998
).
In addition, interaction with Skn7p, a regulator of oxidative stress response
(Krems et al., 1996
;
Alberts et al., 1998
), has been
reported. With respect to signals ensuring cellular integrity, the main
effector of Rho1p is the yeast protein kinase C homologue Pkc1p
(Nonaka et al., 1995
). This
kinase then activates a MAP-kinase cascade consisting of the MAPKKK Bck1p
(Lee and Levin, 1992
), the
MAPKKs Mkk1p and/or Mkk2p (Irie et al.,
1993
) and the MAPK Slt2p
(Torres et al., 1991
), also
referred to as Mpk1p (Lee et al.,
1993b
). Rlm1p (Watanabe et
al., 1995
) and the SBF complex (consisting of Swi4p and Swi6p)
(Madden et al., 1997
) have
been reported as targets of the MAP kinase Slt2p. Rlm1p then regulates
transcription of a specific set of genes
(Jung and Levin, 1999
).
Apart from signalling through the MAPK cascade Pkc1p activity has been
related to the regulation of a variety of cascade-independent functions,
including oligosaccharyl-transferase activity
(Park and Lennarz, 2000),
ribosomal gene transcription (Li et al.,
2000
), nuclear perturbation caused by high osmolarity
(Nanduri and Tartakoff, 2001
),
microtubule function (Hosotani et al.,
2001
) and regulation of phospholipid synthesis
(Sreenivas et al., 2001
).
In addition, Pkc1p is involved in controlling the dynamics of actin
cytoskeleton organisation. In budding yeast cells the cortical actin patches
are polarized correlating with directed growth of the cells
(Adams and Pringle, 1984;
Lew and Reed, 1995
;
Amberg, 1998
). With the change
to isotropic growth at mitosis the patches redistribute over the bud and
mother cell surface and then reorient after cytokinesis to the mother-bud
junction. The asymetric distribution of actin patches can be disturbed by
different stresses such as hypertonic media or heat shock
(Chowdhury et al., 1992
;
Lillie and Brown, 1994
). The
repolarisation of actin during adaptation to growth at higher temperatures has
been shown to be an event dependent on Pkc1p but not on Slt2p activity
(Delley and Hall, 1999
). This
is especially interesting because this repolarisation is antagonistic to the
depolarisation of the actin cytoskeleton that occurs upon the shift to high
temperatures. The latter is also mediated by Pkc1p but in contrast to the
repolarisation it requires an active MAP-kinase cascade
(Delley and Hall, 1999
).
In addition to Rho1p, five other homologues encoding members of the Rho
family were identified in the S. cerevisiae genome
(Garcia-Ranea and Valencia,
1998) namely RHO2, RHO3, RHO4, RHO5 and CDC42.
Only CDC42 and RHO1 are essential genes. Cdc42p participates
in the establishment of cell polarity and bud site assembly
(Johnson and Pringle, 1990
) as
well as in a late step in exocytosis (Adamo
et al., 2001
). Rho3p and Rho4p are also involved in exocytosis and
affect actin cytoskeleton assembly (Adamo
et al., 1999
). Very little information is available on the
function of Rho5p. Until now no phenotype could be assigned either to its gene
deletion or to its overexpression
(Roumanie et al., 2001
). Three
proteins were found to interact with Rho5p: Med1 1p and Ynl099p were
identified in two high-throughput interaction studies
(Schwikowski et al., 2000
;
Ito et al., 2001
); an
additional interactor, Rgd2p was isolated in a systematic screen for GAP
functions (Roumanie et al.,
2001
). Although the latter finding proved that Rho5p in vivo acts
as a small GTPase, no reasonable hint as to its intracellular role has been
provided to date. Because it is known from higher eukaryotes that different
Rho-type GTPases can function in the same pathway
(Anastasiadis and Reynolds,
2001
), we speculated that, like Rho1p, Rho5p may be involved in
the Pkc1p/cell wall integrity pathway of S. cerevisiae.
The data we present in this work indicate that the signal generated by Rho5p has a negative effect on the output of the MAP kinase branch of the Pkc1p-mediated signal transduction pathway.
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Materials and Methods |
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Plasmids, primers and in vitro mutagenesis
Plasmids and primers used in this study are listed in Tables
1 and
2, respectively. In vitro
mutagenesis (Boles and Miosga,
1995) was carried out using primer RHO5Q91Hneu, M13 reverse primer
and pUC19RHO5hin as a template to generate the megaprimer. The product was
purified and used in a second PCR again with M13 reverse primer but together
with pUC19RHO5her as a template. This resulted in an exchange of a T at
position 263 of the RHO5 open reading frame for a C but did not alter
the amino acid sequence. Instead it removes a PstI site to
distinguish between the wild-type and the mutated allele. Additionally the G
at position 273 was exchanged for a C to alter the coding sequence at amino
acid position 91 from glutamine to histidine.
|
|
Video microscopy
The video microscopy setup consisted of an Axioplan 2 microscope (Carl
Zeiss AG, Feldbach, Switzerland), equipped with a 75W/XBO epifluorescence
illumination source, a motorized stage and a Plan-Neofluar 100x/1.3 Oil
PH3 objective. HiQ filter sets were used (Chroma Technology, Brattelboro, VT).
Because the spectrum of a XBO lamp shows a considerable emission in the
infrared range we doubled the normal heat absorption and reflection filters in
the microscope in order to protect the specimen. Fluorescence excitation was
controlled by a shutter controller in combination with a MAC2000 shutter
system (Ludl controller MAC 2000, Ludl Electronics, Hawthorne, CA). We used a
TE/CCD-1000PB back-illuminated cooled CCD camera mounted on the primary port
in combination with the ST-133 controller (Princeton Instruments, Trenton,
NJ). Microscope, camera and fluorescence shutter were controlled by the
Metamorph v. 3.51 software (Universal Imaging, West Chester, PA). Five Z-axis
planes spaced by 0.5 µm were taken for each fluorescence image and one
plane was taken as a phase-contrast image from every sample. The resulting
pictures were deblured using the 2D deconvolution function of the Autodeblur
6.0 (Autoquant Imaging, Watervliet, NY). After deconvolution the pictures were
retransfered into Metamorph and the fluorescence images were combined using
the maximum option. Phase-contrast and fluorescence pictures were than
combined using the overlay command assigning a red look-up table (LUT) for the
phase-contrast and a green look-up table (LUT) for the fluorescence images.
Final size, contrast and brightness of the pictures was adjusted using Adobe
PhotoShop 6.0.
Strains and media
E. coli strain DH5F' (Gibco BRL, Gaithersburg, MD)
was used throughout this study. S. cerevisiae strains are listed in
Table 3. The rho5
deletion was constructed using one step gene targeting with pUG6
(Güldener et al., 1996
)
and primers delRHO5-5 and delRHO5-3. The bem2 deletion was built
similarly but using primers delBEM2-3, delBEM2-5 and pFA6-HIS3mx6
(Longtine et al., 1998
) as a
template. Standard media and growth conditions were employed
(Sherman et al., 1986
). Rich
media were based on 1% yeast extract and 2% bacto peptone (Difco) and
supplemented with 2% glucose (YEPD) or galactose. Yeast transformants were
selected on minimal medium (0.67% yeast nitrogen base, 2% glucose or
galactose) supplemented with amino acids as described
(Zimmermann, 1975
), and
omitting uracil, tryptophane, leucine or histidine when selecting for the
respective markers. To test for Pkc1p pathway-related phenotypes Calcofluor
white, caffeine and Congo red were added to the media at the concentrations
indicated. For serial-dilution patch tests, cells were grown overnight in rich
medium with 1 M sorbitol, diluted to OD600 0.2 and grown to
OD600 0.8. Then dilutions as indicated in the figures were made and
spotted onto the plates.
|
ß-galactosidase assay
For liquid ß-galactosidase assays cells were grown overnight. Cells
were diluted to an OD600 of about 0.2 and grown to an
OD600 of between 0.6 and 1.0. Cells were washed once with Z-buffer
(16 mM Na2HPO4-7H2O, 6 mM
NaH2PO4, 10 mM KCl, 0.1 mM
MgSO4-7H2O) and the pellet was resuspended in 150 µl
of Z-buffer containing 270 µl ß-mercaptoethanol per 100 ml. 50 µl
of chloroform and 20 µl of 0.1% SDS were added. The samples were vortexed
vigorously for 30 seconds. 700 µl of ONPG solution (Z-buffer with
ß-mercaptoethanol and 1 mg/ml o-Nitrophenyl ß-D-galactopyranosid)
were added and the samples were incubated at 30°C. The reactions were
stopped by addition of 500 µl 1M Na2CO3. Samples were
collected by centrifugation at maximum speed in a microcentrifuge for 10
minutes and the optical density of the supernatant at 420 nm was determined.
The activity was calculated by the formula: (OD420 x 1000) /
(OD600 x reaction time x reaction volume).
Actin staining
For actin staining, yeast cells were grown in glucose containing minimal
medium overnight. Cells were inoculated into 50 ml of fresh minimal medium
containing the carbon source indicated and grown for about 4 hours to an
optical density between 0.3 and 0.6 at 30°C. To observe actin dynamics
under heat shock the cultures were shifted to 37°C. At the times
indicated, 10 ml of culture were taken and cells were fixed by addition of 4%
formaldehyde to the medium with subsequent incubation for 1 hour at 30°C
with gentle shaking. After fixation the yeasts were washed twice with 0.1 M
sodium phosphate buffer (pH 7.0) and resuspended in 50 µl of the same
buffer. To this, 2.5 µl of a 3.3 µM Alexa phalloidine solution in
methanol (Molecular Probes, Eugene, OR) and 2.5 µl of 1% Triton X-100 stock
were added and the mixture was incubated for 30 minutes on ice. Finally cells
were washed three times with 0.1 M sodium phosphate buffer. 5 µl of the
cell suspension on a polylysine-treated microscopy slide under a coverslip
were investigated under the microscope.
Slt2p phosphorylation assay
The determination of Slt2p phosphorylation was carried out as dexcribed
previously (Martin et al.,
2000) with some modifications
(Lorberg et al., 2001
).
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Results |
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|
A rho5 deletion strain exhibits increased caffeine
resistance
The observed increase in activity of the Slt2p MAP-kinase pathway in the
rho5 deletion mutant could be explained in two different ways: (1)
the deletion could result in an impaired cellular integrity that in turn is
monitored by the sensors of the pathway signalling for an increased activity;
or (2) the deletion could affect a function that normally downregulates the
pathway. In order to differentiate between these possibilities we determined
the sensitivity of the deletion mutants to different drugs or increased growth
temperatures. As observed previously
(Roumanie et al., 2001), our
rho5 deletion strain grows normally at 37°C (data not shown).
Therefore we also tested for sensitivity against caffeine
(Fig. 1). Interestingly we
observed an increased resistance of the mutant to the drug when compared with
the isogenic wild-type strain. Thus, in contrast to the wild-type control, the
rho5 deletion proved to be resistant to doses of up to 20 mM
caffeine. Together with the results on pathway activity reported above, this
phenotype indicates a role for RHO5 in the downregulation of the
Slt2p MAP-kinase pathway.
|
Phenotypes of an activated rho5 mutant
Mutants defective in the components of the Slt2p-MAPK pathway usually
display sensitivity against drugs such as caffeine, Calcofluor white and Congo
red (Yoshida et al., 1992;
Kirchrath et al., 2000
). To
provide further evidence for Rho5p being a negative regulator of this pathway
we tested the effect of overexpression of an activated allele of RHO5
for sensitivity against these drugs. For this purpose, we took advantage of
the high conservation of essential residues between the members of the Ras
superfamily of small GTPases. Mutations in specific residues have been
reported to result in hyperactive enzymes. Thus the exchange of glutamine at
position 61 in loop 4 of human p21 Ras leads to reduced intrinsic GTP
hydrolysis with a constitutively active protein
(Krengel et al., 1990
). To
construct a similar allele for RHO5 we selected the corresponding
residue (glutamine 91) of RHO5 and exchanged it for a histidine by in
vitro mutagenesis (for details, see Materials and Methods). The resulting
allele was cloned under the control of the GAL1/10 promotor on a
multicopy plasmid to investigate the effect of overexpression
(Fig. 2). Cells grown on
galactose (i.e. under inducing conditions) did not grow at 37°C and were
sensitive against 0.15 mg/ml Calcofluor white, 7 mM caffeine and 0.3 mg/ml
Congo red. These overexpression phenotypes of a constitutively active allele
are again compatible with Rho5p acting as a negative regulator of the Slt2p
MAP-kinase pathway.
|
Partial complementation of kinase module defects by a rho5
deletion
We proceeded with an epistatic analysis of a rho5 deletion by
combining it with other pathway mutants. A double deletion of bck1
(the MAPKK-kinase) and rho5 did not suppress the temperature
sensitivity of the bck1 mutant, but rather exacerbated this phenotype
(Fig. 3A). By contrast, we
observed a suppression of the caffeine sensitivity of the bck1
deletion. These seemingly contradictory phenotypes could be explained by Rho5p
acting downstream of Bck1p but as well by Rho5p being part of a parallel
pathway or by a `crosstalk' with upstream components (for details, see
Discussion). Therefore we also tested the effect of a rho5
deletion in a slt2 deletion background (encoding the MAP-kinase of
the pathway). The double deletion grows at 30°C without osmotic
stabilization in contrast to the single slt2 deletion strain
(Fig. 3B). However, no
suppression was observed when tested for growth at 37°C. Likewise, the
caffeine sensitivity also remained unaltered (data not shown). The suppression
of the growth defect of the slt2 deletion at 30°C suggests that
Rho5p functions downstream of the MAP-kinase cascade. Failure to suppress
under the other growth conditions indicates that Rho5p may serve additional
functions at 37°C, and in the presence of caffeine, that are exerted
independently from the MAP-kinase cascade.
|
Slt2p phosphorylation in a rho5 deletion
The MAPK Slt2p is activated by a dual phosphorylation catalyzed by the
MAPKK pair Mkk1p/Mkk2p (Martin et al.,
2000). To investigate this kind of pathway regulation by the Rho5p
signal, we determined Slt2p phosphorylation in a western blot analysis. Based
on the reporter assay described above, where we observed maximal activation at
37°C, we chose this growth condition for pathway activation. As shown in
Fig. 4 the amount of
phosphorylated Slt2p is not significantly increased in the rho5
mutant when compared with the wild-type. This substantiates the findings of
the suppression experiments described above and places the action of Rho5p
downstream of Slt2p but upstream of Rlm1p (i.e. explaining the increased
activity in the reporter system).
|
Regulation of Rho5p
Bem2p has been proposed to serve a GTPase-activating function for Rho1p as
the upstream activator of protein kinase C
(Peterson et al., 1994).
Although its deletion showed the expected increase in Slt2p phosphorylation
(Martin et al., 2000
) the
phenotypes of bem2 deletions are contrary to those expected for an
activated Slt2p pathway. Mutants are sensitive to several drugs known to
destabilize the cell wall and show a depolarized actin cytoskeleton
(Cid et al., 1998
). These
defects can be suppressed neither by overproduction nor deletion of
SLT2 (Cid et al.,
1998
). Having shown that Rho5p acts downstream of Slt2p, we also
tested whether a rho5 deletion affects any of the phenotypes of a
bem2 deletion. As shown in Fig.
5, in contrast to the bem2
single mutant, the
bem2 rho5 double mutant grows on rich medium at 25°C. The double
mutant also grows at 30°C in the presence of up to 3 mM caffeine. However,
as observed in the phenotypic analysis of slt2 rho5 and bck1
rho5 double deletions, the bem2 rho5 strain still fails to grow
at 37°C. This again suggests an additional function for Rho5p at 37°C
apart from downregulation of the Pkc1p pathway.
|
Defects in regulation of actin dynamics in strains with rho5
deletions and RHO5Q91H overexpression
The proposed function of Rho5p as a negative downstream switch might be
related to events that are regulated by two different branches of the same
signalling pathway. Such a branched function in signalling has been suggested
for Pkc1p. This kinase has been reported to be involved in the control of both
depolarisation and repolarisation of the actin cytoskeleton upon heat shock
(Delley and Hall, 1999). To
see whether the negative regulatory function of Rho5p on the Slt2p MAPK branch
is involved in regulation of actin cytoskeleton polarity, we followed the
distribution of actin in both the rho5 deletion and in a strain
overexpressing the activated RHO5Q91H allele
(Fig. 6). Whereas wild-type
cells showed depolarisation after 45 minutes at 37°C, the rho5
deletion strain displayed no significant changes in actin polarity
(Fig. 6A). In contrast, the
activated RHO5Q91H mutant had its actin depolarised even before the
shift to the higher temperature (i.e. at time 0;
Fig. 6B). This depolarisation
persisted even after 180 minutes. In the latter cells another consequence of
the downregulation of the cellular integrity pathway becomes apparent after
180 minutes: cells expressing the activated allele increase in size, an
observation also reported for pkc1 mutants
(Paravicini et al., 1992
).
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Discussion |
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In order to determine the position of Rho5p within the signalling pathway
we performed epistatic analyses with several different components. We showed
that the caffeine-sensitivity of a bck1 deletion, encoding the MAP
kinase kinase kinase of the pathway, was suppressed by an additional
rho5 deletion. Likewise, the growth defect of a slt2
deletion strain (i.e. lacking the MAP kinase) in the absence of osmotic
stabilization was also suppressed by elimination of Rho5p. This can be
interpreted in two different ways. The first possibility is that Rho5p acts
downstream of Slt2. The second, more indirect possibility would place Rho5p
action in a pathway parallel to the Pkc1p signal transduction, such as the SVG
pathway (Lee and Elion, 1999).
This SVG pathway, like the Pkc1p pathway, regulates cell wall integrity and
complementation of mutations in either pathway by components of the other
pathway has been shown. Both pathways are able to control the transcription of
FKS2, encoding a subunit of the ß-(1,3)-glucan synthase, a
component important for the integrity of the cell wall
(Lee and Elion, 1999
). But in
contrast to the Pkc1p pathway, which controls FKS2 transcription via
Rlm1p, FKS2 transcription relies on Ste12p and Tec1 in the SVG
pathway. Because the reporter assay used in this study is based on changes in
Rlm1p activation, an involvement of Rho5p in the SVG pathway is unlikely. It
therefore is reasonable to place Rho5p action downstream of the MAP kinase
cascade as depicted in Fig. 7.
However, the temperature-sensitivity of bck1- and
slt2-deletions was not alleviated by the rho5 deletion.
Rather, the sensitivity of bck1 strains was exacerbated. This
phenotype could be explained by the need for a highly balanced signal under
heat stress conditions. As observed in the regulation of actin dynamics, first
an up- and than a downregulation would be required. Deletion of a gene
encoding a negative regulatory element (i.e. Rho5p) could result in an
imbalance interfering with the ability of the cell to cope with this specific
stress condition.
|
The inhibitory function of Rho5p downstream of the MAP kinase Slt2p in the signal transduction pathway is also consistent with our observation that Slt2p phosphorylation remains unaffected by a rho5 deletion.
What is the possible advantage of such a downstream switch for the cell? In
view of the branched nature of the Pkc1p-mediated signal transduction pathway
(Lee et al., 1993a;
Li et al., 2000
) one can
imagine that such a switch could turn off the signalling within one branch
while leaving the other unaffected. For the cellular integrity pathway several
such upstream signals that do not require an active MAP-kinase branch have
been reported (see also citations in the Introduction). Among these upstream
signals, some are independent of the signal generated by the MAP kinase and
indeed require a downregulation of the cascade. Thus the depolarisation of
actin is activated by Pkc1p independently of the MAP-kinase cascade, whereas
the repolarisation requires the MAP kinase signal
(Delley and Hall, 1999
).
Therefore, we also tested whether Rho5p is involved in this regulation.
Consistent with the results reported above a deletion mutant of rho5
showed a strongly reduced actin depolarisation after the shift to 37°C.
However, overexpression of an activated RHO5Q91H allele led to a
depolarisation already in the absence of heat stress. Apparently, in the
rho5 deletion the signal for repolarisation is permanently active
(i.e. it cannot be switched off) and the actin cytoskeleton stays polarized.
In contrast, overexpression of RHO5Q91H blocks the repolarisation
signal by switching it off continuously and the actin cytoskeleton remains
mainly depolarized.
Small GTPases are generally regulated by proteins that include GAPs, GEFs
and GDIs (Mackay and Hall,
1998). As stated in the Introduction, for Rho5p only Rga2p has
been identified as a likely GAP function
(Roumanie et al., 2001
). Using
the phenotypes described herein will aid to the identification of more
regulators of this protein. A first hint to such a regulator is provided by
the suppression of bem2 deletion phenotypes by a rho5
deletion. Previous work indicated that Bem2p acts as a GAP for Rho1p as it
shows GAP activity against it in vitro
(Peterson et al., 1994
).
Additionally the deletion of bem2 leads to an increased
phosphorylation of Slt2p (Martin et al.,
2000
). However, the phenotypes of bem2 mutants are
inconsistent with it acting primarily on Rho1p. Thus, cells are more sensitive
to cell wall destabilizing drugs than would be expected. This increased
sensitivity cannot be suppressed by overexpression of SLT2
(Cid et al., 1998
). Also
bem2 mutants display a depolarized actin cytoskeleton
(Cid et al., 1998
), again a
phenotype not consistent with an activated MAP kinase pathway. Taken together
these different results suggest a downregulation of the increased pathway
activity downstream of Mpk1p/Slt2p. Because we could suppress some of the
growth defects of a bem2 deletion by additional deletion of
rho5 it seems likely that Rho5p is responsible for this downstream
block. Consequently, Rho1p and Rho5p would share some of the regulatory
components of the cellular integrity pathway with the possibility of a
negative feedback loop. This model would explain the contradictory results for
Bem2p described above.
In summary, the data presented here provide the first hints as to the biological function of Rho5p in S. cerevisiae. This negative switch apparently acts downstream of the MAP-kinase Mpk1p/Slt2p and is important for the regulation of events that require activated upstream components independently of the signal of the MAP-kinase cascade via Rlm1p itself (e.g. the regulation of actin polarisation and depolaristion under heat stress). Further work to identify other components of this negative regulatory pathway is in progress.
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Acknowledgments |
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