[Cell Cycle 4:7, 961-971; July 2005]; ©2005 Landes Bioscience
Crm1-Mediated Nuclear Export of Cdc14 is Required for the Completion
of Cytokinesis in Budding Yeast
Report
ABSTRACT
The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The
proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic
exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is
released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its
functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In
contrast, less is known about the mechanism by which Cdc14p is released from the nucleus
to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal
(NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p
degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at
non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic
structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated
inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear
export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.
3Department of Biology; Colgate University; Hamilton, New York USA
*Correspondence to: Hongtao Yu; Department of Pharmacology; University of
Texas Southwestern Medical Center; 5323 Harry Hines Boulevard; Dallas, Texas
75390 USA; Tel.: 214.648.9697; Fax: 214.648.2971; Email: hongtao.yu@
utsouthwestern.edu
SC
KEY WORDS
Extensive genetic and biochemical studies in several model organisms have uncovered
the general molecular mechanisms governing the orderly progression through mitosis.1,2
Mitotic cyclin-dependent kinases (Cdks) phosphorylate a large set of cellular proteins,
promoting the onset of mitosis and the subsequent faithful partition of the duplicated
chromosomes and cellular components into the two daughter cells.1,2 After the cells have
accurately segregated their sister chromatids, mitotic Cdks are inactivated largely through
ubiquitin-mediated proteolysis of mitotic cyclins, allowing mitotic exit and cytokinesis.3-7
It is essential to coordinate the inactivation of mitotic Cdks and subsequent dephosphorylation of key mitotic regulators with the execution of mitotic exit and cytokinesis.1,2,8
The molecular mechanisms of mitotic exit and cytokinesis are well understood in
budding and fission yeasts. Two homologous GTPase/kinase signaling networks—the
mitotic exit network (MEN) in budding yeast and the septation initiation network (SIN)
of fission yeast—regulate the final stages of mitosis.9-13 The Cdc14 phosphatases are
critical downstream components of MEN and SIN, and are conserved in higher eukaryotes.9,11-16 Recent structural studies have indicated that the Cdc14 phosphatases prefer to
dephosphorylate phosphoserine/phosphothreonine-proline motifs.14,15 As Cdks are
proline-directed kinases, the Cdc14 phosphatases are appealing candidates for reversing
Cdk-mediated phosphorylation events during mitotic exit and cytokinesis.14,15
Despite the conservation between the molecular components of MEN and SIN, the
MEN and SIN mutants exhibit different phenotypes.9-11,13 For example, budding yeast
cells harboring cdc14 mutations arrest in late anaphase.17,18 They undergo chromosome
segregation, contain elongated spindles, and maintain high levels of mitotic Cdk activity.17,18
The crucial substrates of Cdc14p during mitotic exit include Cdh1p, Sic1p, and Swi5p.17
In particular, dephosphorylation of Cdh1p by Cdc14p is required for the activation of the
anaphase-promoting complex or cyclosome (APC/C), the ubiquitin ligase that mediates
the ubiquitination and degradation of Clb2p.19 In contrast, fission yeast cells containing
mutations in Clp1p/Flp1p, the fission yeast ortholog of Cdc14p, degrade mitotic cyclins
and undergo mitotic exit normally.20,21 Instead, these mutant cells exhibit mild defects in
cytokinesis among other phenotypes.20,21 The apparent phenotypic difference between
S. cerevisiae cdc14 and S. pombe clp1 mutant cells has led to the hypothesis that the Cdc14
IEN
Previously published online as a Cell Cycle E-publication:
http://www.landesbioscience.com/journals/cc/abstract.php?id=1798
INTRODUCTION
CE
Received 02/07/05; Accepted 03/10/05
OT
D
2Department of Animal Biology; University of Pennsylvania; School of Veterinary
Medicine; Philadelphia, Pennsylvania USA
ON
of Pharmacology; University of Texas Southwestern Medical Center;
Dallas, Texas USA
.D
1Department
IST
RIB
UT
E
.
Joshua Bembenek1
Jungseog Kang1
Cornelia Kurischko2
Bing Li1
Jesse R. Raab3
Kenneth D. Belanger3
Francis C. Luca2
Hongtao Yu1,*
ES
ACKNOWLEDGEMENTS
BIO
Cdc14, Crm1, nuclear export signal, Ran,
mitotic exit network, cytokinesis, nucleocytoplasmic
shuttling
©
20
05
LA
ND
We thank D. Kellogg, M. Tyers, O. Cohen-Fix,
and M. Culbertson for reagents, J. Goodman and
the Butow laboratory for help with yeast protocols,
O. Cohen-Fix and J. White for reading the manuscript critically, and members of the Yu laboratory
for helpful discussions. We are grateful to J. White
and the members of LOCI for multiphoton live-cell
imaging. J.B. is supported by a predoctoral training
grant from the National Institutes of Health. H.Y.
is the Michael L. Rosenberg Scholar of Biomedical
Research. This work is supported by the National
Institutes of Health (GM65107 to K.D.B.,
GM60575 to F.C.L., and GM61542 to H.Y.).
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Crm1p-Dependent Nuclear Export of Cdc14p
family of phosphatases performs different tasks at the end of mitosis
in different organisms.9,10
Homologues of Cdc14 have been identified in higher eukaryotes.
In C. elegans, though null mutations of the cdc-14 gene do not result
in mitotic defects, depletion of cdc-14 by RNA interference (RNAi)
in strains with a compromised zen-4 function causes cytokinesis
defects.22-24 Furthermore, ZEN-4 is displaced from the microtubules
of the central spindle by cdc-14 RNAi.25 ZEN-4 is also a direct
substrate of both Cdk1 and CDC-14.25 Two Cdc14 homologues,
hCdc14A and hCdc14B, exist in humans.26 Both human Cdc14
proteins are capable of dephosphorylating Cdh1 and activating
APC/CCdh1 in vitro.27 Furthermore, RNAi-mediated depletion of
hCdc14A in human cells leads to multiple defects, including cytokinesis failure.28 Therefore, Cdc14 has been implicated in both mitotic
exit and cytokinesis in various organisms.
Changes in subcellular localization constitute an important mode
of regulation for Cdc14. In S. cerevisiae, Cdc14p is sequestered in
the nucleolus by Net1p of the RENT complex and kept inactive
during much of the cell cycle.29,30 There are two separate waves of
release of Cdc14p from the nucleolus to the nucleoplasm and cytoplasm
during anaphase.31 The FEAR (Fourteen Early Anaphase Release)
network, consisting of Cdc5p, Spo12p, Esp1p, and Slk19p, initiates
the first wave of Cdc14p release from the nucleolus during early
anaphase, partly by promoting the dissociation of Cdc14p from
Net1p.16,31-36 The MEN network then triggers and maintains the
full release of Cdc14p in late anaphase.16,29,30 Recently, Cdc14p and
components of FEAR have been shown to control the segregation of
nucleolus and ribosomal DNA (rDNA) during anaphase by facilitating the localization of condensin to the rDNA locus.36-42
The Cdc14 proteins in other organisms exhibit similar but distinct
subcellular localization patterns. The S. pombe Clp1p/Flp1p is
sequestered in the nucleolus in interphase while it is released from
the nucleolus and localizes to the SPB, the mitotic spindle, and the
contractile ring during mitosis.20,21 In C. elegans, Cdc14 localizes to
the central spindle in anaphase and to the midbody in telophase.22
In humans, hCdc14A resides on the centrosomes throughout
the cell cycle whereas hCdc14B resides in the nucleolus during
interphase and centrosomes in mitisis.27,28,43 Interestingly,
the subcellular localization of hCdc14A is regulated by
Crm1-dependent nuclear export.28 Mutation of the leucine-rich
nuclear export signal (NES) of hCdc14A or treatment of cells with
leptomycin B, a chemical inhibitor of Crm1, causes the accumulation
of hCdc14A in the nucleolus.28 This suggests that the Ran-dependent
protein trafficking machinery might be important in regulating the
localization and function of Cdc14.
In this study, we have further characterized the role of the Randependent protein transport system in Cdc14 regulation in both
mammalian and budding yeast cells. Using a heterokaryon assay, we
show that hCdc14B shuttles between the nucleolus and the cytoplasm
in mammalian cells and that this nucleocytoplasmic shuttling is
Crm1-dependent and requires a functional NES. We further show
that the budding yeast Cdc14p also contains a functional NES that
associates with Crm1p. Budding yeast cells harboring mutations in
the NES of Cdc14p exhibit a temperature-sensitive (ts) phenotype.
In contrast to the classical ts allele of CDC14, cdc14-1, the cdc14
NES mutant cells degrade Clb2p normally, but undergo abnormal
bud growth and fail to complete cytokinesis at the restrictive temperature. A significant fraction of cdc14 NES mutant cells re-bud in
the absence of cytokinesis at the restrictive temperature. Consistent
with Cdc14p playing a role in cytokinesis, live cell imaging reveals
962
that Cdc14p localizes to the bud neck in late anaphase. The bud
neck localization of Cdc14p is disrupted in the cdc14 NES mutants
at 37˚C and by leptomycin B-treatment. Therefore, our findings
reveal a role of Cdc14p in coordinating mitotic exit and cytokinesis
in budding yeast that is regulated by Crm1p-dependent nucleocytoplasmic transport.
MATERIALS AND METHODS
Plasmids. For mammalian expression plasmids, the open reading frames
(ORF) of hCdc14A, hCdc14B, and the budding yeast CDC14 were amplified
by PCR from human thymus cDNA (Clontech) and yeast genomic DNA,
respectively, and cloned into the pCS2-GFP mammalian expression vectors.
Mutations of the nuclear export signal (NES) motifs of the Cdc14 proteins
were introduced into these mammalian expression vectors using the
QuikChange Site-Directed Mutagenesis Kit (Stratagene) per manufacturer’s
protocols. For expression of Cdc14p in budding yeast, the entire coding
regions of either wild-type or NES mutants of Cdc14p-GFP were amplified
from the pCS2 vectors and subcloned into pRS416 (NEB). The promoter
region of CDC14, 1 kb of genomic DNA upstream of the CDC14 ORF, was
cloned into pRS416-Cdc14-GFP between the SacI and XbaI sites. A transcriptional terminator was also inserted into these vectors between the XhoI
and KpnI sites.
Mammalian cell culture, heterokaryon assay and immunofluorescence.
Human HeLa Tet-On (Clontech) and mouse L929 (ATCC) cells were
grown in Modified Eagle Medium (DMEM; Invitrogen) supplemented
with 10% Fetal Bovine Serum, 2 mM L-glutamine, and 100 µg/ml each of
penicillin and streptomycin. HeLa cells were plated at 50% confluency on
chambered glass slides (Nalge Nunc International) and transfected with
mammalian expression vectors encoding Myc6-tagged hCdc14B using the
Effectene Transfection Reagent (Qiagen) per manufacturer’s protocols. The
transfected HeLa cells were grown for 12-24 hrs to allow for the expression
of Myc6-hCdc14B. To inhibit Crm1, these cells were treated with 300 µM
of Leptomycin B (LC Laboratories) for 5 hrs. The hCdc14B-expressing
HeLa cells were treated with cycloheximide to block protein synthesis and
fused with mouse L929 cells as described previously.44
For immunofluorescence, the fused cells were fixed with ice-cold
methanol for 5 min and permeabilized with PBS containing 0.1% TritonX100 for 5 min. Cells were then incubated with 1 µg/ml anti-Myc in PBS
buffer containing 30 mg/ml BSA and 0.1% Triton-X100 for 90 min. After
three washes with 0.1% Triton-X 100 in PBS for 5 min, samples were incubated for 90 min with a-mouse secondary antibody conjugated to Alexa
Fluor 647 (Molecular Probes) in PBS buffer containing 5% Normal Goat
Serum and 0.1% Triton-X 100. Following three washes with PBS containing
0.1% Triton-X 100 for 5 min, DNA was stained with DAPI for 1 minute,
followed by three additional washes with PBS. Slides were mounted with
Aqua Poly/Mount (Polysciences, Inc.) and visualized with a 63x objective on
a Zeiss Axiovert 200M fluorescence microscope. The images were acquired
with a CCD camera using the Intelligent Imaging software and further
processed with Adobe Photoshop.
Yeast strains and microscopy. The yeast strains used in this study are listed
in Supplementary Table 1. The heterozygous BY4743 CDC14 (YFR028C)
deletion diploid strain was obtained from Research Genetics. This strain was
transformed with the wild-type pRS416-Cdc14-GFP vector. Transformed
diploid cells were sporulated and dissected. The spores containing the
Cdc14-GFP vector were identified by growth on YPD media supplemented
with 50 µg/ml geneticin to select for the resistance gene used for the initial
targeted disruption of the CDC14 ORF. The haploid cdc14∆ strain maintained
by the wild-type pRS416-Cdc14-GFP plasmid was then transformed with
the pRS415 (with LEU2) plasmids containing the coding sequences of
either wild-type or NES mutants of Cdc14p-GFP fusion proteins. Colonies
that grew on SD-Leu plates were then plated onto SD-Leu plates with 1 mg/ml
5-FOA to select for cells that had lost the wild-type pRS416-Cdc14-GFP
plasmid. To test temperature sensitivity, the strains were grown for two days
on SD-Leu plates at 26˚C, 34˚C, or 37˚C. The morphology of the log phase
Cell Cycle
2005; Vol. 4 Issue 7
Crm1p-Dependent Nuclear Export of Cdc14p
Figure 1. Sequence alignment of human Cdc14A, Cdc14B and the budding yeast Cdc14p. Yeast
Cdc14p has two overlapping sequence motifs that fit the canonical NES consensus sequence
(alignments I and II). Yeast Cdc14p mutants used in this study are shown. The three residues critical
for Cdc14p nuclear export and function (see below) are highlighted in red. Cdc14p mutants that
contain mutations of these three residues are denoted with a red asterisk. The green box indicates
the boundaries of the phosphatase domain.
yeast cells grown at 37˚C for 3 hours was analyzed on
slides with the 100X objective on a Zeiss axiovert
200M fluorescence microscope. For time lapse video
microscopy, yeast cells were mounted on an agarose gel
pad (0.5%) containing SD-Leu media, and incubated at
either room temperature or 37˚C with a heated stage.
The images were acquired with a CCD camera using
the Intelligent Imaging software and further processed
with Adobe Photoshop. For 4D microscopy, observation of GFP fluorescence was done on a multiphoton
fluorescence excitation microscope45 with the use of a
100X oil immersion objective and 900-nm excitation
(from a Ti-sapphire laser). Optical sections of ~0.5 µm
were viewed at given time intervals and collected as a
time series. Stacks of images were manipulated in
ImageJ46 using the public domain ImageJ program
(developed at the U.S. National Institutes of Health
and available on the Internet at http://rsb.
info.nih.gov/nih-image/).
Yeast two-hybrid assay. Primers containing 40–50
nucleotides homologous to pJG4-5 and 18–21
nucleotides homologous to CDC14 were used in PCR
reactions to amplify from Cdc14p-WT and Cdc14p-3A
the region of CDC14 encoding amino acid residues
322–398. Yeast strain W303 was cotransformed with the
resulting PCR products and pJG4-5 linearized using
XhoI to generate in-frame fusions of Cdc14p-NES with
A
Figure 2. Human Cdc14A and
Cdc14B undergo Crm1-dependent
nucleocytoplasmic shuttling. (A)
HeLa cells were transfected with
plasmids encoding GFP fusion
proteins of the wild-type hCdc14A
(hCdc14A-WT), the M362A/
I364A mutant of hCdc14A
(hCdc14A-2A), and the I355A/
L356A/L359A mutant of hCdc14A
(hCdc14A-3A). These cells were
treated with DMSO or 250 µM
leptomycin B (LMB; right panel),
fixed with methanol, counterstained
with DAPI, and visualized with a
fluorescence microscope. GFP is
shown in green while DAPI staining
is in blue. The bar indicates 10 µm.
(B) hCdc14B shuttles between the
nucleoli and the cytoplasm in a
Crm1-dependent manner. HeLa
cells were transfected with plasmids
encoding GFP fusion proteins of
wild-type hCdc14B (hCdc14B-WT),
the I400A/I402A mutant of
hCdc14B (hCdc14B-2A), and the
L393A/L394A/V397A mutant of
hCdc14B (hCdc14B-3A). The cells
were treated with cycloheximide,
fused with mouse L929 cells in the
absence or presence of leptomycin
B (LMB), stained with DAPI, and
visualized with a fluorescence
microscope. HeLa nuclei are indicated by arrows. The bar indicates
10 µm.
B
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Crm1p-Dependent Nuclear Export of Cdc14p
the B42 activation domain
(B42AD). The Cdc14p-NESWTB42AD (pKBB358) and Cdc14pNES3A-B42AD (pKBB359) plasmids generated by homologous
recombination were rescued from
Trp+ transformants by EZ yeast
plasmid isolation (GenoTech,
Inc.) per manufacturer’s instructions.
Plasmids pKBB358 and
pKBB359 were co-transformed
with pKW442 [CRM1-LexABD]
into yeast strain RFY206 and
transformants were selected on
SD -Trp-His media.47 RFY206
transformants were mated with
yeast strain EGY48. The resulting
diploids were tested for interactions by plating on SD-Ura-TrpHis-Leu + 2% galactose media.
Cells were incubated at 30˚C for
three days and scored for growth.
A
B
RESULTS
Cdc14p contains a functional
NES. Previous studies have
shown that Crm1-mediated
nuclear export regulates the
subcellular localization of human
Cdc14A.28 Sequence alignment
reveals that, in addition to Figure 3. The budding yeast Cdc14p contains a functional nuclear export signal. (A) HeLa cells were transfected with
hCdc14A, hCdc14B and yeast plasmids that encode Cdc14p-WT-GFP, Cdc14p-3A-GFP, and Cdc14p-∆NES-GFP, fixed with methanol, counterstained
Cdc14p also contain a putative with DAPI, and visualized with a fluorescence microscope. The Cdc14p-WT-GFP-expressing cells were also treated with
leucine-rich nuclear export signal 250 µM. Leptomycin B (LMB). GFP is shown in green while DAPI staining is in blue. The bar indicates 10 µm. (B) The
associates with Crm1p in a yeast two-hybrid assay. Plasmids expressing the NES region of Cdc14p
(NES) (Fig. 1). Due to the low NES of Cdc14p AD
were cotransformed with Crm1p-LexABD or empty LexABD plasmids into two-hybrid reporter yeast
fused
to
the
B42
sequence identities among the
strains. Interactions between Crm1p (Crm1p-BD) and wild-type Cdc14p NES (Cdc14p-NESWT-AD) or mutant Cdc14p
C-terminal regions of the Cdc14
NES (Cdc14p-NES3A-AD) were assayed on media lacking leucine and containing galactose (+GAL –Leu). Growth on
proteins, there are two possible
media containing leucine (+GAL +Leu) confirmed strain viability. Serially diluted cells were photographed after
alignments of the NES motif of
incubation at 30˚C for three days.
Cdc14p that fit the NES consensus (Fig. 1, labeled as I & II).
To test whether the NES motifs of hCdc14B and yeast Cdc14p are accumulation of GFP-hCdc14B in the mouse nuclei was blocked by leptoindeed functional, we assayed the localization of GFP-hCdc14A, GFP- mycin B treatment (Fig. 2B). Thus, hCdc14B contains a functional NES.
hCdc14B, and GFP-Cdc14p fusion proteins in HeLa cells (Fig. 2 and 3A). Despite its apparent localization in the nucleolus during interphase,
Consistent with previous studies,27,28,43 GFP-hCdc14A is localized to the hCdc14B undergoes active Crm1-dependent nuclear export.
centrosomes in interphase cells, as well as diffusive cytoplasmic localization
Unlike in budding yeast, S. cerevisiae Cdc14p-GFP was mostly cytoplasmic
(Fig. 2A). Mutations of the NES motif of hCdc14A lead to the accumula- in HeLa cells (Fig. 3A). Interestingly, mutations of three hydrophobic
tion of GFP-hCdc14A in the nucleoli (Fig. 2A). Similarly, treatment of the residues that were present in putative NES of Cdc14p-GFP in both alignments
transfected cells with leptomycin B, an inhibitor of Crm1, for 4 hours also (Cdc14p-3A) caused its accumulation in the nucleus and nuclear speckles
causes the accumulation of hCdc14A-GFP in the nucleoli (Fig. 2A). This (Fig. 3A). Treatment with leptomycin B, a chemical inhibitor of Crm1, had
indicates that hCdc14A contains a functional NES and is actively exported a similar effect (Fig. 3A). These data suggest that the NES of Cdc14p is also
from the nucleus in a Crm1-dependent manner.
functional, at least in this heterologous system. The budding yeast exportin,
Despite its apparent nucleolar localization, hCdc14B also contains a Crm1p/Xpo1p, mediates nuclear export of cargo proteins through direct
putative NES. We therefore tested whether GFP-hCdc14B undergoes binding to leucine-rich NES sequences.47-49 We used a yeast two-hybrid
nucleocytoplasmic shuttling during interphase using a well-established assay to examine whether Crm1p associated with the NES of S. cerevisiae
heterokaryon assay.44 Briefly, HeLa cells were transfected with plasmids Cdc14p (Fig. 3B). A fragment of Cdc14p (residues 322-398) containing the
encoding GFP-hCdc14B for 12 hours and treated with cycloheximide to wild-type NES sequence (Cdc14p-NESWT) interacted with Crm1p, while
inhibit further protein synthesis. These cells were then fused with untrans- the same domain of Cdc14p containing L359A, I360A and L362A mutations
fected mouse cells and fixed after four hours to detect translocation of the in the NES (Cdc14p-NES3A) did not interact with Crm1p. Cdc14p-NESWTfusion protein into the mouse nuclei. The wild-type GFP-hCdc14B protein B42AD also did not activate the reporter in the absence of Crm1p-LexABD.
was indeed detected in the mouse nuclei, indicating that hCdc14B underwent These data indicate that the leucine-rich NES of S. cerevisiae Cdc14p associactive nucleocytoplasmic shuttling (Fig. 2B). Mutations of the NES motif ates with the exportin Crm1p and that mutation of three residues within the
of hCdc14B largely eliminated its nucleocytoplasmic shuttling (Fig. 2B). NES of Cdc14p disrupts this interaction, consistent with our observation of
This nucleocytoplasmic shuttling of hCdc14B was Crm1-dependent, as the nuclear Cdc14p retention in HeLa cells when Crm1 is inhibited.
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Crm1p-Dependent Nuclear Export of Cdc14p
A
the integrated strains (Fig. 4) and
plasmid-rescued knockout strains
(Fig. 5) yielded comparable
results. Cdc14p-GFP was functional because the wild-type
CDC14-GFP strains were viable at
all temperatures (Fig. 4A and 5A).
On the other hand, strains carrying
cdc14 mutations in the three critical residues of the NES (cdc14DNES, cdc14-3A, cdc14-5A and
cdc14-6A) were not viable at 37˚C
(Fig. 4A and 5A). Unfortunately,
both cdc14-2A and cdc14-2A-2
strains were viable at 37˚C (Fig. 4A
and data not shown). Therefore,
we currently do not know which
alignment of the Cdc14p NES
motif with the NES consensus is
more appropriate. As an important
control, mutations of other
hydrophobic residues in nearby
regions of Cdc14p (cdc14-3A-2)
did not cause lethality at 37˚C
(Fig. 4A and data not shown).
This suggests that efficient
nuclear export of Cdc14p is
important for cell viability at high
temperatures.
We cannot rule out the possibility that, in addition to disrupting its interaction with Crm1p,
the Cdc14p-3A mutation affects
the folding of Cdc14p. However,
the fact that mutations of other
hydrophobic residues in the
C-terminal region of Cdc14p
had no effect on cell viability
argues against this notion. In
addition, the protein level of
Cdc14p-3A-GFP was similar to
that of Cdc14p-WT-GFP (Fig. 5B).
The protein level of Cdc14p-3AGFP remained similar at 26˚C or
37˚C (Fig. 5B), indicating that
the ts phenotype of the cdc14-3A
mutant was not caused by the
instability of Cdc14p-3A at 37˚C.
Lastly and most importantly, the
Cdc14p-3A mutant does not
Figure 4. Mutations of the NES of Cdc14p cause a temperature-sensitive phenotype. (A) Yeast strains carrying the
prevent Clb2p degradation (see
indicated cdc14 mutations integrated at the endogenous CDC14 locus were grown on YPD plates at the indicated
temperatures. (B) Percentage of cells with the indicated morphology. (C) The NES of Cdc14p is not required for Clb2p below), which is inconsistent
degradation. Strains carrying either Cdc14p-WT-GFP or Cdc14p-3A-GFP were arrested with α-factor, released into with a complete loss of function
fresh medium, and grown at 37˚C. Samples were taken at the indicated time points, dissolved with SDS sample buffer, of Cdc14p-3A at the restrictive
temperature. It is possible that
separated on SDS-PAGE, and blotted with the indicated antibodies.
Crm1p-dependent nuclear export
of Cdc14p is not required for cell
Mutations of the Cdc14p NES cause abnormal morphology and cycle progression at low temperature. It is also possible that a Crm1p-indecytokinesis defects at 37˚C. To analyze the function of the Crm1p-dependent pendent mechanism might be responsible for the nuclear export of Cdc14pnuclear export of Cdc14p in budding yeast, we generated various mutations 3A at the permissive temperature. This mechanism might not be sufficient
in CDC14 at the chromosomal locus. We also generated cdc14∆ strains at the restrictive temperature. Finally, there is a remote possibility that mutarescued with low-copy expression plasmids encoding various Cdc14p-GFP tions of the NES of Cdc14p might not completely block its interactions
fusion proteins under the control of the endogenous CDC14 promoter. To with Crm1p. The residual affinity between Crm1p and Cdc14p-3A might
test which alignment of the Cdc14p NES was more appropriate, we also be sufficient for nuclear export of Cdc14p at the permissive temperature.
mutated two pairs of hydrophobic residues that were predicted to be critical The weak interaction between Crm1p and Cdc14p-3A could be disrupted
in only one of the two alignments (Cdc14p-2A and Cdc14p-2A-2). Both at 37˚C, resulting in the ts phenotype of cdc14-3A cells.
B
C
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Crm1p-Dependent Nuclear Export of Cdc14p
Figure 5. The cdc14 NES mutants
degrade Clb2p at the restrictive temperature. (A) The cdc14∆ strains carrying
a plasmid containing the wild-type
CDC14, cdc14-DNES, or cdc14-3A
fused to GFP were grown on YPD
plates at the indicated temperatures.
(B) The cdc14∆ strains carrying either
Cdc14p-WT-GFP or Cdc14p-3A-GFP
were grown at 26˚C or 37˚C.
Samples were dissolved with SDS
sample buffer, separated on SDS-PAGE,
and blotted with the indicated antibodies. (C) cdc14-3A cells with integrated
Tub1p-GFP were grown at 37˚C, fixed
with formaldehyde, stained with DAPI
and phalloidin, and examined under
the microscope. GFP is shown in
green, DAPI in blue, and phalloidin in
red. The bars indicate 5 µm. (D) The
NES of Cdc14p is not required for
Clb2p degradation. The cdc14∆
strains carrying either Cdc14p-WT-GFP
or Cdc14p-3A-GFP and the cdc14-1
strain were grown at 26˚C or 37˚C.
Samples were dissolved with SDS
sample buffer, separated on SDS-PAGE,
and blotted with the indicated antibodies. (E) The cdc14D strains carrying
either Cdc14p-WT-GFP or Cdc14p-3AGFP were arrested with α-factor,
released into fresh medium, and
grown at 37˚C. Samples were taken at
the indicated time points, dissolved
with SDS sample buffer, separated on
SDS-PAGE, and blotted with the
indicated antibodies.
A
B
C
D
We next examined the terminal
phenotypes of cdc14-3A and cdc14-5A
cells (Fig. 4B). Consistent with earlier
findings, 94% of the cdc14-1 (the classical ts allele of CDC14) cells were
large budded cells at 37˚C, characteristic of a late anaphase arrest (data not
shown). At the restrictive temperature,
the morphology of cdc14-3A and
cdc14-5A mutant cells was strikingly
different from that of cdc14-1. A
significant percentage (~35%) of these
cells continued to undergo polarized
growth, forming abnormally elongated
buds (Fig. 4B, phenotype I). A population of cdc14-3A and cdc14-5A cells
(~20%) also re-budded in the absence
of cytokinesis, characteristic of a
cytokinesis defect (Fig. 4B, phenotype
II). There was also a fraction of cells that existed as clusters of four cells (Fig.
4B, phenotype III). The existence of these cells was likely due to a cell-separation defect, as the clustering of these cells was not disrupted by rigorous sonication.
We next stained cdc14-3A cells expressing Tub1p-GFP at 37˚C with
DAPI and phalloidin. As expected, cells with phenotype II re-budded from
the mother at a site proximal to the old bud (Fig. 5C). The majority of cells
with phenotypes I and II also disassembled their spindles (Fig. 5C and data
not shown). Our results indicate that, unlike the cdc14-1 mutant, cdc14-3A
E
966
cells had undergone mitotic exit and exhibit abnormal morphology (phenotype I) and cytokinesis defects (phenotype II) at 37˚C. Thus, the NES motif
of Cdc14p is required for proper coordination between mitotic exit and
cytokinesis. We do not know what exactly are the underlying defects that
cause phenotype I. However, this phenotype is similar to that of the cdc15lyt1 allele.50
The NES of Cdc14p is not required for Clb2p degradation. Cdc14p
activates APC/CCdh1 through dephosphorylation of Cdh1p.19 It is thus
required for the ubiquitin-mediated degradation of Clb2p and consequently
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2005; Vol. 4 Issue 7
Crm1p-Dependent Nuclear Export of Cdc14p
without the completion of cytokinesis. To further examine the
nature of the defect in cdc14-3A
mutants grown at 37˚C, we followed the localization of Myo1pGFP (a component of the actomyosin ring) in these cells after
release from synchronization with
a factor (Fig. 6). The localization
of Myo1p-GFP was largely unaffected in cdc14-3A cells and
appeared normally at the bud neck
after bud emergence. The cdc14-3A
cells progressed into mitosis and
accumulated as large budded cells.
Several cells remained arrested as
large budded cells, and in one
example, lysed (Fig. 6, cell 3). In
several cells that formed elongated
buds (phenotype I) and eventually
lysed (Fig. 6, cell 1), Myo1p localized to the bud neck and the tip
of the elongated bud during
abnormal
morphogenesis.
However, the contractile ring
failed to contract, leading to
polarized cell growth from the
distal tip of the daughter cell
without cytokinesis. Cell 2
formed a second Myo1p ring
indicative of a chain of three cells,
as the daughter cell budded again
while still attached to the mother
(Fig. 6). The two actomyosin
rings of this cell also failed to
contract. These results indicate
that certain components of the
actomyosin ring localize normally
to the bud neck in cdc14-3A
mutant cells, but the actomyosin
Figure 6. cdc14-3A cells fail to contract their actomyosin rings. cdc14-3A cells with integrated Myo1p-GFP were arrestrings fail to contract, leading to
ed with α factor, released into fresh medium, transferred onto an agarose pad at 37˚C, and observed with a fluoresabnormal growth without complecence microscope. As soon as the cells started to bud, images were taken to observe the behavior of Myo1p-GFP at
tion of cytokinesis. As mentioned
10–20 min intervals for 3.5 hrs. Several cells are numbered.
above, this is very reminiscent of
the cdc15-lyt1 allele.50 For reasons
1
the inactivation of Clb2p-Cdc28p. However, it is known that high levels of that are unclear at present, we did not observe cells with phenotype II in this
Clb2p-Cdc28p kinase activity prevent abnormal bud growth and re-budding experiment. One possible explanation is that cells only develop phenotype II
of yeast cells.51-53 Because the cdc14-3A strain was able to form elongated in liquid medium, but not efficiently in agarose pads.
buds and in some cases re-bud in the absence of cytokinesis, it is possible
Cdc14p localizes to the bud neck. To confirm that Cdc14p is indeed
that Clb2p is degraded normally in the cdc14-3A strain at the restrictive exported from nucleus to the cytoplasm after its release from nucleolus, we
temperature. To test this, we examined the levels of Clb2p in wild-type analyzed the localization of the wild-type Cdc14p-GFP protein in live cells
CDC14, cdc14-1 and cdc14-3A strains at 26˚C or 37˚C (Fig. 5D). using time-lapse microscopy. Consistent with earlier reports,29,30 Cdc14pConsistent with earlier reports,18 the cdc14-1 cells arrested at late anaphase GFP localized to the nucleolus during much of the cell cycle and was released
with high levels of Clb2p at 37˚C. In contrast, the cdc14-3A cells contained in late anaphase (Fig. 7 and 8A, early timepoints). We also observed Cdc14plow levels of Clb2p at 37˚C, suggesting that these cells were capable of GFP signals that would be consistent with its SPB and kinetochore localizadegrading Clb2p. To test whether Clb2p was degraded with normal kinetics tions (Fig. 7A, 20' and 25'; Fig. 8A, 0'–9'), as previously reported.54-56
in the cdc14-3A strains, we examined the levels of Clb2p in the wild-type Interestingly, shortly after its release from the nucleolus, Cdc14p-GFP localCDC14 and cdc14-3A strains after the release from α-factor-mediated G1 ized to the bud neck, forming a ring that split into two rings later (Fig. 7B).
arrest at 37˚C. Both the WT and NES mutant strains degraded Clb2p with The bud neck localization persisted for a short time after the majority of
similar kinetics (Fig. 4C and 5E). Thus, the NES of Cdc14p is not required Cdc14p-GFP had returned to the nucleolus (Fig. 7A and Fig. 8A). The
for Clb2p degradation. Instead, it is important for proper coordination Cdc14p-GFP protein in the integrated strain exhibited very similar localizabetween mitotic exit and cytokinesis.
tion patterns (Supplementary Movie 1). In particular, the bud neck localization
The actomyosin rings of cdc14-3A mutant cells fail to contract. Despite of Cdc14p-GFP was also observed (Supplementary Movie 1).
the fact that cdc14-3A mutants had degraded Clb2p and consequently inacMutations in the NES of Cdc14p or leptomycin B treatment prevent
tivated Clb2p-Cdc28p at 37˚C, these cells failed to execute cytokinesis Cdc14p bud neck localization. To determine the effect of NES mutations
properly and continued to undergo polarized bud growth or re-budding on the localization of Cdc14p, we next compared the localization patterns
www.landesbioscience.com
Cell Cycle
967
Crm1p-Dependent Nuclear Export of Cdc14p
B
A
Figure 7. Cdc14p-GFP localizes to the bud neck in late anaphase. (A and B) The cdc14∆ strain carrying the wild-type Cdc14p-GFP was grown on cover
slips at 37˚C. Images were taken at indicated time points. Several z-planes were captured for each time point, deconvolved and stacked. The bud neck
localization is indicated by arrow and magnified in insets. The bar indicates 5 µm.
of Cdc14p-WT-GFP and Cdc14p-3A-GFP by 4D microscopy at 37˚C.
Due to the limitations of epifluorescence microscopy, we used multiphoton
laser-scanning fluorescence microscopy to reduce photobleaching and to
minimize post-acquisition processing of the images (Fig. 8A).57 The cdc14-3A
strain progressed normally into mitosis. Cdc14p-3A-GFP was released from
the nucleolus in late anaphase. However, Cdc14p-3A-GFP failed to accumulate at the bud neck (Fig. 8A, 15' and 18'). Instead, Cdc14p-3A-GFP
appeared to slowly diffuse out of the nucleus and failed to be re-sequestered
in the nucleoli (Fig. 8A, 18' and 21'). The lack of prominent bud neck localization of Cdc14p-3A-GFP at 37˚C may explain why the cdc14-3A cells
failed to complete cytokinesis at the restrictive temperature.
Despite the eventual escape of Cdc14p-3A-GFP to the cytoplasm at the
non-permissive temperature, we did not observe an appreciable accumulation
of Cdc14p-3A-GFP at the bud neck. We hypothesize that efficient export of
Cdc14p from the nucleus by Crm1p is required for efficient bud-neck
targeting. However, our data are also consistent with the possibility that, in
addition to disrupting the interaction between Crm1p and Cdc14p, the
Cdc14p-3A mutation might also affect its binding to a putative anchoring
protein at the bud neck. To further confirm the involvement of Crm1p in
the nuclear export and bud neck localization of Cdc14p, we transformed a
plasmid encoding Cdc14p-WT-GFP into a yeast strain (MNY8) that contains
a leptomycin B (LMB)-sensitive Crm1p allele. LMB treatment also reduced
the cytoplasmic localization of Cdc14p-WT-GFP and prevented its accumulation at the bud neck (Fig. 8B, 45'–63'). Our data indicate that the bud
neck localization of Cdc14p is dependent on Crm1p.
We do not know why mutations of the NES of Cdc14p did not completely prevent its nuclear export. As discussed above, it is possible that
Cdc14p-3A-GFP might still retain some affinity toward Crm1p and result
in its slow release from the nucleus. Alternatively, a Crm1p-independent
mechanism might be responsible for the eventual export of Cdc14p-3AGFP from the nucleus. We also do not know why Cdc14p-3A-GFP is not
re-sequestered in the nucleolus. We speculate that lack of active and concerted
968
Crm1p-dependent export of Cdc14p might perturb the inactivation process
of MEN, resulting in a failure in Cdc14p re-sequestration in the nucleolus.58
DISCUSSION
A role for Cdc14p in cytokinesis in budding yeast. In addition
to their roles in mitotic cyclin degradation and mitotic exit, several
lines of evidence have implicated a role for MEN components in
cytokinesis of budding yeast. First, the mitotic exit defect of Tem1
mutants is circumvented by the net1-1 allele that binds weakly to
Cdc14p and allows Cdc14p release due to inefficient tethering in the
nucleolus.29 The tem1∆net1-1 double mutants exit from mitosis.59
However, they fail to contract their actomyosin ring during cytokinesis
and form chains of cells.59 Second, cells carrying the cdc15-lyt1
mutant allele also bypass the late anaphase arrest and continue to
grow without cytokinesis until their eventual lysis,50 a phenotype
very similar to that of our cdc14 NES mutants. Similarly, overexpression
of truncation mutants of Cdc15p that do not localize to the SPB in
a cdc15∆ background leads to cytokinesis defects.60 Third, overexpression of Sic1p, the mitotic Cdk inhibitor, bypasses the late
anaphase arrest of Mob1 mutants and causes a failure of bud neck
constriction and the formation of chains of connected cells.52 Fourth,
Cdc5p, a component of both the MEN and FEAR networks, is also
required for cytokinesis.61 Finally, the disassembly of the mitotic
spindle and the contraction of the actomyosin ring are uncoupled in
lte1∆ mutant cells.62
Our data presented herein are consistent with Cdc14p playing a
role during cytokinesis of budding yeast. Previous findings have also
implicated the Cdc14 family phosphatases in cytokinesis in other
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2005; Vol. 4 Issue 7
Crm1p-Dependent Nuclear Export of Cdc14p
Figure 8. The bud neck localization of Cdc14p requires an intact NES and Crm1p. (A) Cdc14p-3A-GFP fails to localize to the bud neck in late anaphase.
The cdc14∆ strains carrying Cdc14p-WT-GFP or Cdc14p-3A-GFP were grown on cover slips at 37˚C and examined by 4D microscopy using a multiphoton fluorescence microscope. Images were stacked in ImageJ using the Sum method. The position of the bud neck is indicated by arrow. (B) Inhibition of
Crm1p by leptomycin B (LMB) prevents the accumulation of Cdc14p at the bud neck. A plasmid encoding Cdc14p-WT-GFP was transformed into the MNY8
strain that contains an LMB-sensitive Crm1p allele. This strain (grown with and without LMB) was examined by 4D microscopy using a multiphoton fluorescence microscope. Images were stacked in ImageJ using the Sum method. The position of the bud neck is indicated by arrow. Two cells undergoing cell
division are numbered and their bud necks are indicated.
organisms.20-22,28 We propose that the requirement for MEN components during cytokinesis is masked by the inability of these mutants to
properly degrade mitotic cyclins and to inactivate Clb2p-Cdc28p.
Specific mutant alleles of MEN components, such as cdc14-3A,
allow the down-regulation of Clb2p-Cdc28, thus revealing the
cytokinesis function of these components. Interestingly, we show
that Cdc14p localizes to the bud neck in late anaphase. Cdc14 has
also been observed at the site of cell cleavage in other organisms.20-22,63
Furthermore, a component of the centralspindlin complex, MKLP1,
has recently been shown to be a substrate of hCdc14A.25 It will be
important to identify the targets of Cdc14p at the bud neck that
might regulate the constriction of the actomyosin ring and cytokinesis in budding yeast.
Regulation of Cdc14p by nucleocytoplasmic shuttling. The
Cdc14 phosphatases undergo nucleocytoplasmic shuttling in
mammalian cells and in budding yeast. Nucleocytoplasmic shuttling
may help explain the puzzling fact that the Cdc14 family of phosphatases
exhibits distinct patterns of subcellular localization in different
organisms. Our findings suggest that the localization of Cdc14 proteins
is a result of the delicate balance between two dynamic Ran-dependent
processes—nuclear import and nuclear export—during interphase.
The relative strength of these two processes partially determines the
Cdc14 localization at steady state. Consistent with this notion, mutawww.landesbioscience.com
tion in Kap104p, a member of the karyopherin b family of nuclear
import factors, causes a partial release of Cdc14p from the nucleolus
during interphase in budding yeast.64 Though it is unclear whether
Kap104p directly mediates the nuclear import of Cdc14p, it is
possible that a weakened nuclear import of Cdc14p is responsible for
its delocalization from the nucleolus. Analysis of the FEAR-dependent
release of Cdc14p has revealed that the MEN is required for the
cytoplasmic localization of Cdc14p, suggesting that these regulatory
networks influence the nucleocytoplasmic shuttling of Cdc14p.31
The SIN of fission yeast has recently been shown to be required for
the cytoplasmic release of Clp1p/Flplp under certain conditions.65
Finally, C. elegans CDC-14 is sequestered in the nucleolus in
terminally differentiated cells whereas it localizes to cytoplasmic
structures in actively dividing cells.24 Sine higher eukaryotic cell
breakdown and disperse the nucleolus prior to anaphase, nucleolar
sequestration is not expected to be the sole mechanismfor Cdc14
regulation. Our results suggest that nucleocytoplasmic shuttling of Cdc
14 is a conserved, and perhaps central mode if its regulation.
Understanding how the regulatory pathways of mitotic exit and
cytokinesis operate during an open mitosis will be an interesting topic
of future studies.
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969
Crm1p-Dependent Nuclear Export of Cdc14p
CONCLUSION
In this study, we have characterized the function of Crm1pdependent nuclear export of Cdc14p in budding yeast. Cells harboring
mutations in the nuclear export signal of Cdc14p exhibit a temperature-sensitive (ts) phenotype. In contrast to the classical ts allele of
CDC14, cdc14-1, the cdc14 NES mutant cells degrade Clb2p
normally at the restrictive temperature, but fail to complete cytokinesis due to an inability to contract the actomyosin ring and continue
to undergo abnormal bud growth and re-budding. Thus, disruption
of the concerted nuclear export of Cdc14p appears to allow mitotic
exit, but prevents the completion of cytokinesis. Mutations of the
NES of Cdc14p have uncoupled the essential roles of Cdc14p in
mitotic exit from its functions in cytokinesis. Consistent with Cdc14p
playing a role in cytokinesis, live cell imaging reveals that Cdc14p
localizes to the bud neck in late anaphase. The bud neck localization
of Cdc14p is disrupted in the cdc14 NES mutants at 37˚C and by
inhibition of Crm1p with leptomycin B. Therefore, our findings are
consistent with a role of Crm1p-dependent nucleocytoplasmic transport of Cdc14p in coordinating late mitotic events in budding yeast.
Supplementary Material
Supplementary Movie 1: Time lapse video microscopy of yeast
cells with Cdc14p-WT-GFP integrated into the CDC14 locus.
During the course of the experiment, several cells show bud neck
localization of Cdc14p-GFP. www.landesbioscience.com/journals/
cc/bembenekCC4-7-movie.avi.
Supplementary Table 1: Yeast strains used in this study.
www.landesbioscience.com/journals/cc/bembenekCC4-7-sup.pdf.
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