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J. Cell Biol.
Cite by DOI: 10.1083/jcb.201108108 JCB 1 of 16
Correspondence to Douglas R. Kellogg: firstname.lastname@example.org
Eukaryotic cells show extraordinary diversity in size and shape,
and they can maintain the same size even as their rate of growth
changes. The mechanisms that underlie size control are largely
unknown. It seems likely that these mechanisms are as an-
cient and conserved as the cell cycle because they would have
been necessary for survival of the earliest eukaryotic cells.
If so, there must be universal mechanisms for cell size con-
trol that are robust and adaptable so that they can function in
cells of diverse shape and in cells that differ by many orders
of magnitude in size. Although several proteins are known to
be required for cell size control, it has not yet been possible
to identify conserved core mechanisms that control cell size
(Jorgensen and Tyers, 2004).
Cell size checkpoints play an important role in cell size
control (Rupes, 2002; Kellogg, 2003; Jorgensen and Tyers,
2004). These checkpoints ensure that key cell cycle transitions
are initiated only when sufficient growth has occurred. A cell
size checkpoint that operates at entry into mitosis is thought to
be mediated by the Wee1 kinase and the Cdc25 phosphatase
(Nurse, 1975; Nurse et al., 1976). Wee1 delays mitosis by phos-
phorylating and inhibiting Cdk1 (Gould and Nurse, 1989). Cdc25
promotes entry into mitosis by removing the inhibitory phos-
phorylation (Russell and Nurse, 1986; Dunphy and Kumagai,
1991; Gautier et al., 1991; Kumagai and Dunphy, 1991). Early
work in fission yeast discovered that Wee1 mutants enter mitosis
before sufficient growth has occurred, leading to abnormally
small cells (Nurse, 1975). Conversely, Cdc25 mutants delay
entry into mitosis and become abnormally large (Nurse,
1975; Russell and Nurse, 1986). These observations led to the
hypothesis that Wee1 delays mitosis until cells have reached a
The budding yeast homologues of Wee1 and Cdc25 are
called Swe1 and Mih1. Loss of Swe1 causes premature mitosis
and a reduced cell size (Lim et al., 1996; Jorgensen et al., 2002;
Harvey and Kellogg, 2003; Harvey et al., 2005; Rahal and
Amon, 2008). Loss of Mih1 causes delayed mitosis and an in-
creased size (Russell et al., 1989; Jorgensen et al., 2002; Pal
et al., 2008). Thus, the key functions of Wee1 and Cdc25 in fission
yeast have been conserved in budding yeast, which suggests the
existence of a conserved checkpoint. However, a role for Wee1
and Cdc25 family members in cell size control has been contro-
versial because mutants may cause cell size defects indirectly
by allowing more or less time for growth before entry into
mitosis. Moreover, an alternative model has been proposed in
which Wee1 and Cdc25 family members mediate a morphogen-
esis checkpoint that monitors the shape of the cell via the actin
cytoskeleton (Lew and Reed, 1995a; Gachet et al., 2001; Lew,
2003; McNulty and Lew, 2005). The checkpoint functions of
membrane traffic causes a mitotic checkpoint arrest via
Wee1-dependent inhibitory phosphorylation of Cdk1.
Checkpoint signals are relayed by the Rho1 GTPase,
protein kinase C (Pkc1), and a specific form of protein
phosphatase 2A (PP2ACdc55). Signaling via this pathway is
dependent on membrane traffic and appears to increase
gradually during polar bud growth. We hypothesize that
ddition of new membrane to the cell surface
by membrane trafficking is necessary for cell
growth. In this paper, we report that blocking
delivery of vesicles to the site of bud growth generates
a signal that is proportional to the extent of polarized
membrane growth and that the strength of the signal
is read by downstream components to determine when
sufficient growth has occurred for initiation of mitosis.
Growth-dependent signaling could explain how mem-
brane growth is integrated with cell cycle progression.
It could also control both cell size and morphogenesis,
thereby reconciling divergent models for mitotic check-
A link between mitotic entry and membrane growth
suggests a novel model for cell size control
Steph D. Anastasia, Duy Linh Nguyen, Vu Thai, Melissa Meloy, Tracy MacDonough, and Douglas R. Kellogg
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064
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T H E J O U R N A L O F C E L L B I O L O G Y
on March 27, 2012
Published March 26, 2012
This article has original data in the JCB Data Viewer
JCB 2 of 16
bind to PP2ACdc55 and target it to Mih1 but are not required for
the activity of PP2ACdc55 against Swe1 (Wicky et al., 2011). A
model that could explain these observations is that hyperphos-
phorylation of Mih1 early in the cell cycle reflects the action
of a checkpoint that keeps Mih1 inactive before mitosis. In
this model, dephosphorylation of Mih1 by PP2ACdc55 relieves
inhibition of Mih1 to promote mitotic entry. Although it has
not yet been possible to assay the activity of differently phos-
phorylated forms of Mih1, genetic data support the idea that
hyperphosphorylation of Mih1 is inhibitory (Pal et al., 2008;
Wicky et al., 2011).
The dramatic cell cycle–dependent changes in phosphory-
lation of Mih1 and Swe1 likely reflect the action of upstream
checkpoint signals that control their activity, yet the cellular
events that send checkpoint signals to Mih1 and Swe1 are poorly
understood. Here, we have explored the cellular events that
send checkpoint signals to Mih1 and Swe1. A starting point for
these analyses was the connection between Yck1/2 and Mih1.
We found this connection to be intriguing because Yck1/2 are
transported to the site of bud growth on secretory vesicles (Babu
et al., 2002). Moreover, inactivation of Yck1/2 causes defects in
bud growth and Cdk1 inhibitory phosphorylation (Robinson
et al., 1993; Pal et al., 2008). These observations suggested that
control of Cdk1 inhibitory phosphorylation could be linked to
membrane traffic. In principle, mechanisms that link cell cycle
progression to membrane traffic must exist to ensure that growth
is coordinated with the cell cycle. We therefore set out to test
for a connection between membrane traffic and control of Mih1
Blocking membrane traffic triggers a rapid
Swe1-dependent checkpoint arrest
We analyzed the effects of disrupting membrane traffic on pro-
gression through mitosis in synchronized rapidly dividing cells.
To disrupt membrane traffic, we used a temperature-sensitive
mutant of SEC6 (sec6-4) because previous work found that it
causes a rapid arrest of membrane traffic. Sec6 is a component
of the exocyst complex, which is required for docking and fu-
sion of secretory vesicles at the site of membrane growth in the
bud (TerBush et al., 1996). To assess mitotic progression, we
assayed cleavage of the cohesin Mcd1, which normally occurs
at the metaphase to anaphase transition, as well as levels of the
mitotic cyclin Clb2.
Wild-type and sec6-4 cells were released from a G1 arrest
and shifted to the restrictive temperature at 30 min after release,
which was before bud growth had been initiated. Growth of a
new bud was blocked in sec6-4 cells because membrane traf-
fic is required for bud growth. Mcd1 cleavage failed to occur
in sec6-4 cells, which indicated that they arrested before ana-
phase (Fig. 1 A). To determine whether the arrest was caused
by Swe1-dependent inhibitory phosphorylation of Cdk1, we
also assayed Mcd1 cleavage in sec6-4 swe1 cells (Fig. 1 A).
The extent and timing of Mcd1 cleavage were similar in wild-
type and sec6-4 swe1 cells, which revealed that swe1 caused
complete checkpoint failure. As expected, the sec6-4 cells
Wee1 and Cdc25 are uncertain because we lack a clear under-
standing of the upstream signals that control their activity.
Elucidation of these signals is thus an essential step toward
understanding G2/M checkpoints and conserved mechanisms
that control entry into mitosis.
Recent work has led to a new understanding of the func-
tion and regulation of Wee1 and Cdc25 family members. In
both vertebrates and yeast, Wee1 and Cdc25 function in a systems-
level mechanism that generates and maintains a low level of
Cdk1 activity during early mitosis (Deibler and Kirschner,
2010; Harvey et al., 2011). The underlying mechanism is best
understood in yeast. Swe1 is initially phosphorylated by Cdk1
associated with mitotic cyclins, which stimulates Swe1 to bind,
phosphorylate, and inhibit Cdk1 (Harvey et al., 2005, 2011).
The initial phosphorylation of Swe1 is opposed by protein
phosphatase 2A associated with the Cdc55 regulatory subunit
(PP2ACdc55; Harvey et al., 2011). The opposing activity of
PP2ACdc55 sets a threshold that limits activation of Wee1 by
Cdk1, thereby allowing a low level of Cdk1 activity to escape
Wee1 inhibition in early mitosis. A key early mitotic event
that is initiated via low level activation of Cdk1 is a positive
feedback loop in which Cdk1 promotes transcription of the
mitotic cyclin Clb2, which leads to a rapid rise in Clb2 levels
(Amon et al., 1993; Harvey et al., 2011). A second key event
is a switch in the pattern of bud growth. Growth of the bud
initially occurs in a polar manner, but when the mitotic cyclins
appear, they trigger a switch to isotropic growth, in which
growth occurs over the entire surface of the bud (Lew and Reed,
1993). The mitotic cyclins induce this switch by repressing
transcription of the G1 cyclins Cln1 and Cln2, which drive
polar bud growth (Lew and Reed, 1993; Amon et al., 1994;
McCusker et al., 2007).
After the initial phosphorylation of Swe1 in early mitosis,
subsequent phosphorylation events lead to full hyperphos-
phorylation of Swe1, which inactivates Swe1 and is likely
necessary for full entry into mitosis (Harvey et al., 2005). In
vertebrates and yeast, mitotic Cdk1 is capable of full hyper-
phosphorylation and inactivation of Wee1 family members
when it is present at sufficiently high levels (Tang et al., 1993;
Mueller et al., 1995; Harvey et al., 2005). However, multiple
kinases are required for full hyperphosphorylation of Wee1
family members in vivo, and their relative contributions are
poorly understood (Coleman et al., 1993; Wu and Russell, 1993;
Shulewitz et al., 1999; Sreenivasan and Kellogg, 1999; Sakchaisri
et al., 2004; Asano et al., 2005).
Cdc25 family members are also regulated by phosphory-
lation. Phosphorylation of vertebrate Cdc25 by mitotic Cdk1
stimulates Cdc25 activity in a feedback loop that promotes
entry into mitosis (Kumagai and Dunphy, 1992; Izumi and
Maller, 1993). A similar feedback loop may also work on Mih1
in budding yeast (Pal et al., 2008). Mih1 is also controlled
by casein kinase 1, which is encoded by a pair of redundant
genes called YCK1 and YCK2 (Pal et al., 2008). Early in the
cell cycle, Mih1 undergoes hyperphosphorylation that is
dependent on Yck1/2. During entry into mitosis, Yck1/2-
dependent phosphorylation of Mih1 is removed by PP2ACdc55.
A pair of redundant regulatory proteins called Zds1 and Zds2
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Published March 26, 2012
3 of 16Entry into mitosis linked to membrane traffic • Anastasia et al.
in the daughter bud (Balasubramanian et al., 2000; Pereira
et al., 2000). Membrane traffic is required for cytokinesis
(Xu et al., 2002).
Together, these observations demonstrate that blocking
membrane traffic triggers a Swe1-dependent checkpoint arrest.
They also suggest that the checkpoint blocks low level acti-
vation of Cdk1 in early mitosis, which leads to a failure
in short spindle assembly and a failure to activate the posi-
tive feedback loop that promotes Clb2 transcription (Amon
et al., 1993).
Normal signaling to Mih1 and Swe1 fails to
occur when membrane traffic is blocked
A cell cycle arrest could be induced by preventing activation
of Mih1, inactivation of Swe1, or both. We therefore tested
whether blocking membrane traffic sends signals to Mih1 or Swe1.
Mih1 and Swe1 undergo dramatic changes in phosphorylation
arrested with a single nucleus, whereas the sec6-4 swe1 cells
underwent nuclear division within the unbudded mother cell to
form binucleate cells (Fig. 1 B).
To further characterize the arrest, we analyzed accumu-
lation of the mitotic cyclin Clb2. The sec6-4 mutant caused a
severe delay in Clb2 accumulation that was rescued by
swe1 (Fig. 1 C). We also analyzed mitotic spindles. In bud-
ding yeast, assembly of a short mitotic spindle is initiated in
early mitosis by a low level of mitotic Cdk1 activity (Rahal
and Amon, 2008). During anaphase, the spindle elongates to
segregate the chromosomes. The sec6-4 mutant caused de-
layed and reduced assembly of short spindles as well as a
complete block to spindle elongation (Fig. 1, D and E). Clb2
levels remained elevated, and long spindles did not disas-
semble normally in sec6-4 swe1 cells, which may be caused
by a checkpoint that prevents exit from mitosis when cytokine-
sis is blocked by a checkpoint that monitors spindle orientation
Figure 1. Blocking membrane traffic triggers a checkpoint arrest. (A) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C)
at 30 min after release. Cleavage of Mcd1-6×HA was assayed by Western blotting. (B) Cells were released from a G1 arrest and shifted to the restrictive
temperature (34°C) at 30 min after release. DNA staining was used to determine the percentage of cells with multiple nuclei. (C) Cells were released from
a G1 arrest and shifted to the restrictive temperature (34°C) at 45 min after release. Levels of Clb2 were assayed by Western blotting. (D and E) Cells were
released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. The percentage of cells with short or long spindles was
determined. Error bars represent SEMs for three biological replicates. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.
on March 27, 2012
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JCB ? of 16
the normal full hyperphosphorylation that is associated with
inactivation of Swe1 (Fig. 2 B). It also failed to undergo de-
struction. The partial phosphorylation of Swe1 observed in
sec6-4 and sec6-4 cdk1-Y19F cells likely corresponds to the
initial activation of Swe1 by Cdk1 that occurs during entry into
mitosis (Harvey et al., 2005, 2011).
Wee1 family members are inactivated when they are fully
hyperphosphorylated, and it is thought that dephosphorylation
of Mih1 plays a role in its activation (Mueller et al., 1995;
Harvey et al., 2005; Pal et al., 2008; Wicky et al., 2011). Thus,
these observations suggest that the checkpoint blocks entry into
mitosis by coordinately preventing inactivation of Swe1 and
activation of Mih1.
Blocking membrane traffic during
early mitosis triggers rapid
signaling to Mih1
We next examined the effects of inactivating membrane traffic
during early mitosis. Wild-type and sec6-4 cells were released
from a G1 arrest and shifted to the restrictive temperature
at 70 min after release. At this time, bud emergence was
complete, and Mih1 dephosphorylation was just beginning.
Because Mih1 dephosphorylation is initiated when the mitotic
cyclin Clb2 first appears, the presence of dephosphorylated
forms of Mih1 indicated that cells were initiating the G2 to
M transition (Pal et al., 2008). Blocking membrane traffic at
this point caused rapid reversal of Mih1 dephosphorylation
(Fig. 3 A). The response was remarkably rapid: Mih1 hyper-
phosphorylation was complete within 5 min. The response
time includes the time required for the cultures to reach the re-
strictive temperature and for protein inactivation to occur, so
it is likely that the actual response time is even more rapid. In-
activation of sec6-4 at this time did not cause a rapid change in
Swe1 phosphorylation, although at the time of the temperature
shift, Swe1 was in the partially phosphorylated active form
and had not yet undergone full hyperphosphorylation that is
associated with Swe1 inactivation.
The rapid hyperphosphorylation of Mih1 was not af-
fected by the cdk1-Y19F allele, which indicated that it was
caused by signals upstream of Mih1 (Fig. 3 A). Dephosphory-
lated forms of Mih1 reappeared in sec6-4 cdk1-Y19F cells at
longer times after the shift to the restrictive temperature. This
is consistent with the experiment in Fig. 2 A, which showed
that dephosphorylated forms of Mih1 appear in later time
points in sec6-4 cdk1-Y19F cells shifted to the restrictive tem-
perature in G1. One explanation for these observations is that
mitotic Cdk1 activity may trigger a positive feedback loop
that contributes to Mih1 dephosphorylation, but only when it
reaches high levels.
To test whether rapid hyperphosphorylation of Mih1 is a
general response to an arrest of secretion, we tested a mutant
that affects intra-Golgi transport (sec7-4). The sec7-4 mutant
caused hyperphosphorylation of Mih1 as rapidly as the sec6-4
mutant (Fig. 3 B). We also tested mutants that affect endo-
cytosis, including a conditional allele of END3 (end3-1) and
deletions of SYP1 and EDE1. None of the mutants caused
hyperphosphorylation of Mih1.
during entry into mitosis that provide a readout for signals that
control their activity or localization (Sreenivasan and Kellogg,
1999; Harvey et al., 2005; Pal et al., 2008). Phosphorylation
of Swe1 and Mih1 can be assayed by Western blotting, which
detects shifts in their electrophoretic mobility. In principle,
changes in phosphorylation of Mih1 or Swe1 during a check-
point arrest could be a cause of the arrest, or they could be an
indirect consequence of the arrest. To test for the latter possi-
bility, we included a control in which phosphorylation of
Swe1 and Mih1 was assayed in sec6-4 cdk1-Y19F cells. The
cdk1-Y19F allele lacks the conserved tyrosine that is targeted
by Swe1, so cells carrying this allele fail to undergo check-
Wild-type, sec6-4, and sec6-4 cdk1-Y19F cells were re-
leased from a G1 arrest and shifted to the restrictive tempera-
ture at 30 min after release. The dephosphorylation of Mih1 that
normally occurs during entry into mitosis failed to occur nor-
mally in sec6-4 cells (Fig. 2 A). Limited dephosphorylation of
Mih1 occurred in sec6-4 cdk1-Y19F cells, but it was delayed
and diminished. Swe1 underwent partial hyperphosphorylation
in sec6-4 and sec6-4 cdk1-Y19F cells but failed to undergo
Figure 2. Normal signaling to Mih1 and Swe1 fails to occur when mem
brane traffic is blocked. (A and B) Cells were released from a G1 arrest
and shifted to the restrictive temperature (34°C) at 30 min after release. The
behavior of Mih1 and Swe1 were assayed by Western blotting. Numbers
shown next to the Western blots indicate molecular mass in kilodaltons.
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? of 16 Entry into mitosis linked to membrane traffic • Anastasia et al.
Arndt, 1995; Minshull et al., 1996; Yang et al., 2000; Pal et al.,
2008; Yasutis et al., 2010; Harvey et al., 2011; Wicky et al.,
2011). PP2ACdc55 is regulated by a pair of redundant proteins
called Zds1 and Zds2 that associate with PP2ACdc55 and target
it to Mih1 (Queralt and Uhlmann, 2008; Yasutis et al., 2010;
Wicky et al., 2011). It appears that Zds1/2 can play both activat-
ing and inhibitory roles in regulation of PP2ACdc55 (Pal et al.,
2008; Queralt and Uhlmann, 2008). There are also hints that
Zds1/2 inhibit the activity of PP2ACdc55 against Swe1 (Wicky
et al., 2011).
Previous work found that overexpression of Zds2 can
override the checkpoint arrest caused by depolymerization
of actin (Yasutis et al., 2010). The ability of Zds2 to override
the checkpoint was dependent on Cdc55, which indicated that
it works through PP2ACdc55 (Yasutis et al., 2010). We found
that overexpression of Zds1 drove cells through the check-
point arrest caused by blocking membrane traffic, as revealed
by cleavage of Mcd1 (Fig. 5 A). Moreover, overexpression of
The effects of blocking membrane traffic
do not appear to be caused by indirect
effects on actin
Previous work reached the conclusion that Swe1 mediates a
checkpoint that monitors bud morphogenesis. This was based
on the observation that depolymerization of actin causes a
Swe1-dependent checkpoint arrest (Lew and Reed, 1995a;
Lew, 2003; Keaton and Lew, 2006). Because actin is required
for bud morphogenesis, it was proposed that the checkpoint
monitors bud morphogenesis. However, actin is required for
delivery of vesicles to the growing bud, and depolymerization
of actin therefore causes rapid cessation of growth (Mulholland
et al., 1997; Karpova et al., 2000). Thus, depolymerization of
actin could activate a checkpoint arrest indirectly by block-
ing membrane traffic. Conversely, blocking membrane traffic
causes defects in actin organization, which suggests that blocking
membrane traffic could cause a checkpoint arrest indirectly by
causing defects in actin organization (Finger and Novick, 1997;
Pruyne et al., 2004).
To learn more about the relative effects of depolymerizing
actin versus blocking membrane traffic, we tested whether actin
depolymerization caused rapid hyperphosphorylation of Mih1.
Wild-type cells were released from a G1 arrest, and latrunculin
A was added at 70 min after release to depolymerize actin. The
effects on Mih1 phosphorylation were indistinguishable from
the effects caused by blocking membrane traffic (Fig. 4 A).
We next tested whether blocking membrane traffic caused
rapid defects in the organization of actin. Wild-type and sec6-4
cells were shifted to the restrictive temperature, and phalloidin
staining was used to monitor actin organization. There were no
detectable effects on the organization of actin cables or patches
after 5 min, when effects on Mih1 phosphorylation were maxi-
mal (Figs. 3 and 4 B).
Together, these observations suggest that the checkpoint
arrest caused by actin depolymerization may be a consequence
of a block to membrane traffic. However, we cannot rule out the
possibility that sec6-4 causes rapid defects in actin organization
that cannot be detected by fluorescence microscopy.
Overexpression of Zds1 drives cells
through the checkpoint arrest caused by
blocking membrane traffic
We next searched for the signals that control Swe1 and Mih1 in
response to arrest of membrane traffic. PP2ACdc55 was a good
candidate because it controls both Swe1 and Mih1 (Lin and
Figure 3. Blocking membrane traffic triggers rapid signal
ing to Mih1. (A and B) Cells were released from a G1 arrest
and were shifted to the restrictive temperature (34°C) at
70 min after release, when Mih1 dephosphorylation was
being initiated. Mih1 phosphorylation was assayed by
Western blotting. Numbers shown next to the Western blots
indicate molecular mass in kilodaltons.
Figure 4. The response to arrest of membrane traffic is not a con
sequence of indirect effects on actin. (A) Wild-type cells were released from
a G1 arrest at room temperature. A sample was taken at 70 min after
release (t = 0), and the cells were divided into two aliquots. Latrunculin A
(LatA) was added to one aliquot, and solvent (DMSO) was added to the
other as a control. Mih1 phosphorylation was assayed by Western blot-
ting. (B) Cells were grown to log phase in YPD media and then shifted
to the restrictive temperature (34°C) for 5 min. Cells were then fixed and
stained with FITC-phalloidin. Bar, 5 µm. Numbers shown next to the West-
ern blot indicate molecular mass in kilodaltons.
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JCB 6 of 16
and Tartakoff, 2001). Finally, the closest homologue of Pkc1 in
vertebrates is PRK2 (PKC-related kinase 2). Depletion of PRK2
by RNAi causes a G2/M block, most likely due to a failure to
properly regulate Cdc25 (Roelants et al., 2004; Schmidt et al.,
2007). Pkc1 is localized to the site of membrane growth and is
therefore well positioned to relay signals regarding the status
of growth (Andrews and Stark, 2000).
We first used coimmunoprecipitation to confirm the re-
ported two-hybrid interaction between Zds2 and Pkc1. Pkc1
could be coprecipitated with PP2ACdc55-3×HA in wild-type cells but
not in zds1 zds2 cells, consistent with an interaction between
Pkc1 and Zds1/2 (Fig. 6 A). Pkc1 was hyperphosphorylated
in extracts made from zds1 zds2 cells, which suggests that
PP2ACdc55 may oppose phosphorylation of Pkc1 (Fig. 6 A).
We next analyzed the effects of inactivating Pkc1. The
commonly used temperature-sensitive allele of PKC1 (pkc1-1)
has a restrictive temperature of 37°C, which causes transient
heat shock effects in wild-type cells that affect phosphorylation of
Mih1 and Swe1. We therefore isolated a collection of 40 new
pkc1 temperature-sensitive alleles to find alleles that cause rapid
inactivation of Pkc1 at 34°C, which does not cause heat shock
effects. We also hoped to identify mutants that preferentially
affect different functions of Pkc1, which could provide new in-
formation on Pkc1 function.
We first screened the collection of alleles for mutants that
affect mitosis. We reasoned that if Pkc1 relays signals that in-
activate Swe1 or activate Mih1, loss of Pkc1 could cause cells to
become elongated because mitotic Cdk1 suppresses polar bud
growth (Booher et al., 1993; Lew and Reed, 1995b; Ma et al.,
1996). Five pkc1 mutants caused a significant fraction of cells
to become elongated when grown at semirestrictive tempera-
tures. In each case, the elongated cell phenotype was eliminated
by swe1, which indicated that it was caused by a failure to
control Cdk1 inhibitory phosphorylation. The strongest pheno-
type was observed in pkc1-14 cells grown at a semirestrictive
temperature of 30°C. The elongated phenotype became severe
when cells were grown to high density, which suggests that
pkc1-14 compromises functions of Pkc1 that are important when
growth is slowed by nutrient limitation (Fig. 6 B). pkc1-14 was
recessive to wild-type PKC1.
We next assayed Mih1 dephosphorylation in pkc1-14 cells.
Cells were released from a G1 arrest and shifted to the restrictive
temperature at 75 min after release, when bud emergence was
complete and cells were entering mitosis. Mih1 dephosphoryla-
tion failed to occur as Clb2 levels increased, consistent with a role
for Pkc1 in controlling Mih1 phosphorylation (Fig. 6 C). In both
wild-type and pkc1-14 cells, Swe1 remained in the partially phos-
phorylated form. We were not able to analyze a more complete
cell cycle in pkc1 mutants because they caused cell lysis after
longer times at the restrictive temperature, as reported previously
for other pkc1 alleles (Levin and Bartlett-Heubusch, 1992).
Pkc1 controls Mih1 phosphorylation
We next analyzed the effects of a PKC1 gain-of-function allele.
A constitutively active form of Pkc1 (referred to as Pkc1*) can
be created by mutating an autoinhibitory phosphorylation site
Zds1 in checkpoint-arrested cells caused dephosphorylation
of Mih1 and hyperphosphorylation of Swe1 (Fig. 5, B and C).
These observations suggest that PP2ACdc55 coordinately regu-
lates Swe1 and Mih1 and that Zds1/2 are key regulators of the
checkpoint functions of PP2ACdc55. An attractive model is that
Zds1/2 coordinately regulate Swe1 and Mih1 by shifting the
activity of PP2ACdc55 away from Swe1 to Mih1. This could
initiate full hyperphosphorylation and inactivation of Swe1 as
well as dephosphorylation of Mih1, which is likely necessary
for Mih1 activation.
Pkc1 controls entry into mitosis
To better understand how arrest of membrane traffic triggers
a checkpoint arrest, we searched for proteins that regulate
PP2ACdc55-Zds1/2. Pkc1, the budding yeast member of the atyp-
ical PKC family, was a good candidate because it associates
with Zds2 in a two-hybrid assay (Uetz et al., 2000; Drees et al.,
2001; Yasutis et al., 2010). Moreover, Pkc1 functions in a sig-
naling pathway that blocks ribosome biogenesis when mem-
brane traffic is blocked, which demonstrates that it mediates a
response to arrest of membrane traffic (Li et al., 2000; Nanduri
Figure 5. Overexpression of Zds1 drives cells through the checkpoint ar
rest. (A–C) Cells were grown in YEP with 2% raffinose and arrested in G1
with -factor at 25°C. Cells were released from the arrest into YEP with 2%
raffinose at 25°C. When 10% of cells had undergone bud emergence, they
were shifted to the restrictive temperature (34°C) to induce the checkpoint
arrest. After 30 min, galactose was added to induce expression of ZDS1.
Cleavage of Mcd1-3×HA and phosphorylation of Mih1 and Swe1 were
assayed by Western blotting. The asterisks denote background bands that
appear with some batches of purified anti-Mih1 antibody. Numbers shown
next to the Western blots indicate molecular mass in kilodaltons.
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? of 16Entry into mitosis linked to membrane traffic • Anastasia et al.
Full hyperphosphorylation of Swe1 failed to occur in rho1-2
cells (Fig. 8 B). In addition, rho1-2 caused cells to arrest in mito-
sis, as revealed by a failure to degrade the mitotic cyclin Clb2
(Fig. 8 C). The arrest was dependent on Swe1. These observa-
tions are consistent with a role for Rho1 in signaling to Mih1
and Swe1. However, bud emergence and growth were delayed
in rho1-2 cells at the semirestrictive temperature. Thus, defects
in mitotic events caused by rho1-2 could be caused by defective
signaling to Mih1 and Swe1 or to defects in bud growth.
To test for a role for Rho1 under conditions in which
cell growth was not a complicating factor, we assayed the effects
of a constitutively active form of Rho1 that was created by
mutating glutamine 68 to histidine (referred to as Rho1*;
Delley and Hall, 1999). Expression of RHO1* from the GAL1
promoter caused dephosphorylation of Mih1 in rapidly growing
cells and in checkpoint-arrested cells (Fig. 8, D and E). Ex-
pression of RHO1* failed to induce Mih1 dephosphorylation
in cells carrying a temperature-sensitive allele of PKC1 (pkc1-21;
Fig. 8 D). Together, these data establish that Rho1 signals to
Mih1 via Pkc1.
Rho1 is transported to the site of bud growth in association
with secretory vesicles (McCaffrey et al., 1991; Abe et al., 2003;
Forsmark et al., 2011). Rho1 associated with secretory vesicles
is inactive; activation of Rho1 occurs at the site of membrane
growth and is dependent on fusion of vesicles with the plasma
membrane (Abe et al., 2003). A guanine nucleotide exchange
factor that activates Rho1 is localized to the site of bud growth
independent of membrane traffic (Abe et al., 2003). The fact
(Watanabe et al., 1994). Expression of Pkc1* from the GAL1
promoter caused rapid dephosphorylation of Mih1 (Fig. 7 A).
Pkc1* did not cause dephosphorylation of Mih1 in cells carry-
ing a temperature-sensitive allele of CDC55 (cdc55-4), which
demonstrated that it acts via PP2ACdc55 (Fig. 7 B). Pkc1* was
able to drive dephosphorylation of Mih1 in checkpoint-arrested
cells (Fig. 7 C).
In previous work, we found that a fraction of Zds1 under-
goes dephosphorylation during entry into mitosis (Wicky et al.,
2011). Because Pkc1 associates with Zds1/2, we tested whether
Pkc1* caused effects on Zds1 phosphorylation. We found that
Pkc1* caused rapid dephosphorylation of Zds1 (Fig. 7 D).
Together, the effects of loss-of-function and gain-of-function
mutants demonstrate that Pkc1 relays signals via PP2ACdc55-Zds1/2
that control Mih1 phosphorylation and entry into mitosis.
Rho1 signals to Mih1 via Pkc1
An important upstream regulator of Pkc1 is the Rho1 GTPase;
the active GTP-bound form of Rho1 directly binds and acti-
vates Pkc1 (Kamada et al., 1996). A previous study identified
temperature-sensitive alleles of RHO1 that appear to be defec-
tive in activation of Pkc1 (Saka et al., 2001). We assayed Mih1
phosphorylation in one of these alleles (rho1-2) and found that
dephosphorylation of Mih1 failed to occur when cells were
grown at a semirestrictive temperature (Fig. 8 A). A hyperphos-
phorylated form of Mih1 appeared in the rho1-2 cells that
was not detected in control cells, which is consistent with a
role for Rho1 in activation of PP2ACdc55 (Fig. 8 A, arrowhead).
Figure 6. Pkc1 associates with PP2ACdc55 and is required for Mih1 dephosphorylation. (A) Anti-HA antibodies were used to immunoprecipitate PP2ACdc55-3×HA
from wild-type, zds1 zds2, and untagged control cells. Coprecipitation of Pkc1 was assayed by Western blotting with anti-Pkc1 antibodies. Crude
extract samples were electrophoresed longer than the immunoprecipitated samples to resolve phosphorylation forms. (B) Cells were inoculated into YPD
media at low density and grown at 30°C until they reached an OD of 1.7. Bar, 10 µm. (C) Cells were released from a G1 arrest and shifted to the
restrictive temperature (34°C) at 75 min after release. Mih1 phosphorylation and Clb2 levels were assayed by Western blotting. Numbers shown next to
the Western blots indicate molecular mass in kilodaltons.
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JCB 8 of 16
molecular marker for the switch from polar to isotropic bud
growth and entry into mitosis (Amon et al., 1993; Lew and
Reed, 1995b). The time course was performed at 22°C to slow
down the cell cycle, which provided a better resolution of cell
Pkc1 began to undergo hyperphosphorylation when Cln2
first appeared (Fig. 9 A). Interestingly, the peak of Pkc1 phos-
phorylation was reached at 80 min. At this point, Cln2 levels
were declining, and Clb2 was beginning to accumulate, which
corresponds to the switch from polar to isotropic bud growth.
Thus, Pkc1 hyperphosphorylation was correlated with polar
The fact that peak Pkc1 phosphorylation was not corre-
lated with peak levels of Cln2 or Clb2 suggested that it is not
controlled by direct signals from either of these cyclins. To deter-
mine whether Pkc1 phosphorylation is dependent on membrane
traffic, we released wild-type and sec6-4 cells from a G1 arrest
that activation of Rho1 is dependent on fusion of vesicles at the
site of bud growth suggests a direct connection between mem-
brane growth and signals that control Mih1 phosphorylation.
Signaling to Pkc1 may be proportional to
the extent of polar bud growth
To further investigate the link between membrane traffic and
mitosis, we explored the nature of the signals that control Pkc1.
We raised an antibody against Pkc1 and used it to assay the
behavior of Pkc1 during the cell cycle after release from a
G1 arrest. We also assayed levels of a G1 cyclin (Cln2) and
a mitotic cyclin (Clb2) in the same samples. Cln2/Cdk1 is
required for initiation and maintenance of polar bud growth;
Cln2 therefore provides a molecular marker for the period of
polar bud growth (Cross, 1990; McCusker et al., 2007). Clb2/
Cdk1 represses Cln2 transcription and induces the switch from
polar growth to isotropic growth; Clb2 therefore provides a
Figure 7. Pkc1 signals to Mih1 via PP2ACdc55Zds1/2. (A) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Galactose
was added, and cells were shifted to 30°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. (B) Cells were grown to log phase in YEP
media containing 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 25 or 34°C at t = 0. Mih1 phosphorylation was assayed
by Western blotting. (C) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Cells were shifted to 34°C for 60 min to
induce a checkpoint arrest, and galactose was then added. Mih1 phosphorylation was assayed by Western blotting. (D) Cells were grown to log phase
in YEP with 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 30°C at t = 0. Zds1 phosphorylation was assayed by Western
blotting. The asterisks denote background bands that appear with some batches of purified anti-Mih1 antibody. Numbers shown next to the Western blots
indicate molecular mass in kilodaltons.
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? of 16
Entry into mitosis linked to membrane traffic • Anastasia et al.
(Kamada et al., 1996; Uetz et al., 2000; Drees et al., 2001;
Queralt and Uhlmann, 2008; Yasutis et al., 2010; Wicky et al.,
2011). Rho1, Pkc1, Zds1, and Cdc55 are all localized to the site
of membrane growth, so they are well positioned to relay check-
point signals (Yamochi et al., 1994; Andrews and Stark, 2000;
Gentry and Hallberg, 2002; Rossio and Yoshida, 2011). It is not
known whether they are found together in a single complex, as
shown in the hypothetical model in Fig. 10 B, or whether they
assemble as dynamic subcomplexes.
Signaling to Mih1 could be detected within minutes upon
inactivation of Sec6. The rapidity of the response suggests that
the checkpoint monitors membrane traffic events, rather than
events that are disrupted as a secondary consequence of a block
to membrane traffic. The Rho1–Pkc1 signaling axis suggests
further connections to membrane traffic. Rho1 is transported on
post-Golgi vesicles in an inactive form and becomes activated
at the site of membrane growth (Abe et al., 2003). A mutant that
blocks vesicle fusion at the site of growth also blocks Rho1
activation (Abe et al., 2003). In addition, Rom2, a guanine
nucleotide exchange factor known to activate Rho1, is localized
to the site of bud growth independently of membrane traffic
and shifted them to the restrictive temperature at 70 min after
release. Pkc1 phosphorylation was lost within 5 min, which is
the same time scale observed for loss of Mih1 phosphorylation
under these conditions (Figs. 3 and 9 B).
A Rho1–Pkc1 signaling axis links membrane
traffic to entry into mitosis
It has long been known that Wee1 and Cdc25 family members
mediate a checkpoint that controls entry into mitosis, yet the
cellular events that are monitored by the checkpoint have re-
mained poorly understood. Here, we report that blocking mem-
brane traffic causes a Swe1-dependent checkpoint arrest. The
arrest is triggered via concerted effects on the regulation of both
Swe1 and Mih1. PP2ACdc55 appears to be the agent of concerted
checkpoint control. Signals regarding the status of membrane
traffic are relayed to PP2ACdc55 via a signaling axis that includes
Rho1, Pkc1, and Zds1/2. Fig. 10 A summarizes dependency
relationships in the axis defined by loss- and gain-of-function
mutants. Fig. 10 B summarizes known binding interactions
Figure 8. Rho1 signals to Mih1 via Pkc1. (A) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 35 min after
release. Mih1 phosphorylation was assayed by Western blotting. The arrowhead marks a hyperphosphorylated form that appears in the rho1-2 cells. At
34°C, rho1-2 cells grow slowly but are viable. (B) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 45 min after
release. Swe1 phosphorylation was assayed by Western blotting. (C) Cells were released from a G1 arrest and shifted to a semirestrictive temperature
(34°C) at 45 min after release. Clb2 levels were assayed by Western blotting. (D) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol.
Galactose was added, and the cells were shifted to 34°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. The pkc1-21 allele was used
because it was found to cause rapid inactivation of Pkc1. (E) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol. The cells were shifted
to 34°C for 60 min to induce a checkpoint arrest, and galactose was then added. Mih1 phosphorylation was assayed by Western blotting. Numbers
shown next to the Western blots indicate molecular mass in kilodaltons.
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JCB 10 of 16
that inactivation of septins, or proteins that regulate the septins,
causes a Swe1-dependent arrest or delay later in mitosis with
high levels of mitotic cyclin (Carroll et al., 1998; Barral et al.,
1999; Sreenivasan and Kellogg, 1999; Harvey et al., 2011).
Together, these observations suggest that Swe1 may control
multiple steps during entry into mitosis.
Key questions remain: How does phosphorylation con-
trol the activity or localization of Mih1? How does Pkc1 acti-
vate PP2ACdc55 to dephosphorylate Mih1? What is the functional
significance of Zds1 phosphorylation? What are the kinases
that phosphorylate Zds1? How do Zds1/2 target PP2ACdc55
to Mih1? These questions will be important directions for
A growth-dependent signaling hypothesis
for the G2-M checkpoint
Sec mutants block membrane traffic, which is essential for cell
growth. Indeed, it is likely that the most ancient and conserved
function of the secretory pathway is to generate membranes for
cell growth. It is therefore tempting to speculate that the check-
point monitors membrane growth to ensure that cell cycle pro-
gression is integrated with membrane growth. The checkpoint
could also monitor membrane growth as part of a mechanism
that controls cell size. Our observations, combined with pre-
vious observations, suggest interesting possibilities for how this
could work. The fact that Rho1 is transported on vesicles and
becomes activated at the site of membrane growth suggests a
mechanism by which a signal could be generated that is depen-
dent on and proportional to membrane growth: as more and
more vesicles are delivered to the site of growth, the Rho1 sig-
nal could increase in strength (Fig. 10 C). Downstream compo-
nents could read the signal and flip a switch when it reaches a
threshold, thereby triggering cell cycle progression when suffi-
cient growth has occurred. This model suggests that a cell size
checkpoint could operate by monitoring the amount of growth
that has occurred, rather than the absolute size of the cell. We
refer to this as a growth-dependent signaling hypothesis for
Interestingly, blocking membrane traffic causes rapid
repression of ribosome biogenesis via Pkc1 (Li et al., 2000;
Nanduri and Tartakoff, 2001). Thus, membrane traffic is linked
to ribosome biogenesis. It is therefore conceivable that diverse
aspects of cell growth and the cell cycle are regulated by signals
generated via membrane growth.
There are several attractive features of a growth-dependent
signaling hypothesis. A mechanism that monitors the extent
of growth, rather than absolute cell size, would be adaptable to
cells of diverse sizes and shapes. By linking signaling to the site
of growth, the checkpoint could control the extent of growth at
specific locations. In previous work, we found that the Swe1-
dependent checkpoint specifically monitors the size or growth
of the bud, which is consistent with the idea that the checkpoint
can monitor growth at a specific site (Harvey and Kellogg, 2003).
Growth-dependent signaling could also work in cells that do not
increase their size via polar growth. In this case, growth at mul-
tiple sites over the surface of the cell could generate a signal
that is read by downstream components. Another attractive
(Abe et al., 2003). Active Rho1 directly binds Pkc1 and induces
Pkc1 autophosphorylation, and Pkc1 undergoes hyperphos-
phorylation during the period of bud growth that is dependent
on vesicle fusion. Pkc1, in turn, binds to PP2ACdc55-Zds1/2,
which directly controls the phosphorylation states of Mih1
and Swe1 (Uetz et al., 2000; Drees et al., 2001; Pal et al., 2008;
Yasutis et al., 2010; Wicky et al., 2011). Together, these obser-
vations connect Sec6-dependent vesicle fusion at the site of
polar membrane growth to entry into mitosis.
A role for Rho1, Pkc1, and PP2ACdc55 in controlling entry
into mitosis may be conserved. The closest human homo-
logue of Pkc1 is called PRK2 (also called PKN2). PRK2 was
found to control entry into mitosis, likely via regulation of Cdc25
(Schmidt et al., 2007). In Drosophila melanogaster, a close rel-
ative of Pkc1 controls proliferation and asymmetric division
of neuroblasts and interacts with PP2ATwins (Chabu and Doe,
2009). Twins is the Drosophila homologue of Cdc55 and con-
trols mitosis (Chen et al., 2007).
Previous genetic analysis suggested that the dephos-
phorylation of Mih1 is an important step in mechanisms re-
quired for activation of Mih1 during entry into mitosis (Pal et al.,
2008; Wicky et al., 2011). The experiments reported here
strengthen the link between Mih1 dephosphorylation and entry
into mitosis, and they support a model in which hyperphos-
phorylation of Mih1 reflects the action of a checkpoint that
keeps Mih1 inactive early in the cell cycle. It is not yet known
whether hyperphosphorylation of Mih1 controls its localiza-
tion or phosphatase activity. Thus far, it has not been possible
to assay the activity of differently phosphorylated forms of
Mih1 because it is a low abundance protein and shows poor
solubility (unpublished data).
Disrupting membrane traffic caused cells to undergo a
Swe1-dependent checkpoint arrest in early mitosis with low
levels of mitotic cyclin. Interestingly, previous work has shown
Figure 9. Signaling to Pkc1 is dependent on membrane traffic. (A) Cells
were released from a G1 arrest at room temperature. The behavior of
Pkc1, Cln2-3×HA, and Clb2 were assayed by Western blotting. All blots
are from the same samples, so timing of events may be directly compared.
(B) Wild-type and sec6-4 cells were released from a G1 arrest. A sample
was taken at 70 min (t = 0), and the cells were then shifted to the restrictive
temperature (34°C). Pkc1 phosphorylation was assayed by Western blot-
ting. Numbers shown next to the Western blots indicate molecular mass
on March 27, 2012
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11 of 16Entry into mitosis linked to membrane traffic • Anastasia et al.
is required for both cell growth and morphogenesis, the check-
point could effectively monitor both events.
An appealing model is that the checkpoint controls the
duration of polar growth by determining when the switch from
polar to isotropic growth occurs. Because mitotic Cdk1 induces
the switch, the timing of Cdk1 activation determines the timing
of the switch. It is likely that the switch is induced early in
mitosis by a low level of Cdk1 activity because G1 cyclins, which
drive polar growth, begin to decline as soon as the mitotic
cyclins appear (Fig. 9 A). Recent work defined a systems-level
mechanism that generates and maintains low level Cdk1 activa-
tion in early mitosis (Deibler and Kirschner, 2010; Harvey et al.,
2011). Interestingly, mathematical modeling suggests that low
level activation of Mih1 is required for low level activation
of Cdk1 (Harvey et al., 2011). Therefore, an interesting possi-
bility is that hyperphosphorylation of Mih1 early in the cell
cycle keeps it inactive. Pkc1-dependent dephosphorylation of
Mih1 could then relieve inhibition of Mih1 and allow it to
become active at a basal level that promotes low level activa-
tion of Cdk1/Clb2, thereby triggering the switch from polar to
We found no evidence that expression of constitutively
active Rho1 or Pkc1 from the GAL1 promoter could trigger
destruction of Mcd1 or hyperphosphorylation of Swe1 in
checkpoint-arrested cells. This may be an uninformative nega-
tive result. For example, overexpression of constitutively active
Rho1 or Pkc1 may activate PP2ACdc55 to an artificially high
level that blocks hyperphosphorylation of Swe1. Alternatively,
the function of the Rho1–Pkc1 pathway may be to initiate early
feature of growth-dependent signaling is that the proposed
components are highly conserved and could carry out similar
functions in all eukaryotic cells.
Although growth-dependent signaling is an appealing
hypothesis, the data are currently consistent with alternative
hypotheses. For example, the checkpoint may simply monitor
whether membrane addition at the site of cell growth is occur-
ring, rather than the extent of membrane growth. Another pos-
sibility is that the checkpoint monitors the concentration of
active Rho1 or Pkc1 associated with the daughter bud mem-
brane as a means of measuring absolute bud size. We also
cannot yet rule out the possibility that blocking membrane traf-
fic causes indirect effects on other cellular events that more
directly trigger the checkpoint arrest.
Reconciling divergent views of the
Pioneering work in fission yeast reached the conclusion that
Wee1 and Cdc25 mediate a cell size checkpoint (Nurse, 1975).
Subsequent studies found that budding yeast Swe1 and Mih1
are required for cell size control, which suggested the existence of
a conserved cell size checkpoint (Russell et al., 1989; Jorgensen
et al., 2002; Harvey and Kellogg, 2003; Pal et al., 2008). How-
ever, it has also been proposed that Swe1 and Mih1 mediate a
checkpoint that monitors bud morphogenesis rather than size
(Lew and Reed, 1995a; Lew, 2003; Keaton and Lew, 2006).
The discovery that disrupting membrane traffic at the site of
bud growth causes a checkpoint arrest suggests a way to recon-
cile these divergent views. Because localized membrane traffic
Figure 10. A model for signals that link mitotic entry to membrane growth. (A) Dependency relationships in the Rho1–Pkc1 signaling axis. (B) Known
binding interactions in the Rho1–Pkc1 signaling axis. (C) A hypothetical model for generation of a signal that is proportional to membrane growth. Rho1
is activated at the site of membrane growth by a guanine nucleotide exchange factor (GEF). As more Rho1-bearing vesicles are delivered to the site of
growth, the amount of active Rho1 increases. Downstream components of the signaling axis read the signal and flip a switch to initiate mitosis when the
signal reaches a threshold level.
on March 27, 2012
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JCB 12 of 16
unified understanding of their functions in diverse cell types as
well as a better understanding of the mysterious mechanisms
that control cell growth, size, and shape.
Materials and methods
Yeast strains and culture conditions
The genotypes of the strains used in this study are listed in Table 1. All
strains are in the W303-1A background (leu2-3,112 ura3-1 can1-100
ade2-1 his3-11,15 trp1-1 bar1 GAL+) except where noted. Cells were
grown in YPD (yeast extract-peptone-dextrose) media supplemented with
40 mg/liter adenine, except where noted. To facilitate moving the sec6-4
mutant allele into different strain backgrounds, the kanMX6 marker cas-
sette was integrated 256 base pairs downstream of the sec6-4 allele
in strain RSY2786 to create strain DK1473 (oligonucleotides [oligos]
TCCCCGGGTTAATTAA-3 and 5-GCTCCGTCTAGAGAATGACACGAAA
CCTCACTCGTTGTTTCCGAATTCGAGCTCGTTTAAAC-3). To transfer the
sec6-4 mutant allele into different strain backgrounds, PCR was used to
amplify sec6-4 along with the KanMX6 cassette and flanking DNA, and
the resulting product was used to transform cells (oligos 5-CCACCACA-
TTGGAACATATTTGAAGTATAC-3 and 5-CATTGCTACCAAGCTAACA-
AAAGGATCAGG-3). Transformants were selected on G418 and screened
for temperature sensitivity to identify which ones recombined the sec6-4
mitotic events, including shutting off polar growth, without
triggering full entry into mitosis.
A role for Wee1 and Cdc25 family members in determin-
ing the duration of polar growth could explain the paradoxical
finding that loss of Wee1 causes a severe phenotype in fission
yeast but only a mild phenotype in budding yeast. Fission yeast
are completely dependent on polar growth. Thus, Wee1 mutants
that disrupt the duration of polar growth should have severe
consequences. In contrast, budding yeast have a short polar
growth phase, which is followed by isotropic growth. Thus, loss
of Swe1 would be expected to cause a mild decrease in cell size
and formation of cells that are more round, which is the ob-
Cell growth, size, and shape are of fundamental impor-
tance, so it seems likely that they are controlled by conserved
core mechanisms that appeared early in evolution. Wee1 and
Cdc25 family members are highly conserved, yet a clear picture
of their conserved functions in diverse cell types has remained
surprisingly elusive. Further analysis of the signals that control
Wee1 and Cdc25 family members is likely to lead to a more
Table 1. Strains used in this study
Altman and Kellogg, 1997
Altman and Kellogg, 1997
Harvey et al., 2011
Pal et al., 2008
Saka et al., 2001
Saka et al., 2001
MATa his3-200 leu2-3,112 trp1-1 ura3-52 sec6-4 BAR1
MATa his3-200 leu2-3,112 trp1-1 ura3-52 sec6-4::kanMX6 BAR1
MATa sec6-4::kanMX6 cdc28Y19F-HA::URA3
MATa sec6-4::kanMX6 swe1::His3MX6
MATa MCD1-6×HA::natNT2 sec6-4::kanMX6
MATa MCD1-6×HA::natNT2 sec6-4::kanMX6 swe1::His3MX6
MATa sec7-4 his3-11 leu2-3/112 ura3-1
MATa cdc55-4::His3MX6 BAR1
MATa pkc1-14::His3MX6 swe1::URA3
MATa sec6-4::His3MX6 pDN3 [GAL1-RHO1-Q68H, URA3]
MATa pDK20 [GAL1, URA3]
MATa pSH32 [GAL1-3XHA, URA3]
MATa pDN4A [GAL1-PKC1*-3×HA, URA3]
MATa pDN5A [GAL1-PKC1-3×HA, URA3]
MATa MCD1-6×HA::natNT2 sec6-4::kanMX6 pSH32 [GAL1-3×HA, URA3]
MATa MCD1-6×HA::natNT2 sec6-4::kanMX6 pDN4A [GAL1-PKC1*-3×HA, URA3]
MATa BAR1 pDN4A [GAL1-PKC1*-3×HA, URA3]
MATa cdc55-4::His3MX6 pDN4A [GAL1-PKC1*-3×HA, URA3] BAR1
MATa sec6-4::kanMX6 MCD1-6×HA::natNT2 pGAL1-ZDS1 [GAL1-ZDS1, URA3]
CDC55-3×HA::His3MX6 zds1 zds2
pDN3 [GAL1-RHO1*, URA3]
pkc1-21 pDN3 [GAL1-RHO1*, URA3]
ura3 trp1 ade2 lys2 leu2 his3 gal-
rho1::HIS3 ade3::rho1-2::LEU2 ura3 trp1 ade2 lys2 leu2 his3 gal-
aUniversity of California, Berkeley, Berkeley, CA.
on March 27, 2012
Published March 26, 2012
13 of 16Entry into mitosis linked to membrane traffic • Anastasia et al.
peptone) containing 2% raffinose or 2% glycerol and 2% ethanol. Expres-
sion was initiated by addition of galactose to 2%.
At each time point, 1.6-ml samples were collected in screw-cap
tubes. The cells were rapidly pelleted, the supernatant was removed, and
250 µl of glass beads was added before freezing on liquid nitrogen. To lyse
cells, 140 µl of sample buffer (65 mM Tris-HCl, pH 6.8, 3% SDS, 10% glycerol,
5% -mercaptoethanol, 50 mM NaF, and 100 mM -glycerophosphate)
was added. 2 mM PMSF was added to the sample buffer immediately
before use from a 100-mM stock made in 100% ethanol. To lyse cells,
tubes were placed in a disrupter (Multibeater-8; BioSpec) at top speed
for 2 min. The tubes were immediately removed, centrifuged for 13 s
in a microfuge (5415C; Eppendorf), and then placed in a boiling water
bath for 5 min. After boiling, the tubes were centrifuged again for 5 min,
and either 5 µl (for Zds1) or 20 µl was loaded on a gel. To assay nuclear
division, 250-µl samples were collected, and the cells were pelleted and
fixed by resuspending in 250 µl of 70% ethanol and 30% 50 mM Tris-HCl,
pH 8.0. Nuclei were stained with Sytox green, and the presence of multiple
nuclei was assayed by microscopy. To assay mitotic spindle assembly, cells
were fixed with formaldehyde and stained with antitubulin as previously
described (Harvey and Kellogg, 2003). At each time point, a minimum of
200 cells was scored for the presence of short or long spindles.
PAGE and Western blotting were performed as previously described
(Anderson et al., 1973; Harvey et al., 2011). All gels were run at 20 mA on
the constant current setting. For Mih1 and HA Western blots, electrophore-
sis was performed on a 10% polyacrylamide gel until a 29-kD prestained
marker ran to the bottom of the gel. For Swe1 Western blots, electropho-
resis was performed on a 10% polyacrylamide gel until a 66.5-kD marker
ran to the bottom of the gel. For Zds1 Western blots, electrophoresis was
performed on a 9% polyacrylamide gel until a 65-kD marker ran to the
bottom of the gel. For Pkc1 Western blots, electrophoresis was performed
on a 9% polyacrylamide gel until a 57.6-kD marker ran to the bottom of
the gel. Western blots were transferred for 90 min at 800 mA at 4°C in a
transfer tank (Hoeffer) in a buffer containing 20 mM Tris base, 150 mM
glycine, and 20% methanol. Blots were probed overnight at 4°C with
affinity-purified rabbit polyclonal antibodies raised against a Mih1, Swe1,
Zds1, Pkc1, or HA peptide. Blots were probed with an HRP-conjugated
donkey anti–rabbit secondary antibody (GE Healthcare).
Coimmunoprecipitation of Pkc1 and PP2ACdc55-Zds1/2 was assayed as
previously described with the following modifications (Mortensen et al.,
2002). Strains DK186 (untagged control), HT195 (CDC55-3×HA), and
DK354 (CDC55-3×HA zds1 zds2) were grown to an OD of 0.7 in YPD
media at room temperature. Cells from 50 ml of each cell culture were pel-
leted, resuspended in 1 ml YPD, pelleted again in a 2-ml tube, and frozen
on liquid nitrogen.
Immunoaffinity beads were made by binding mouse anti-HA mono-
clonal antibodies (Santa Cruz Biotechnology, Inc.) to protein A beads (Bio-
Rad Laboratories) overnight at 4°C on a rotator (Labquake Rotisserie;
Barnstead Thermolyne). For each immunoprecipitation, 10 µg anti-HA anti-
body was bound to 15 µl protein A beads in the presence of phosphate-
buffered saline containing 500 mM NaCl and 0.1% Tween 20.
Cell extracts were made by adding 300 µl of acid-washed glass
beads to frozen cell pellets followed by 300 µl of cold lysis buffer (50 mM
Hepes-KOH, pH 7.6, 75 mM B glycerol phosphate, 50 mM NaF, 1 mM
MgCl2, 1 mM EGTA, 5% glycerol, 0.25% Tween 20, and 1 mM PMSF).
The tubes were immediately placed into a disrupter (Multibeater-8) and
shaken at top speed for 25 s. The tubes were briefly spun at 14,000 rpm
in a microfuge to collect the sample at the bottom of the tube and then
placed in an ice-water bath for 5 min. 250 µl supernatant was transferred
to a new 1.5-ml tube and replaced with 250 µl lysis buffer. The tubes were
beaten again for 25 s. 250 µl supernatant was removed, pooled with the
first supernatant, centrifuged at 14,000 rpm in a microfuge for 10 min
at 4°C, and then added to the immunoaffinity beads equilibrated in lysis
buffer. A 10-µl sample of the extract was taken before treatment with
antibody and frozen in liquid nitrogen for analysis by Western blotting.
The tubes were rotated gently end over end at 4°C for 1 h and 45 min and
then washed three times batchwise with 400 µl lysis buffer without PMSF.
Cdc55-3×HA and associated proteins were eluted from the beads by the
addition of 150 µl elution buffer (50 mM Hepes-KOH, pH 7.6, 1 M NaCl,
1 mM MgCl2, 1 mM EGTA, 5% glycerol, and 0.5 mg/ml HA dipeptide) at
room temperature. The beads were incubated for 15 min and gently mixed
every few minutes to allow for mixing of the beads. The beads were pel-
leted in a microfuge, and 125 µl of the supernatant was removed, taking
allele into the genome. This approach was used to put the sec6-4 allele in
strains DK186 and SH761, thereby creating strains DK1475 and DK1606,
respectively. Strain DK936 was generated by switching the mating type of
strain AFY39 to MATa (Deitz et al., 2000). Strains DK1731, DK1732, and
DK1733 were generated by putting a 6×HA tag at the C terminus of MCD1
in DK186, DK1475, and DK1600 using a PCR-based approach (oligos
GCTGCAGGTCGAC-3 and 5-TTGGGTCCACCAAGAAATCCCCTCG-
GCGTAACTAGGTTTTAATCGATGAATTCGAGCTCG-3; Janke et al., 2004).
Strain DK1895 was generated by digesting plasmid pGAL-ZDS1 with
NcoI to target integration at the URA3 locus in strain DK1732. Strain
DK1729 was created by digesting pDN3 with StuI to target integration at
the URA3 locus in strain DK1440. Strains DK1725 and DK1786 were cre-
ated by digesting pDK20 with StuI to target integration at the URA3 locus
in strains DK1440 and DK186. Strain DK1788 was created by digesting
pSH32 with ApaI to target integration at the URA3 locus in strain DK186.
Strains DK1790, DK1807, and DK1809 were created by digesting pDN4A
with ApaI to target integration at the URA3 locus in strains DK186, DK1496,
and DK177. Strain DK1834 was created by digesting pDN5A with ApaI
to target integration at the URA3 locus in strain DK186.
Plasmid construction and generation of antiPkc1 antibodies
An integrating plasmid that expresses ZDS1 from the GAL1 promoter was
created by amplifying the ZDS1 open reading frame by PCR and clon-
ing into the EcoR1 and BamH1 sites of pDK20 to create pGAL1-ZDS1
(oligos 5-GCGGAATTCATGTCCAATAGAGATAACGAGAGC-3 and
5-GCGGGATCCTCAGGGTTGTTGTTGTTGTTGTTG-3). This plasmid can
be cut with Nco1 to target integration at URA3. An integrating vector that
expresses RHO1 from the GAL1 promoter was created by amplifying the
RHO1 open reading frame and cloning into the HindIII and EagI sites of
pDK20 to create pDN1 (oligos 5-GCGCAAGCTTATGTCACAACAAGTT-
GGTAACAGTATCAGAAGA-3 and 5-CGCCGGCCGCTATAACAAGA-
CACACTTCTTCTTCTTCTTTTCAGTAGT-3). This plasmid can be cut with Stu1
to target integration at URA3. To create a plasmid that expresses constitu-
tively active RHO1 (RHO1*) from the GAL1 promoter, site-directed muta-
genesis was used to convert glutamine 68 to histidine in pDN1 to create
pDN3 (oligos 5-TGGGATACCGCTGGTCACGAAGATTATGATAGA-3 and
5-TCTATCATAATCTTCGTGACCAGCGGTATCCCA-3; Delley and Hall,
1999). An integrating plasmid that expresses constitutively active PKC1
(PKC1*) tagged with 3×HA was created by amplifying PKC1* from pGAL1-
PKC1* (Delley and Hall, 1999) and cloning into the Eag1 and Xho1 sites
of pSH32 to create pDN4A (oligos 5-GCGCTCGAGATGAGTTTTTC-
ACAATTGGAGCAGAACATTAAAAAAAAGA-3 and 5-GGAAGTGA-
An integrating plasmid that expresses wild-type PKC1 from the GAL1
promoter was created by mutagenizing PKC1* in pDN4A back to wild
type to create pDN5A (oligos 5-GCAGTTGATGGGTGGACTACATCGT-
CATGGTGCTATTATCAATAGG-3 and 5-CCTATTGATAATAGCACCAT-
GACGATGTAGTCCACCCATCAACTGC-3). To make an antibody that
recognizes Pkc1, the N terminus of PKC1 was amplified and recombined
into the vector pDONR221 (Gateway; Invitrogen) to create pDK117 (oligos
GAAC-3 and 5-GTCAAGAAAGCTGGGTCTCACGGTTGTTGATTATC-
CATTATGTCATT-3). The N terminus was then recombined into pDEST15
to create pDK118, which allows expression as a GST fusion. The GST-Pkc1
fusion was purified and used to immunize a rabbit using standard proto-
cols. The serum was run over a GST column to deplete anti-GST antibodies
and was then run over a GST-Pkc1 column to purify anti-Pkc1 antibodies.
To test the specificity of the antibody, we probed extracts from wild-type
cells and from cells in which Pkc1 was fused to CFP. The Pkc1-CFP fusion
was shifted above untagged Pkc1, which revealed that there are no back-
ground bands in the vicinity of Pkc1 (Fig. S1). Cells carrying the Pkc1-CFP
fusion protein as their sole source of Pkc1 grew slowly at low temperatures
and were nearly inviable at 37°C, which indicated that the protein was
not fully functional. This could explain why multiple phosphorylation forms
of the Pkc1-GFP fusion protein were not observed.
Cell cycle time courses
Cells were grown overnight at room temperature to an OD600 of 0.65 and
were then arrested in G1 by the addition of -factor to either 0.5 µg/ml
(for bar1 strains) or 15 µg/ml (for BAR1 strains) for 3 h. Cells were re-
leased from the arrest by washing 3× with fresh YPD media at room tem-
perature. For time courses involving a temperature shift, cultures were
incubated at room temperature before the shift and were transferred to
a shaking water bath to shift the temperature. For expression of galactose-
inducible promoters, cells were grown overnight in YEP (yeast extract
on March 27, 2012
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care to avoid the antibody-containing beads. This process was repeated
once more with the exception that 150 µl of the supernatant was removed.
The supernatants were pooled and precipitated by the addition of trichloro-
acetic acid to 10%. The resulting pellet was resuspended in 25 µl protein
sample buffer; 20 and 2 µl were loaded onto a 9% polyacrylamide gel
and used for a Western blot probing for Pkc1 and Cdc55-3×HA, respec-
tively. For Western blotting of the crude extracts, 90 µl protein sample
buffer was added to 10 µl of crude extract, the samples were boiled, and
15 µl was loaded onto an SDS-PAGE gel.
Generation of temperaturesensitive alleles of PKC1
To generate a collection of PKC1 temperature-sensitive alleles, we first
used a PCR-based approach to integrate the His3MX6 marker cassette
downstream of the PKC1 gene in the wild-type strain DK186, creating
strain DK1631 (oligos 5-GCCGTATGTTCAACAATGCGCATTCTGTTTA-
CATTATTAACGGATCCCCGGGTTAATTAA-3 and 5-CTGTCAACTTTA-
Genomic DNA was purified from this strain and used as a template to
amplify the PKC1-His3MX6 fragment and flanking DNA by PCR using Pfu
polymerase (oligos 5-GCTATTCAATTTGGCTGAGTAG-3 and 5-GCTTT-
GAGTATAGTCGAATGTG-3). The PCR product was gel purified and
used as a template to carry out a large-scale PCR reaction with the error-
prone Taq polymerase (oligos 5-GATAGCCGTCGAAGAAAATA-3 and
5-GATTCGTCCATTTATGCCGTAT-3). The PCR reaction was then trans-
formed into DK186 and plated onto –His plates at 150 colonies per
plate to screen 4,500 colonies for temperature sensitivity by replica
plating. To eliminate mutants in the HIS gene, candidates were also tested
for temperature sensitivity on YPD plates, and mutants were further tested
for rescue by wild-type PKC1 on a centromere plasmid. The screen identi-
fied 36 temperature-sensitive alleles of PKC1. To screen the collection for
defects in cell growth, mutants were struck on YPD plates and left to grow
at room temperature, 34, or 37°C for 2 d. Cell morphology was exam-
ined by light microscopy.
Yeast cells were fixed with formaldehyde and photographed using a
microscope (Axioskop; Carl Zeiss) fitted with a 100× Plan-Neofluar 1.3
NA objective and a camera (AxioCam HRm; Carl Zeiss). Images were
acquired using AxioVision software (Carl Zeiss). Actin was stained with
Latrunculin A treatment
For Fig. 4, cells were released from an -factor arrest into fresh YPD media
at room temperature. After 70 min, the presence of small buds was verified
by microscopy. The culture was then split in half, and latrunculin A was
added to a final concentration of 100 µM to one half of the culture,
whereas an equal volume of DMSO was added to the other half.
Online supplemental material
Fig. S1 shows the specificity of the anti-Pkc1 antibody. Online supple-
mental material is available at http://www.jcb.org/cgi/content/full/jcb
We thank members of the laboratory and Needhi Bhalla for helpful advice
and critical reading of the manuscript.
This work was supported by the University of California Cancer
Research Coordinating Committee and the National Institutes of Health
(grant GM069602). V. Thai was supported by a National Institutes of
Health Postdoctoral Fellowship (grant F32GM087103-02).
Submitted: 18 August 2011
Accepted: 23 February 2012
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on March 27, 2012
Published March 26, 2012