The small G-protein Cdc42 functions in many
eukaryotic signal transduction pathways. In the budding yeast
Saccharomyces cerevisiae, cells with defective Cdc42 fail
to induce mating-specific genes in response to mating factor and to
adopt the proper morphology for conjugation. Here we show that the
failure of mating factor-induced transcription is largely the indirect
result of arrest at a specific cell cycle position and/or the
accumulation of high levels of the Cln1/2-Cdc28 kinase, a known
repressor of mating factor signal transduction. Cdc42-defective
cells with restored transcriptional induction have a
partially restored mating ability but are still defective in the
morphological response to mating factor. These results show that Cdc42
is not required for transduction of the mating factor signal per
se but that it is essential for proper mating factor-induced
morphogenesis.
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INTRODUCTION |
Cdc42 is a member of the Rho family of small G-proteins, which is
essential for many signal transduction and morphogenic processes in
eukaryotic cells (1, 2). Some of its biological effects result from the
binding to and the activation of members of the p21-activated kinase
(PAK)1 family of protein
kinases (3). Like other G-proteins, Cdc42 can exist in an active,
GTP-bound state and an inactive, GDP-bound state (4). In the budding
yeast Saccharomyces cerevisiae, the transition from inactive
to the active state is catalyzed by the guanylate exchange factor Cdc24
(5), and several GTP-ase activating proteins are thought to promote the
formation of the inactive GDP-Cdc42 state (5, 6). The GTP-bound form of
Cdc42 can interact with the yeast PAK family members Ste20 and Cla4
(7-9). Ste20 by itself is essential for mating factor signal
transduction, haploid invasive growth, and pseudohyphal
differentiation, and it shares an essential function at cytokinesis
with Cla4 (for reviews see Refs. 3 and 10).
The in vitro kinase activity of Ste20 is stimulated by
GTP-bound Cdc42 (7), and cells with inactive Cdc42 have strongly reduced mating factor signal transduction activity (7, 11). These
observations suggested that an interaction of Cdc42 with Ste20 is
required for full Ste20 kinase activity, a likely prerequisite for the
various functions of Ste20. Recently, however, biologically active
Ste20 mutants were constructed that are specifically defective in their
interaction with Cdc42 by deletion of the so called CRIB domain
(Cdc42 regulatory and interaction
box) (12-14). Such Ste20 mutants are defective in the
cytokinesis, haploid-invasive, and pseudohyphal growth functions of
Ste20, but they have normal in vitro kinase activity and are
proficient in mating functions (13, 14). Apparently, the interaction of
Cdc42 with Ste20 is required for some but not for all Ste20
functions.
Because the Cdc42-Ste20 interaction is not required for mating factor
signal transduction, it is unclear why cells with temperature-sensitive cdc42 and cdc24 alleles are defective in mating
factor-induced transcription and other mating functions (7, 11, 15).
One possibility is that Cdc42 interacts with a factor other than Ste20 that is essential for mating factor signal transduction. This idea is
supported by the recent finding in mammalian cells that Cdc42 does not
only interact with PAKs but also with the MEKK1 and MEKK4 protein
kinases (16), which have homologs in yeast. Another potential
explanation for the signal transduction defect of cdc42 and
cdc24 cells at restrictive temperature is based on the
following observations: (a) the signaling defect of
cdc42 and cdc24 cells is observed most strongly
in cells that are fully arrested at the
cdc24/cdc42 block (7, 11), (b) it has
been shown that high levels of Cln1/2-Cdc28 kinase accumulate at such blocks (17), and (c) we have previously shown that high
levels of Cln1/2-Cdc28 can repress the mating factor signal
transduction pathway (18). We tested whether the signaling defect of
cdc24 and cdc42 cells was due to repression of
mating factor signaling by the high levels of Cln1/2-Cdc28 kinase at
the cdc24/cdc42 block.
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MATERIALS AND METHODS |
Yeast Strains and Growth Conditions--
The genotypes of yeast
strains used in this study are given in Table
I. All strains were isogenic to
BF264-15D (trp1-1a leu2-3, 112 ura3 ade1 his2) or, in the
case of cdc24-1 and cdc42-1 strains, backcrossed
to the BF264-15D strain background at least five times. Strains were
constructed by standard methods, and cells were grown in YEP (yeast
extract peptone) media with 2% dextrose or 3% galactose as described
(19). Treatment of cells with mating factor was at concentrations of
0.1 µM or higher. Quantitative mating assays were carried
out essentially as described previously (18).
Northern and Morphological Analysis--
Northern analysis was
as described (18). Cell morphology was examined by phase contrast
optics, and negatives of pictures were scanned into a digital format.
Contrast and brightness were further adjusted using the Adobe Photoshop
computer program.
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RESULTS |
Repression of Mating Factor Signal Transduction by Cln1/2-Cdc28
Kinase Contributes to Poor Signaling of cdc42 and cdc24 Cells--
To
test whether the defect in signal transduction activity observed at a
cdc42 or cdc24 block could be due to repression
of the mating factor signaling pathway by the G1-cyclins
CLN1 and CLN2, we compared the signal
transduction activity under restrictive conditions of cdc42
and cdc24 strains with and without CLN1/2. As
shown before (7, 11), cells with temperature-sensitive alleles of
cdc42 and cdc24 at restrictive temperature showed
little transcriptional induction by mating factor of the reporter gene FUS1 (Fig. 1A).
Deletion of CLN1 and CLN2 in the cdc42
and cdc24 strains resulted in a significant increase in
signaling activity at restrictive temperature; cdc24 or
cdc42 cells without CLN1/2 had about a 3-fold
increase in signal transduction activity when compared with cells with
CLN1/2 (Fig. 1A). Some defect in signal transduction activity, however, was still observed. At permissive temperature none of these strains showed a major defect in signaling activity (Fig. 1A). These observations suggest that part of
the signaling defect at the cdc42 and cdc24
arrest points is due to the presence of CLN1 and
CLN2.

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Fig. 1.
Mating factor signal transduction in
cdc42 and cdc24 cells with different CLN
genotypes. In all experiments samples for Northern analysis were
taken before and after 15 min of treatment with mating factor. Northern
blots were probed with a TCM1 fragment to check for loading,
and with a FUS1 fragment to monitor transcriptional signal
transduction activity. Data were quantitated with a PhosphorImager,
and only values that are normalized for loading are given. Mating
factor-induced FUS1 levels in wild type (wt)
cells at 24 (A) or 36 °C (B and C)
were arbitrarily chosen as 100 units. Other values are given in
relation to this value. A, cells of the indicated genotypes
were grown in YEP dextrose medium at 24 °C, and a portion was then
shifted to 37 °C for 3 h. The strains used were: wild type
(BOY391), cdc24-1 (BOY1248), cln1 cln2 cdc24-1
(BOY1235), cdc42-1 (BOY1251), and cln1 cln2
cdc42-1 (BOY1240). B, cells were grown in YEP galactose
medium at 24 °C, and dextrose was added to one part of the culture
for 2.5 h. A portion of each culture was then shifted to 37 °C
for 2.5 h. The strains used were:
cln GAL1::CLN1 (BOY836),
cln cdc24-1 GAL1::CLN1
(BOY1074), and cln cdc42-1
GAL1::CLN1 (BOY1076). C, cells were grown in
YEPD medium at 24 °C and then shifted to 37 °C for 3 h. The
strains used were: wild type (BOY921), cdc24-1 (BOY1001),
cdc24 cln1 (BOY1005), cdc24
cln2 (BOY1004), and cdc24 cln1 cln2
(BOY1007).
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Mating Factor Signal Transduction in cdc42 and cdc24 Cells Blocked
the START Cell Cycle Position (20)--
Mutant cdc42 and
cdc24 cells arrest at a specific cell cycle position as
large unbudded cells (20). It is at this position that the signaling
defect of these cells was observed (7, 11). We wanted to eliminate
potential cell cycle effects that might contribute to the defect in
signal transduction activity. The possibility of such cell cycle
effects is suggested by observations in cdc34-1 mutant
cells, which like cdc24 and cdc42 arrest at a
post-START cell cycle position (20). Just like cdc24 and
cdc42 cells, cdc34-1 blocked at restrictive
temperature are defective in pheromone signal transduction activity
(data not shown). Unlike cdc42 and cdc24,
however, the defect in signal transduction activity of these
cdc34 cells could not be alleviated by deletion of
CLN1 and CLN2 (data not shown), indicating that
besides CLN1/2-mediated repression of signal transduction,
another effect, possibly related to the post-START cell cycle position,
strongly contributes to the signaling defect of cdc34-1
cells. To test for potential cell cycle position effects that might
contribute to the signaling defect of cdc24 and
cdc42 cells, we used cln
cdc24-1 GAL1::CLN1 and
cln
cdc42-1 GAL1::CLN1
cells. These cells were grown on galactose medium and then arrested at
START by addition of glucose, which results in CLN
deprivation by repression of the GAL1 promoter (21). Cdc24
or Cdc42 were then inactivated by elevation of the temperature, and
mating factor signal transduction activity was determined by monitoring
FUS1 transcriptional induction. At this different cell cycle
position, signal transduction activity of the cdc42 and
cdc24 mutant cells was similar to that of wild type cells at
both permissive (data not shown) and restrictive temperature (Fig.
1B). The same cells kept in galactose and arrested at the cdc arrest points (with high CLN1 expression
levels) were strongly defective in signal transduction activity (Fig.
1B). Deletion of STE5 (a gene required for mating
factor signal transduction (22)) in cln
cdc24-1 GAL1::CLN1 cells eliminated all mating
factor-induced signal transduction activity of CLN-blocked
cells at restrictive temperature (data not shown). This indicates that
transcriptional induction of the FUS1 reporter gene by
mating factor is not due to some artifactual activation but occurs
through bona fide activation of the mating factor signal transduction
pathway. Taken together, these data demonstrate that the previously
observed signaling defect of cdc24 and cdc42
cells can be explained largely by cell cycle position effects in
combination with high level CLN1/2-associated kinase
activity.
In Vivo, CLN2 Appears to Contribute More to Repression than
CLN1--
Our observations on the signaling defects of
cdc24 and cdc42 cells allowed us to test the
in vivo contribution of CLN1 and CLN2
expressed from their own promoters in signaling repression. For this
purpose, cdc24 cells with different CLN genotypes
were tested for their signaling activity. Cdc24 cells at restrictive temperature, which were deleted for CLN1 had a more severe
signaling defect than cells deleted for CLN2, which in turn
signaled worse than cells deleted for both (Fig. 1C).
Although these observations are made in a somewhat artificial
situation, this indicates that both CLN1 and CLN2
can, when expressed from their own promoter, by themselves reduce
mating factor signal transduction activity. The observation
that CLN2 seems more effective in repression (Fig. 1C) might help explain why expression of
CLN2 from the GAL1 promoter is particularly
effective in repression of signal transduction (18).
Cdc42 Function Is Required for Mating Factor-induced
Morphogenesis--
It has been observed that cdc42 and
cdc24 cells at restrictive temperature are defective in
mating factor-induced morphogenesis and have a strong mating defect
(15, 23-25). It is unclear, however, whether this reflects a genuine
requirement for Cdc42 function in morphogenesis and mating or whether
these observations are an indirect result of the strong signal
transduction defect at the cdc24/cdc42 blocks.
Because inactivation of Cdc42 function in START-arrested cells did not
result in a major signal transduction defect, we could separate direct
from indirect effects and directly examine the requirement of Cdc42 for
mating factor-induced morphogenesis and mating. We used thermosensitive
cdc24 mutants because the cdc24-1 allele has been
observed to be "tighter" than the cdc42-1 allele (Ref.
17 and data not shown); it is therefore expected to be better suited
for these experiments that require extended incubations at restrictive
temperature.
To test the involvement of Cdc24 in mating factor-induced
morphogenesis, cln
cdc24
GAL1::CLN1 cells were arrested at START by
CLN deprivation, and a portion of the cells was then shifted
to the restrictive temperature of 36 °C. Fig.
2 shows the morphology of START-arrested cells with and without mating factor treatment. In response to mating
factor, both wild type and cdc24 cells at permissive
temperature formed the typical mating projections called "shmoos."
At the elevated temperature, however, only the wild type cells formed mating projections, whereas the cdc24 cells in the presence
of mating factor displayed a round, nonpolarized morphology. Because START-arrested cdc24 cells at 36 °C are proficient in
signal transduction activity, this shows that Cdc24 and by inference
Cdc42 are required for proper mating factor-induced morphogenesis.

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Fig. 2.
Mating factor-induced morphogenesis in
cdc24 cells arrested at a cln block. Cells
were grown in YEP galactose medium at 24 °C, and dextrose was added
for 2 h before a portion of the cultures was shifted to 37 °C
for 2 h. Half of each culture was then treated with mating factor.
After 4 h, samples were taken for morphological examination. The
strains used were: cln
GAL1::CLN1 (BOY836) and
cln cdc24-1 GAL1::CLN1
(BOY1074).
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We then tested the mating ability of cdc24 cells in which
mating factor signal transduction activity was restored by
CLN deprivation. As shown in Fig.
3, cdc24 cells blocked at
restrictive temperature displayed a marked mating defect, although in
our strain background and under these experimental conditions, the
defect did not appear as large as reported previously by others (15).
In cdc24 cells in which signal transduction activity was
restored by prior CLN deprivation, there was a partial
restoration of the mating defect at restrictive temperature. The
cdc24 cells at a CLN block still appeared to have
a somewhat reduced mating efficiency when compared with wild type
cells.

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Fig. 3.
Mating efficiencies. cdc24-1 and
control cells were grown in YEP galactose medium at 23 °C. Glucose
was added to a portion of the culture for 3 h. Cultures were split
again, and one was switched to 36 °C for 2.5 h. Cells were then
mixed with an equal number of mating tester, filtered, and incubated at
permissive or restrictive temperature on either YEP dextrose
("blocked" cells) or YEP galactose plates. After 6 h of
incubation, cells on the filters were resuspended in water, and 10-fold
serial dilutions were plated on YEP galactose plates to count the total
number of cells in the reaction and on YEP minimal plates to determine
to number of diploids formed. Colonies were counted after 3 days, and
mating frequencies were calculated by dividing the number of diploid
cells by the total number of cells in the suspension. The strains used
were the same as those for Fig. 2.
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DISCUSSION |
We demonstrate that repression of the mating factor signal
transduction pathway by the Cln1/2-cdc28 kinase in combination with
cell cycle position effects can largely explain the previously observed
signaling defect of cdc42 and cdc24 cells blocked
at restrictive temperature (7, 11). There clearly is no absolute requirement for Cdc42 or Cdc24 in mating factor signal transduction. This is consistent with the observations that there is no requirement for an interaction of Cdc42 with Ste20 for mating factor signal transduction (11, 13, 14). Moreover, these data show that there is not
some other step in the mating factor signal transduction route that
requires Cdc42 function.
Even though cdc24 cells that were arrested at START by
CLN deprivation had normal mating factor signal transduction
activity, they failed to form projections for conjugation. This
requirement of Cdc42 and Cdc24 in morphogenesis during the sexual cycle
of budding yeast is in keeping with the crucial importance of these proteins in morphogenesis during the vegetative cell cycle (23-25) and
with their morphogenic role in other eukaryotes (1, 2). An involvement
of Cdc24/Cdc42 specifically in mating factor-induced morphogenesis is
also supported by a recent study describing the generation of
CDC24 alleles that are only defective in mating factor-induced morphogenesis, whereas other vegetative and sexual functions of Cdc24 are unaffected (26). When compared with activation of PAK family members from other organisms (7, 27), Cdc42 binding to
the budding yeast Ste20 or Cla4 kinases results only in, at best, a
moderate stimulation of kinase activity (7, 9, 28), whereas for Ste20
it appears to be critical for in vivo localization of the
kinase (13, 14). The primary mode of activation of Cla4 or Ste20 by
Cdc42 may be localization of the kinases to the proper site for
function rather than stimulation of in vivo kinase activity.
Such a localization mechanism accounts for a large part of the
activation of Raf kinase by Ras (29, 30).
The mating defect of cdc24 cells at restrictive temperature
could be partially overcome by blocking cells at a different cell cycle
position with low CLN kinase activity. Because cells have normal mating factor signal transduction activity at that position, this suggests that part of the previously observed mating defect of
cdc24 cells (15) can be attributed to a signal transduction defect. That mating in these cells was not as efficient as that of wild
type cells, might be due to the fact that they failed to form
projections for conjugation. This indicates that the poor mating
phenotype of cdc24 cells that was observed previously can be
attributed to a combination of signal transduction and morphogenic defects.
We thank D. Lew for strains, B. Benton for
useful discussions, and J. Liu for excellent technical assistance.