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Molecular and Cellular Biology, September 2001, p. 6292-6311, Vol. 21, No. 18
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.18.6292-6311.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mechanisms Controlling Subcellular Localization of
the G1 Cyclins Cln2p and Cln3p in Budding Yeast
Mary E.
Miller
and
Frederick R.
Cross*
The Rockefeller University, New York, New
York 10021
Received 1 May 2001/Returned for modification 7 June 2001/Accepted 25 June 2001
 |
ABSTRACT |
Different G1 cyclins confer functional specificity to
the cyclin-dependent kinase (Cdk) Cdc28p in budding yeast. The Cln3p G1 cyclin is localized primarily to the nucleus, while
Cln2p is localized primarily to the cytoplasm. Both binding to Cdc28p
and Cdc28p-dependent phosphorylation in the C-terminal region of Cln2p are independently required for efficient nuclear depletion of Cln2p,
suggesting that this process may be physiologically regulated. The
accumulation of hypophosphorylated Cln2 in the nucleus is an
energy-dependent process, but may not involve the RAN GTPase. Phosphorylation of Cln2p is inefficient in small newborn cells obtained
by elutriation, and this lowered phosphorylation correlates with
reduced Cln2p nuclear depletion in newborn cells. Thus, Cln2p may have
a brief period of nuclear residence early in the cell cycle. In
contrast, the nuclear localization pattern of Cln3p is not influenced
by Cdk activity. Cln3p localization requires a bipartite nuclear
localization signal (NLS) located at the C terminus of the protein.
This sequence is required for nuclear localization of Cln3p and is
sufficient to confer nuclear localization to green fluorescent protein
in a RAN-dependent manner. Mislocalized Cln3p, lacking the NLS, is much
less active in genetic assays specific for Cln3p, but more active in
assays normally specific for Cln2p, consistent with the idea that Cln3p
localization explains a significant part of Clnp functional specificity.
 |
INTRODUCTION |
Cyclin-dependent kinases (Cdks) are
serine/threonine protein kinases that are required for key events in
the cell division cycle. Cdk activity depends on conformational changes
induced by physical association with cyclin proteins and subsequent
cyclin-dependent targeting of Cdk activity to potential substrates
(3, 49, 50, 56). A single Cdk can carry out multiple
functions, dependent on the identity of its cognate cyclin partner. In
budding yeast, the Cdk Cdc28p is required for all key cell cycle
events. When bound by any one of three G1 (Cln)
cyclins, Cdc28p supports cell cycle entry (12). When bound
by one of six B-type (Clb) cyclins, Cdc28p supports later cell cycle
events, such as DNA replication and mitosis (54). For both
G1 Cln cyclins and B-type Clb cyclins, specific
cyclins provide functional specificity to the Cdk complex independent
of expression patterns and abundance of associated kinase activity
(14, 30, 38).
Deletion of all three G1-type cyclin
CLN genes results in a cell cycle arrest before cell cycle
initiation, as unbudded cells with 1C DNA content. CLN1,
CLN2, and CLN3 are functionally redundant, since
all double cln deletions result in viability. However, there are significant differences in the functional specificity of
CLN2 (representative of the highly homologous CLN1
CLN2 gene pair) and CLN3. Functional analysis of
CLN2 and CLN3 indicates that CLN3 is
primarily required for the expression of genes during late
G1, including CLN1 and
CLN2. This Cln3p activity requires the SCB transcription
complex, consisting of Swi4p and Swi6p (34). Once
expressed, Cln2p is believed to trigger events required for cell cycle
progression, such as bud emergence and activation of Clb-Cdc28p
complexes (7, 12, 15, 40). Bck2p also triggers SCB-dependent transcription in G1, most likely
working in parallel with Cln3p (17, 21). We have
characterized the subcellular localization patterns of the
G1 cyclins Cln2p and Cln3p and find strikingly
distinct localization patterns. These patterns are consistent with the
idea that Cln2p and Cln3p confer functional specificity to the Cdk
Cdc28p and support cell cycle entry by distinct mechanisms. Cln3p,
thought to be the physiologically relevant cyclin for activation of
transcription (19, 38, 70), is localized primarily to the
nucleus (45). Cln2, thought to trigger other events, such
as bud emergence (7, 12, 15, 40), is localized primarily
to the cytoplasm (45).
Differences in functional specificity between Cln2p and Cln3p can be
assayed in specific genetic backgrounds. For example, CLN2
is able to support viability in the absence of Swi4p, while CLN3 is not (38, 55, 57). This suggests that
the ability of CLN3 to rescue a cln1 cln2 strain
requires Cln3p- and Swi4p-dependent transcription. The PCL1
and PCL2 gene pair (encoding cyclin homologues that can bind
to and activate the Pho85p Cdk) is also essential for Cln3p-dependent
viability in the absence of cln1 and cln2 (38, 43, 57). PCL1 and PCL2 are
transcribed under the control of Swi4p (43, 57),
suggesting that at least some of the Swi4p requirement in the
cln1 cln2 background may be due to the Pcl1p, Pcl2p
requirement. This requirement may involve promoting functions important
for morphogenesis (37).
Studies of higher eukaryotic cyclins have established the importance of
spatial control for regulation of cyclin and/or Cdk activity. The most
extensively studied are the mitotic cyclin B1 and the
G1 cyclin D1. When unphosphorylated, cyclin B1
localizes to the cytoplasm through physical association with the
exportin CRM1. The physical association between cyclin B1
and CRM1 is abolished when cyclin B1 is phosphorylated, resulting in
localization to the nucleus, possibly through interactions with the
karyopherin
-
complex (46, 58, 82). Cyclin B1 may
also enter the nucleus through physical interactions with cyclin F or
independently through physical interactions with importin
(71). Phosphorylation of cyclin B1 has also been
implicated in triggering nuclear import, independent of inhibition of
binding to CRM1 (25, 83). Physical association of cyclin
B1 with Cdk does not significantly contribute to nuclear import
(46). In vitro studies show that both the cyclin B1-Cdk1
complex, mitogen-activated protein kinase, and casein kinase II are
able to phosphorylate cyclin B1 in the region that associates with CRM1
(28, 42). The D-type cyclins function at early stages in
the cell division cycle, responding to growth factor stimulation and
facilitating the activation of cyclin-Cdk2 complexes required for entry
into the phase of the cell cycle at which DNA replication occurs. The
shift in localization of cyclin D1 from the nucleus to the cytoplasm at
the onset of DNA replication is linked to the proteosomal degradation
of cyclin D1 and requires phosphorylation by glycogen synthase
kinase-3
on Thr-286. This shift in localization reflects
phosphorylation-dependent export of cyclin D1 (18). Two
different human B-type cyclins, B1 and B2, exhibit different
subcellular localization (to microtubules and to the Golgi apparatus)
(29). Interestingly, chimeras constructed that switch the
localization signal of cyclin B1 and cyclin B2 also influence their
ability to function, demonstrating that differences in subcellular
localization influence the functional specificity of these cyclins
(20).
In this paper, we analyze mechanisms that regulate localization of
Cln2p and Cln3p. We address the role of Cdc28p in regulated localization of G1 cyclins in budding yeast and
characterize the distinct mechanisms used to import different
G1 cyclins into the nucleus.
 |
MATERIALS AND METHODS |
The relevant genotypes of the yeast strains used are shown in
Table 1. All strains (except for the
grr1 strain, MMY42 strain, ACY212, MMY103, MMY106, and
controls) are congenic with BF264-15D (59). YPD
(dextrose), YPG (galactose), and synthetic complete (SC) media for
yeast growth were prepared as described previously (63).
Yeast cells were transformed with plasmid DNA as described previously
(24).
Plasmids.
The 9× myc-epitope-tagged CLN3 CEN/ARS
plasmid pMM99, Cln2mycp integrating construct
pMM56, and CEN/ARS plasmid pMM82
(CLN3::CLN2myc) have been
described previously (45). The 9× myc tag is engineered at the COOH terminus of the cyclin proteins in these constructs.
The genomic copy of CLN2 was tagged by a COOH-terminal,
in-frame integration of a PCR-derived DNA fragment encoding the
immunoglobulin G (IgG) binding domain of protein A. This DNA fragment
was amplified from the plasmid pProtA/HIS5, which carries
the Schizosaccharomyces pombe HIS5 gene as its selectable
marker (75), as described previously (1).
This results in the expression of protein A (PA)-tagged
CLN2 from the CLN2 promoter. Correct integration
of the tag was confirmed by PCR analysis of genomic structure and immunoblotting. Initial integration was carried out in the diploid strain W303d, resulting in strain MMY17. This strain was sporulated, and haploid cells expressing PA-tagged CLN2 were obtained by
tetrad dissection. The diploid strain MMY42, expressing both the PA- and 9× myc epitope-tagged versions of CLN2, was made by
mating haploid MMY17 to haploid W303 strain expressing
CLN2myc (constructed with integration
vector pMM56 as described previously [45]).
The Cln3-
22mycp expression plasmid (pMM98) was
constructed by PCR amplification of CLN3 coding sequences
lacking the last 66 bp. The 3' primer for the
cln3-
22 amplifications was
5'-CTAGCGGCCGCTCAGTTGGGTGGG GGCAGAGCATGGGTG-3', and the 5'
primer was 5'-CTTACATTCCATTGCATCTCCC-3'. The plasmid pMM99
(45) was used as the template for PCRs. PCR products were
subsequently cloned into TOPOII vector with topoisomerase-based ligations (Invitrogen). EcoRI-NotI cassettes from
the TOPOII clones were used to replace the
EcoRI-NotI CLN3 sequences of pMM45 to give rise to pMM96. The 9× myc epitope tag NotI cassette
from pAG4 (A. Gartner) was cloned into the NotI site
following the CLN3 sequences in pMM96 to give rise to pMM98.
The pMM98 plasmid was sequenced to confirm the absence of the last 66 bp of CLN3 coding sequence followed in frame by the 9× myc
epitope tag. The nuclear localization signal (NLS), mutant
nonfunctional NLS (mnls), nuclear export signal (NES), and mutant
nonfunctional NES (mnes) Cln3-
22p-expressing plasmids were made by
replacing the ClaI-EcoRI fragment of pMM98 with
the ClaI-EcoRI fragments of pMM83
(NLS-CLN3-1), pMM84
(mnls-CLN3-1), pMM85
(NES-CLN3-1), and pMM86
(mnes-CLN3-1) to give rise to pMM104, pMM105,
pMM106, and pMM107, respectively (45). The cln3
mutant cln3-A13 contains two amino acid substitutions of
K106A and R108A. These mutations abolish detectable interactions with
Cdc28p assayed by coimmunoprecipitation assay (unpublished data).
The CLN2-4T3S mutant integration vector pMM68 was
made by replacing the 3× hemagglutinin (HA) tag of pMT480 (M. Tyers)
with the NotI 9× myc epitope tag. The resulting clone was
sequenced to ensure identity and that the Cln coding sequences were
followed in frame by the myc epitope tags. The
CLN3myc integration vector pMM162 was made
by inserting the SalI-SacII cassette (containing
the CLN3 promoter-driven 9× myc-tagged CLN3) of
pMM99 into the integration vector pRS404. Strain MMY32 was produced by
integration of this vector into strain 1255-5C.
The CLN3 promoter-driven 9× myc-tagged Cln2-4t3sp
expression plasmid (pMM108) was constructed by replacing the
SpeI-NotI carboxy-terminal CLN2
fragment of pMM82 (45) with the
SpeI-NotI carboxy-terminal CLN2
fragment of pMM68 and subsequent addition of the NotI
fragment containing the 9× myc tag. The CLN2
promoter-driven 9× myc-tagged Cln2p(pMM159) and Cln2-4t3sp(pMM151)
expression plasmids were constructed by replacing the
SalI-ClaI CLN3 promoter fragments of
pMM82 and pMM108, respectively, with the
SalI-ClaI CLN2 promoter fragment from
pKL007 (38). The CLN3 promoter-driven 9×
myc-tagged Cln2-KAEAp expression plasmid (pMM66) was constructed by
replacing the NotI cassette containing the 3× HA tag of
pKL005 (38) with the NotI cassette containing
9× myc of pMM99. The CLN2 promoter-driven 9× myc-tagged
Cln2-KAEAp (pMM167) and Cln2-KAEA-4t3sp (pMM166) expression plasmids
were constructed by replacing the ClaI-BclI N-terminal CLN2 fragments of pMM159 and pMM151,
respectively, with the ClaI-BclI N-terminal
CLN2 fragment of pMM66. Plasmids were sequenced to ensure
the presence of KAEA mutations.
Plasmids expressing green fluorescent protein (GFP)-CLN fusions were
constructed by using pKW431 (69). These plasmids encode fusion proteins consisting of the simian virus 40 (SV40) NLS (the nonfunctional mutant protein kinase inhibitor [PKI] NES) and two GFP
molecules. The HindIII-EcoRI SV40 NLS
fragment of pKW431 was replaced with the CLN3 NLS by
homologous recombination in vivo, resulting in pMM131. The
HindIII-CLN3 NLS-EcoRI fragment was produced by PCR amplification with the forward primer
5'ATACAATCT GCACAATATTTCAAGCTATACCAAGCATACAATAAGCTTATGAAAA AGAGATCTACTTCCTCTGTGGAT3'
and the reverse primer
5'TTGTTGA TATCAAGACCTGCTAATTTCAAGGCTAATTCATTGAATTCGCGAGTT TTCTTGAGGTTGCTACT3'.
Nonfunctional Cln3 mnls-GFP fusion proteins were made by identical
means, except the primers contain base changes that result in alanine
substitution of the basic clusters at the beginning and end of the
Cln3p NLS. Mutation of both clusters resulted in plasmid pMM133.
Mutation of the first basic cluster resulted in pMM135, and mutation of
the second basic cluster resulted in pMM137. The nonfunctional PKI NES
in these constructs was presumably inert. Substitution of the
functional PKI NES in the Cln3 NLS construct resulted in loss of
nuclear accumulation of Cln3 NLS-PKI NES-GFP fusion (data not shown).
Indirect immunolocalization and GFP localization.
Exponentially growing cultures expressing the myc-tagged Cln proteins
were fixed in growth medium with formaldehyde (1:10 dilution) for 60 min at 30°C with rotation. When indicated, cells were fixed by
"fast fixation." In this case, cells were harvested by filtration
and fixed in buffer (0.1 M
KH2PO4, 0.5 M
MgCl2, 40 mM KOH) with formaldehyde (1:10
dilution) for 5 min. The extended fixation time (up to 2 h) did
not change localization patterns with the fast fixation method, and
fast fixation gave essentially identical results to standard fixation.
Cells were prepared for indirect immunofluorescence as described
previously (45). For detection of the myc epitope, primary monoclonal antibody 9E10 (Santa Cruz Biotechnology) was diluted 1:200
in blocking solution, cleared for 2 min by centrifugation in a
microcentrifuge, and incubated on the cells for 2 h. Secondary antibody (Cy3-conjugated antimouse IgG; Jackson Immunochemicals) was
diluted 1:200 in blocking solution, cleared, and incubated on the wells
for 2 h. The negative control for integrated myc-tagged Cln
proteins was the wild-type strain 1255-5C. The negative controls for
plasmid-based expression of myc-tagged Cln proteins were the vector
controls pRS414 and pRS416 (66).
Cells expressing the GFP fusion proteins were grown to log phase,
sonicated, placed on polylysine-coated slides, and visualized microscopically. To confirm the localization of Cln3 NLS-GFP signal in
the nucleus, cells were sonicated briefly, washed in 1×
phosphate-buffered saline (PBS), fixed for 12 min in 70% ethanol at
room temperature, washed in 1× PBS, stained for 12 min with 1:1,000
dilution of 1-mg/ml 4',6'-diamidino-2-phenylindole (DAPI) at room
temperature, washed in 1× PBS, and assayed for fluorescence. This
procedure allows simultaneous detection of DAPI and GFP signal.
Standard indirect immunofluorescence and GFP experiments were done on
an Axoplan universal microscope (Carl Zeiss, Inc.) with a Hamamatsu
digital camera. Images were collected with Openlab software version 1.2 (Improvision) and processed with Photoshop version 4.0 (Adobe systems).
When noted, cells were analyzed with the DeltaVision restoration
microscopy system (Applied Precision, Inc.) with a Roper CH350
full-frame KAF1401E 12-bit high-resolution charge-coupled device (CCD)
camera. The DeltaVision system was mounted on an Olympus 1X-70
microscope, and the UplanApo100X oil 1.35NA objective was used. When
using the DeltaVision system, images were collected, processed, and
deconvolved by using softWoRx V2.5 software (Applied Precision, Inc.).
All images presented in a single figure were captured and processed in
parallel by identical means when noted.
Functional assays.
Cell viability and volume assays were
carried out as described previously (45). Mutant strains
(Table 1) were transformed with the following plasmids: pMM82, pMM99,
pMM98, pMM104, pMM105, pMM106, pMM107, pMM60, pMM61, pMM92, pMM93 and
pRS416. All plasmids are episomal centromeric low-copy plasmids and
carry the TRP1 gene. The data presented are representative
of data from at least two independent experiments.
Protein extraction, immunoprecipitations, cellular fractionation,
and immunoblotting.
Total cellular protein lysates were obtained,
immunoprecipitated, and immunoblotted as previously described
(38, 45). Cellular fractionation was carried out exactly
as previously described (45). The monoclonal antibody
HA.11 was used for immunoprecipitation of HA-tagged Cdc28p. Polyclonal
HA.11 antibody was used for Western blot detection of
Cdc28hap present in immunoprecipitates. The
polyclonal antibody
-myc A-14 (Santa Cruz Biotechnology) was used to
detect the myc epitope in Western blot analysis. Detection was by
enhanced chemiluminescence (ECL) with the Super Signal ECL kit (Pierce).
Elutriation.
The diploid strain MMY42 contains
CLN2myc at one CLN2 locus and
CLN2PA at the other CLN2 locus;
both expressed from the CLN2 promoter. MMY42 was grown to
log phase (optical density at 660 nm [OD660] ~0.9) in YEPD at 30°C (1-liter culture), harvested by filtration, resuspended in icewater, sonicated, and separated on the basis of cell
size by centrifugal elutriation with a Beckman J6-M elutriating rotor
with a 40-ml chamber. Elutriation was carried out with 0°C water, and
the rotor chamber was maintained at 4°C throughout the elutriation.
The rotor was maintained at the speed of 2,400 rpm, and the first
fraction was taken at the flow rate of 70 ml/min. Four-hundred-milliliter fractions were collected with 10% increments in flow rates between fractions. The first fraction was saved when the
OD660 of the sample rose above 0.02. After
collection of fractions, equivalent amounts (as determined by OD) of
each fraction were concentrated by filtration, resuspended in ice-cold YPD medium, and immediately subjected to indirect immunofluorescence analysis and protein extraction. Unfixed samples were subjected to
Coulter counter analysis to determine the size of the fractions and
used to measure the OD660. Subsequent
immunoblotting analysis was carried out as described above.
Metabolic poisoning.
Metabolic poisoning was carried out as
described in reference 65. Exponential cultures (50 ml)
were harvested by filtration, washed with an equal volume of water, and
resuspended in 6 ml of metabolic poison (10 mM NaN3, 10 mM deoxyglucose
in YEP medium lacking glucose). Cells were incubated at 30°C with
rotation for 45 min. Cells were removed, collected by filtration, fast
formaldehyde fixed (see above), and processed for indirect
immunofluorescence (see above). The poison was washed out of the
culture by filtration followed by washing with an equal volume of
water. Cells were allowed to recover by resuspending the cells in YPD
medium and incubation at 30°C with rotation. Samples were collected
and fast formaldehyde fixed for indirect immunofluorescence after
various times of recovery.
 |
RESULTS |
Localization of Cln2p is regulated by Cdc28p-dependent
phosphorylation.
We showed previously that Cln2p was localized
primarily to the cytoplasm (45) (Fig.
1). Since Cdc28p binds to Cln2p,
phosphorylates Cln2p, and also localizes to the cytoplasm
(78; our unpublished data), we investigated the
possibility that Cdc28p activity might be important for Cln2p
cytoplasmic localization. We assayed the localization pattern of a
Cln2mycp mutant in which all seven potential
Cdc28p phosphorylation sites have been mutated to alanine
(Cln2-4t3smycp) (36, 62). Cln2-4t3sp
binds Cdc28p, but the mutant Cln2p is not phosphorylated
(36). myc-tagged CLN2-4T3S was
integrated at the CLN2 locus in a wild-type strain.
Wild-type Cln2p localizes primarily to the cytoplasm with clearly
detectable "holes" of nuclear depletion, while the
Cln2-4t3smycp mutant is no longer depleted in the
nucleus and shows some nuclear accumulation (Fig. 1). We note that this
nuclear accumulation is not complete, and a population of
Cln2-4t3smycp remains in the cytoplasm (Fig. 1).
It is unclear if the punctate nature of the Clnp staining in this assay
results from fixation or reflects a relevant localization pattern in
vivo. Consistent with these data, Cln2p also accumulates in the nucleus
when CDC28 has been inactivated by using the
temperature-sensitive allele cdc28-4 (Fig.
2); Cln2p phosphorylation
is strongly reduced under these conditions (data not
shown).

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FIG. 1.
Indirect immunolocalization of Cln2mycp,
Cln2-4t3smycp, and Cln3mycp. Strains expressing
wild-type Cln2mycp (MMY1) (A), mutant
Cln2-4t3smycp (MMY4) (B), or wild-type Cln3mycp
(MMY32) (C) were assayed for indirect immunolocalization as described
in Materials and Methods. Images were collected, processed, and
deconvolved by using the DeltaVision restoration system (Applied
Scientific) as described in Materials and Methods. Shown are three
sections taken at 0.2-µm intervals labeled 1, 2, and 3. The DAPI
signal indicates the position of the nucleus (blue), the -myc signal
indicates the position of the myc-epitope-tagged cyclin (red), and the
merge combines these two images. The negative control (no myc-tagged
cyclin) strain 1255-5C gave no detectable signal (data not shown).
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FIG. 2.
Indirect immunolocalization of Cln2mycp.
cdc28-4 and cdc34 mutant
cells expressing integrated myc-epitope-tagged Cln2p from the
CLN2 promoter were assayed by indirect
immunofluorescence as described in Materials and Methods. Cells were
grown to log phase at 23°C. Cultures were split, and half was
incubated at 23°C and half was incubated at 37°C for 2 h. (A) Quantitation
of Cln2 localization. The percentage of cells showing nuclear depletion
(gray bars), signal throughout the cell (white bars), or nuclear
accumulation (black bars) is shown. A cell was scored positive for
nuclear depletion when the position of the nucleus (DAPI signal) could
be estimated correctly due to a reduction in Cln2mycp
signal. A cell was scored positive for nuclear accumulation when the
position of the nucleus (DAPI signal) could be estimated correctly due
to an increase in Cln2mycp signal. When the position of the
nuclei could not be determined based on Cln2mycp
localization, the cells were scored as having a signal throughout the
cell. Note that this procedure is unbiased in that the position of Cln2
depletion is determined from immunostaining before the position of the
nucleus is determined from DAPI staining. Cln2 depletion from the
vacuole will not be scored as nuclear depletion by this procedure. Data
for Cln2 localization in the presence of mutant
cdc28-4 and mutant cdc34
incubated at 23 and 37°C are shown. No fewer than 200 cells were
counted with the DeltaVision microscopy restoration system. (B and C)
Indirect immunolocalization of Cln2mycp expressed in mutant
cdc28-4 (B) and mutant
cdc34 (C) strains. The first row shows the indirect
immunofluorescence with monoclonal anti-myc antibody 9E10 ( -myc),
and the second row shows DAPI staining of DNA. Images were collected by
standard microscopy.
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Hypophosphorylated Cln2-4t3sp is stabilized because
Cdc28p-dependent phosphorylation is required for Skp1-Cdc34-F-box
(SCF)-dependent ubiquitination and subsequent degradation
(36). We find that increased protein stability is not
sufficient for the nuclear accumulation of Cln2p. CDC34
encodes the ubiquitin-conjugating component of SCF (6, 16,
77), and GRR1 is an SCF component that binds
specifically to phosphorylated Cln2p, and this binding is required for
SCF-dependent ubiquitination of Cln2p (6, 33, 41, 67, 68).
Inactivation of CDC34 or GRR1, resulting in stabilization of Cln2p, does not alter Cln2p localization, since nuclear depletion is still observed, and no nuclear accumulation occurs
(Fig. 2) (data not shown). Nuclear exclusion of Cln2p in the
absence of CDC34 function is still dependent on
CDC28-dependent phosphorylation, because Cln2p accumulates
in the nucleus in a cdc28-4 cdc34 double mutant
just as it does in a cdc28-4 single mutant (Fig.
2) (data not shown).
To confirm that hypophosphorylated Cln2p accumulates in the nucleus,
nuclear and cytoplasmic components of the cell were separated and
analyzed for Cln2mycp localization in cellular
fractionation experiments. Wild-type Cln2mycp
localizes primarily to the cytoplasm (Fig. 1) (45) and
largely cofractionates with cytoplasmic marker Pgk1p (Fig.
3) (45). The
Cln2-4t3smycp mutant shows an increase in nuclear
localization (Fig. 1), and a subfraction cofractionates with nuclear
marker Nop1p (Fig. 3). Consistent with the indirect immunofluorescence
experiments, a significant amount of
Cln2-4t3smycp cofractionates with the cytoplasmic
Pgk1p (Fig. 3). Our ability to easily visualize the smaller amount of
Cln2-4t3smycp accumulated in the nucleus is most
likely due to the smaller size of the nuclear compartment compared to
the cytoplasm, effectively concentrating the nuclear signal. (Note that
the protein gels for the fractionation experiment in Fig. 3 are loaded
in cell-equivalent amounts, not equal amounts of protein from the
different fractions, to give a better representation of the overall
distribution.) In both indirect immunofluorescence and cellular
fractionation experiments, the
Cln2-4t3smycp mutant shows an increase in
nuclear localization compared with wild-type
Cln2mycp. (Although these fractionation
experiments were somewhat hampered by variable nuclear breakage,
as indicated by recovery of the nuclear marker Nop1p in inappropriate
fractions, concentration of Cln2-4t3smycp
compared to that of wild-type Cln2mycp in the
authentic nuclear fraction was reproducibly observed in multiple
fractionations [data not shown].)

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FIG. 3.
Localization of Cln2mycp and
Cln2-4t3smycp by subcellular fractionation. Fractions were
prepared as described in Materials and Methods. Duplicate samples of
each fraction were separated by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (12% polyacrylamide) and analyzed by Western
blotting. Fractions are indicated at the top of each set of blots.
Fraction 1 corresponds to the crude cytoplasmic fraction, fraction 2 corresponds to the surface of the sucrose gradient, fraction 3 corresponds to the surface-to-2.01 M sucrose interface, fraction 4 corresponds to the 2.01-to-2.1 M-sucrose interface, fraction 5 corresponds to the 2.1-to-2.3 M sucrose interface, and fraction 6 corresponds to the remaining pellet of the sucrose gradient. (A)
Immunoblot analysis of fractions from a strain expressing
Cln2mycp from the CLN2 promoter (MMY1). The
position of Cln2mycp is indicated by "Cln2."
Corresponding immunoblot analysis of the Nop1p and Pgk1p fractionation
is shown below the Cln2p blot. The nucleolar protein Nop1p detected
with the monoclonal antibody D77 (4) indicates which
fractions contain nuclear proteins. The cytoplasmic protein Pgk1p
detected with the anti-Pgk1p polyclonal antibody (Molecular
Probes) indicates which fractions contain cytoplasmic proteins.
(B) Immunoblot analysis of fractions from a strain expressing
Cln2-4t3smycp from the CLN2 promoter
(MMY4). The Cln2-4t3smycp, Nop1p, and Pgk1p
immunoblots are shown. A moderate level of nuclear breakage apparently
occurred in both fractionation experiments, as indicated by the leakage
of Nop1p into the intermediate and cytosolic fractions.
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These data show a role of Cln2p phosphorylation by Cdc28p in regulated
Cln2p localization.
Regulated subcellular localization of Cln2p requires binding to
Cdc28p.
To ask if Cdc28p binding is necessary for Cln2p
cytoplasmic localization, we made use of a Cln2p mutant
(Cln2-KAEAp) with crippling mutations (K129A E183A) in the cyclin
box region that mediates interaction with Cdc28p (27, 32,
38). The localization pattern of
Cln2-KAEAmycp differs from that of wild-type
Cln2mycp. Nuclear depletion is observed in
approximately 60% of the cells expressing
Cln2mycp, while it is observed in approximately
10% of the cells expressing Cln2-KAEAmycp (Fig.
4). Cln2p localization was also assayed
in a strain expressing a mutant Cdc28p,
Cdc28csr1-1p (cdc28-Q188P).
Cdc28csr1-1p shows 100-fold reduced binding to
wild-type Cln2p upon immunoprecipitation (39). Similar to
the KA, EA cln2 mutation, the
cdc28csr1-1 mutation causes
a drop in the percentage of cells with nuclear depletion of
Cln2mycp (Fig. 4). Residual binding between
mutant Cdc28csr1-1p and wild-type
Cln2p may occur, accounting for the remaining cells (approximately
30%) showing nuclear depletion. This residual binding may be lost in
the case of the Cln2-KAEAmycp mutant
(39). Using this mutant, we observed only a low level of
nuclear depletion in the presence of either wild-type or mutant Cdc28csr1-1p. These data suggest that the
physical association between Cln2p and Cdc28p is required for efficient
nuclear exclusion of Cln2mycp.


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FIG. 4.
Requirement for binding to Cdc28p for regulated
Cln2mycp localization. (A) Strain 1255-5C containing
plasmids that express Cln2mycp (pMM159), mutant
Cln2-4t3smycp (pMM151), mutant Cln2-KAEAmycp
(pMM167), or mutant Cln2-KAEA-4t3smycp (pMM166) was assayed
for indirect immunolocalization as described in Materials and Methods.
Images were collected, processed, and deconvolved with the DeltaVision
restoration system (Applied Scientific) as described in Materials and
Methods. All plasmids are episomal CEN (low copy number) and express
the myc-tagged Cln2 protein from the CLN3 promoter.
Shown are three sections taken at 0.2-µm intervals labeled 1, 2, and
3 for each set. The DAPI signal indicates the position of the nucleus
(blue), the -myc signal indicates the position of the myc
epitope-tagged cyclin (red), and the merge combines these two images.
The negative control (no myc-tagged cyclin) strain 1255-5C containing
plasmid pRS416 gave no detectable signal (data not shown). (B)
Quantitation of Cln2 localization. Percentages of cells showing nuclear
depletion (gray bars), signal throughout the cell (white bars), or
nuclear accumulation (black bars) were determined as described in the
legend to Fig. 2. Data for Cln2 localization in the presence of
wild-type Cdc28p (CDC28) and Cdc28csr1-1p
(CDC28csr1-1) are shown. No
fewer than 200 cells were counted for each experiment. The data shown
are the average of three independent transformants with standard
deviation indicated by the error bar. Images in this figure were
captured and processed in parallel.
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The hypophosphorylated Cln2-4t3smycp mutant shows
nuclear accumulation in approximately 80% of the cells assayed. These
data indicate that the physical interaction between Cln2p and Cdc28p is
not sufficient for Cln2p cytoplasmic localization, since Cln2-4t3sp binds Cdc28p (36) and accumulates in the nucleus (Fig. 1,
3, and 4). Interestingly, the hypophosphorylated Cln2-4t3sp also requires binding to Cdc28p for regulated localization, since when the
Cdc28p binding of the hypophosphorylated
Cln2-4t3smycp is crippled (because of the
cln2-KAEA mutation, the
cdc28csr1-1 mutation, or both),
we no longer observe nuclear accumulation of
Cln2-4t3smycp (Fig. 4). This may explain the lack
of strong nuclear accumulation of KAEAmycp (Fig.
4), which is hypophosphorylated like
Cln2-4t3smycp, but unlike
Cln2-4t3smycp binds to Cdc28p inefficiently
(38; data not shown).
These results on nuclear exclusion of Cln2p can be accounted for by
proposing that, first, only the localization of Cln2p-Cdc28p complex is
regulated, but the Cln2p unbound to Cdc28p is distributed throughout
the cell; and, second, that C-terminal phosphorylation of Cln2p in the
Cln2p-Cdc28p complex is required for its efficient nuclear exclusion.
The results with cdc28-4 (Fig. 2) are somewhat complicated to interpret in this framework because we don't know how
much Cdc28-4p-Cln2p binding occurs in vivo.
We have been unable to identify an NLS or NES by inspection of the
Cln2p sequence. Fusion of the wild-type or phosphorylation mutant Cln2p
C terminus (residues 291 through 545, containing the seven Cdk
phosphorylation sites) to GFP does not result in import of GFP to the
nucleus (data not shown) and therefore gives no indication of an import
activity encoded within this region of Cln2p. Also, no cytoplasmic
export of GFP was observed upon fusing the Cln2p C terminus to an SV40
NLS-GFP fusion (data not shown). Thus, we have no evidence that this
region of Cln2p provides signals for import or export. However, these
regions of Cln2p do not include the Cdc28p binding domain, and these
results might reflect a requirement for physical association with Cln2p
for regulated localization, as suggested above.
Energy-dependent nuclear localization of Cln2p.
We assayed
cells expressing wild-type or mutant Cln2mycp for
energy-dependent localization by incubating the cells in metabolic poison and assaying Cln2p localization by indirect immunofluorescence. Exponential cultures were treated with metabolic poison as described previously (65). We find that after poison treatment, no
difference in the localization pattern of wild-type
Cln2mycp is observed (Fig.
5). In contrast, the
Cln2-4t3smycp showed a dramatic change in
localization, failing to accumulate in the nucleus in the presence of
the poison (Fig. 5). Additionally, nearly 50% of the cells show some
nuclear depletion of Cln2-4t3smycp (Fig. 5). We
find that the maximal shift in localization occurred at 45 min of
incubation with poison and did not change with longer incubations (up
to 2 h). We were able to reconstitute nuclear accumulation by
washing out the poison and incubating the cells in rich medium at
30°C. The nuclear accumulation of Cln2-4t3smycp
after the poison was washed out was rapid and maximized between 15 and
20 min (Fig. 5). Steady-state protein levels of
Cln2mycp did not change significantly when
incubated with poison (data not shown), though the population of
hypophosphorylated Cln2mycp increased. Upon
recovery, there was some reduction in Cln2mycp
levels combined with some recovery of Cln2mycp
phosphorylation. Cln2-4t3smycp abundance did not
change significantly when incubated with poison or during recovery
(data not shown). Overall, poison effects on Cln2p abundance seem
unlikely to account for the localization results.


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FIG. 5.
Energy-dependent localization of Cln2mycp.
Strains expressing wild-type untagged Cln2 (1255-5C), wild-type
Cln2mycp (MMY1), Cln2-4t3smycp (MMY4), or
Cln2-KAEAmycp (pMM167 in strain 1255-5C) were treated with
metabolic poison and assayed for localization as described in Materials
and Methods. (A) Indirect immunofluorescence of strains growing
exponentially (log) after 45 min of treatment with metabolic poison
(poison) and 20 min after removal of poison and incubation in rich
medium at 30°C (recovery). Cells express untagged Cln2 (no tag),
wild-type Cln2mycp (Cln2), or hypophosphorylated Cln2
mutant (Cln2-4t3smyc). Negative controls (no tag) incubated
with poison and after recovery gave background signal identical to data
shown for exponentially growing cells. (B) Quantitation of
Cln2mycp localization during treatment with metabolic
poison. Percentages of cells showing nuclear depletion (gray bars),
signal throughout the cell (white bars), or nuclear accumulation (black
bars) are shown as follows: exponentially growing cells expressing
wild-type Cln2mycp without poison treatment (Cln2 poison), cells expressing Cln2mycp after 45 min of
treatment with poison (Cln2 + poison), cells expressing
Cln2mycp after poison had been washed out and allowed to
grow in rich medium for 20 min (Cln2 recovery 20), exponentially
growing cells expressing mutant Cln2-4t3smycp without the
addition of poison (Cln2-4t3s poison), cells expressing
Cln2-4t3smycp after 45 min of treatment with poison
(Cln2-4t3s + poison), cells expressing Cln2-4t3smycp after
poison had been washed out and allowed to grow in rich medium for 5, 10, and 20 min (Cln2-4t3s recovery 5, recovery 10, and recovery 20, respectively), cells expressing Cln2-KAEAmycp without
addition of poison (Cln2-KAEA poison), cells expressing
Cln2-KAEAmycp after 45 min of treatment with poison
(Cln2-KAEA + poison), and cells expressing Cln2-KAEAmycp
after poison had been washed out and allowed to grow in rich medium for
20 min (Cln2-KAEA recovery 20). Quantitation was performed as described
in the legend to Fig. 2. Essentially identical results were obtained
with plasmid-based and genomic expression of Cln2p and Cln2-4t3sp. No
fewer than 200 cells were counted for each experiment. The data shown
are the average of two independent experiments with standard deviation
indicated by the error bar. Images in this figure were captured and
processed in parallel.
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These data suggest that the nuclear accumulation of hypophosphorylated
Cln2p occurs by energy-dependent active import. Nuclear depletion after
incubation with poison introduces the possibility that nuclear export
of Cln2-4t3smycp occurs in an energy independent
manner, although we cannot exclude some contribution of nuclear
degradation of Cln2p under these conditions. It is possible that once
the Cln2-4t3smycp mutant moves into the
cytoplasm, it is retained in the cytoplasm by an energy-independent
mechanism, for example, by interaction with cytoplasmic proteins.
Consistent with this, wild-type Cln2p continues to show nuclear
depletion after up to 2 h of incubation in poison (data not shown).
Crippling the physical interaction between Cln2p and Cdc28p
by introducing the cln2-KAEA or
cdc28csr1-1 mutation results in
apparent deregulation of Cln2p localization, with the majority of cells
(approximately 80%) showing Cln2p signal throughout the cell (Fig. 4).
To determine if the subpopulation of unbound Cln2p present in the
nucleus entered by energy-dependent import, we assayed localization
of the Cln2-KAEAmycp mutant after
treatment with metabolic poison (Fig. 5). After poison treatment,
Cln2-KAEAmycp showed nuclear depletion in
approximately 50% of the cells assayed. Washing out the poison rapidly
reconstituted localization of Cln2-KAEAp throughout the cell.
Essentially identical results were obtained with
Cln2-KAEA-4t3smycp and with the
cdc28csr1-1 mutant (data not shown). These data
suggest a constant energy-dependent nuclear import of Cln2p that occurs
independent of Cln2p phosphorylation or Cln2p-Cdc28p complex formation.
Nevertheless, interaction with Cdc28p appears to be required for
efficient and phosphorylation-regulated Cln2p localization.
The metabolic energy requirement for nuclear accumulation of
Cln2-4t3smycp could result from a requirement for
GTP hydrolysis by the RAN GTPase (44, 47, 61). To test
this possibility, we assayed wild-type Cln2mycp
and mutant Cln2-4t3smycp localization in a
temperature-sensitive ran mutant strain expressing the
gsp1-1 and gsp1-2 alleles
(79). No change in wild-type
Cln2mycp localization was observed at permissive
and nonpermissive temperatures (data not shown) (Fig.
6). In this strain, the
Cln2-4t3smycp shows a localization pattern
primarily throughout the cell, differing significantly from that
observed with wild-type Cln2mycp. No
RAN-dependent change in the localization pattern of Cln2-4t3sp was
observed at permissive and nonpermissive temperatures (Fig. 6).
Additionally, inactivation of the transport factors Crm1p, Msn5p,
Los1p, Sxm1p, Mtr10p, Nmd5p, Kap104p, and Kap114p individually does not
significantly alter wild-type or Cln2-4t3sp localization (data not
shown). Localization of Cln2p might also be regulated by physical
interactions with non-transport proteins, such as members of the 14-3-3 family (reviewed in reference 22). However, none of the
potential phosphorylation sites mutated in this study clearly falls
within described 14-3-3 binding motifs (52, 80).

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FIG. 6.
Immunolocalization of Cln2mycp and
Cln2-4t3smycp in the absence of Ran activity.
GSP1 deletion strains carrying a plasmid that expresses
either the wild-type GSP1, mutant
gsp1-1, or mutant
gsp1-2 alleles and integrated myc
epitope-tagged Cln2p from the CLN2 promoter were assayed
by indirect immunofluorescence as described in Materials and Methods.
Cells were grown to log phase at 23°C. Cultures were split, and half
was incubated at 23°C and half was incubated at 37°C for 2 h.
(A) Indirect immunolocalization of Cln2mycp and
Cln2-4t3smycp expressed in wild-type GSP1
strain (top row), gsp1-1 strain (second
row) and gsp1-2 strain (third row)
incubated at 37°C. The first column shows the indirect
immunofluorescence with monoclonal anti-myc antibody 9E10 (red), the
second column shows DAPI staining of DNA (blue), and the third column
shows the merge of these two images. Images were collected with the
DeltaVision microscopy restoration system. (B) Quantitation of
Cln2 localization. The percentage of cells showing nuclear
depletion (gray bars), signal throughout the cell (white bars), or
nuclear accumulation (black bars) is shown. Quantitation was
performed as described in the legend to Fig. 2. Data for
Cln2mycp and Cln2-4t3smycp localization in the
presence of wild- type and mutant GSP1
incubated at 37°C are shown. No fewer than 200 cells were counted
with the DeltaVision microscopy restoration system.
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These data suggest that the nuclear accumulation of hypophosphorylated
Cln2p-Cdc28p occurs by a Ran-independent, yet energy-dependent form of
active transport (Fig. 5 and 6). Export of Cln2p-Cdc28p from the
nucleus may occur in an energy-independent manner (Fig. 5).
Cln2p nuclear depletion may be inefficient early in the cell
cycle.
We analyzed Cln2p phosphorylation and subcellular
localization in a population of rapidly growing cells separated by
centrifugal elutriation into fractions of different cell size. The
smallest (newborn) cells obtained from the elutriation contain
Cln2p that is significantly underphosphorylated compared
to larger unbudded cells (Fig.
7A). This is true for 9×
myc-epitope-tagged Cln2p, as well as for protein A-tagged Cln2p (in
both cases integrated at the CLN2 locus). Indirect
immunofluorescence shows little or no detectable depletion of
Cln2pmyc from the nucleus of the smallest
unbudded cells (Fig. 7B and C), consistent with the idea that Cln2p
shuttles between the cytoplasm and nucleus. These data also suggest the
possibility that shuttling is differentially regulated, with more
nuclear Cln2p present very early in the cell cycle.

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FIG. 7.
Cell-cycle-regulated nuclear depletion of Cln2p. Rapidly
growing cultures of diploid strain MMY42 were chilled and subjected to
centrifugal elutriation to obtain fractions of cells of increasing size
as described in Materials and Methods. (A) Fractions were subjected to
protein extraction and immunoblot analysis to detect 9× myc
epitope-tagged or PA epitope-tagged Cln2p, each expressed from a
different allele from the CLN2 promoter. Nop1p levels
were monitored as a loading control. (B) Fractions were assayed by
indirect immunofluorescence to detect Cln2mycp
localization. The bar graph shows cells that were scored for the
presence of nuclear depletion as described in the legend to Fig. 2. No
nuclear accumulation was observed in these fractions. The percentage of
budded cells is the percentage of cells with buds in the population of
cells in each fraction. The percentage of the total is the percentage
of cell mass present in a fraction of the total amount of cell mass as
determined by OD of samples. (C) Representative immunofluorescence
results of two MMY42 elutriations (sets 1 and 2). Cells were assayed by
indirect immunofluorescence as described in Materials and Methods. The
first row shows differential interference contrast (DIC) images
(cells), the second row shows the indirect immunofluorescence with
monoclonal anti-myc antibody 9E10 ( -myc), and the third row shows
DAPI staining of DNA. The untagged control from this experiment
consisted of exponentially growing cells of strain W303d. The amounts
of cells of fractions 1, 2, 3, and 4 were 66, 73, 78, and 89 fl,
respectively, as determined by identification of peak channels by
Coulter Counter analysis of cells.
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We showed previously that addition of an NES to Cln2p significantly
weakened its biological activity (45), implying that Cln2p
might spend some time in the nucleus and that this nuclear residence
might be important for its function. Our present results suggest that
physical association with Cdc28p and phosphorylation may regulate
nuclear accumulation of Cln2p; this binding or phosphorylation may be
cell cycle regulated, leading to the potential for Cln2p to function in
the nucleus specifically very early in the cell cycle.
Another hypothesis that could explain loss of nuclear exclusion
of Cln2p in cells early in the cell cycle would be that
Cln2p-Cdc28p complex formation is inefficient at this time. We do
not yet have information on this point, because the extremely low
yield of cells at this point in the cell cycle from the elutriating
rotor (Fig. 7) makes a definitive analysis of Cln2p-Cdc28p complex
formation in these cells technically challenging.
Hypophosphorylated Cln2p could occur early in the cell cycle because of
low kinase activity or because of the activity of a phosphatase. We
tested the idea that the Cdc14p phosphatase, which is known to reverse
other Cdk phosphorylations (31, 73), might also
dephosphorylate Cln2p. This idea seemed reasonable, since we detect
hypophosphorylated Cln2p early in the cell cycle, not long after Cdc14p
has been released from the nucleolus late in mitosis (5,
64). We found, though, that strong overexpression of Cdc14p from
the GAL1 promoter sufficient to arrest the cell cycle
(73) or deletion of the Cdc14p regulator CFI1
(74) has little or no effect on Cln2p phosphorylation or
localization (data not shown).
Cln3p localization does not require Cdc28p activity or
binding.
In contrast to Cln2p localization, the localization of
Cln3p does not appear to be influenced by Cdc28p activity. Cln3p
remains nuclear when expressed in the cdc28-4
strain at permissive and nonpermissive temperatures (data not shown).
Additionally, mutations within the cyclin box of Cln3p that eliminate
detectable interaction with Cdc28p (M. Miller, unpublished
observations) do not alter Cln3p nuclear localization (data not shown).
Nuclear localization of both wild-type and cyclin box-defective Cln3p
was also observed in the presence of Cdc28csr1-1p
mutant Cdc28p (data not shown); this Cdc28 mutation reduces immunoprecipitable Cln3p-Cdc28p complexes by about 10-fold
(39). Thus, we were unable to detect any Cdc28p
requirement for Cln3p localization.
Carboxy-terminal Cln3p bipartite NLS.
The
CLN3-1 truncation lacks the C-terminal 177 amino
acids of Cln3 and shows a partial increase of cytoplasmic localization when compared to full-length Cln3p (45). These data
suggest that sequences important for the nuclear localization are
present in the carboxy-terminal tail. The last 22 amino acids of Cln3p contain a potential bipartite NLS (this was proposed as a candidate NLS
previously, although it was not tested functionally
[81]). Deletion of the last 22 amino acids of Cln3p
(Cln3-
22p) resulted in a primarily cytoplasmic localization, unlike
the nuclear localization of Cln3mycp (Fig.
8). Thus, the last 22 amino acids are
required for nuclear localization of Cln3p.

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FIG. 8.
Indirect immunolocalization of
Cln3- 22mycp. Wild-type (1255-5C) cells were transformed
with plasmids pRS414 (vector), pMM99 (Cln3mycp), and pMM98
(Cln3- 22mycp). All plasmids are episomal CEN (low copy
number) and express the myc-tagged Cln3 protein from the
CLN3 promoter. Transformants were assayed by indirect
immunofluorescence as described in Materials and Methods. The first row
shows DIC images (cells), the second row shows indirect
immunofluorescence with monoclonal anti-myc antibody 9E10 ( -myc),
and the third row shows DAPI staining of DNA. Images in this figure
were captured and processed in parallel.
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To determine if the putative Cln3p NLS is sufficient for nuclear
localization, the last 22 amino acids of Cln3p were fused to the N
terminus of the GFP expressed from the transport assay plasmid pKW431
(69). Since this sequence consists of two basic clusters
separated by 15 amino acids (diagrammed in Fig.
9A), we also constructed mutant versions
of the Cln3p NLS fused to GFP, in which the clusters of basic residues
are mutated to alanine singly or together (Cln3p KA, AK, or AA NLS).
The GFP proteins containing the Cln3p NLS show a staining pattern in
living cells consistent with nuclear localization, while the GFP
fusions containing the mutant Cln3p mnls sequences were cytoplasmic
(Fig. 9B). To ensure that the Cln3p NLS shifts the localization of GFP
into the nucleus, cells were fixed and assayed for both GFP
localization and DAPI staining. The Cln3p-NLS-GFP fusion colocalized
with the nuclear DAPI signal, although this procedure weakened the GFP signal significantly (data not shown).

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FIG. 9.
Cln3-NLS-GFP localization. (A) Amino acid sequence of
the bipartite-type Cln3 NLS. The mutant Cln3 nls AA substitutes the
basic residues in the first and second basic clusters to alanine. The
mutant Cln3 nls AK substitutes the first basic cluster with alanines,
and the mutant Cln3 nls KA substitutes the second basic cluster with
alanines. Mutated residues are underlined. (B) Immunofluorescent
localization of the various Cln3 NLS-GFP in living cells. The top row
shows DIC images (cells), and the bottom row shows GFP signal in an
identical field of cells. Images in this figure were captured and
processed in parallel.
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Taken together, these data indicate that the CLN3p NLS is a bipartite
NLS, which is necessary and sufficient for nuclear localization.
Cln3-
22p protein characterization.
To determine if deletion
of the Cln3 NLS alters steady-state expression levels, Western blot
analyses were carried out on a wild-type strain expressing
Cln3mycp and
Cln3-
22mycp.
Cln3-
22mycp was expressed at levels as high as
wild-type Cln3mycp, and in 3 out of 5 experiments
Cln3-
22mycp was expressed at higher levels
than wild-type Cln3mycp (Fig.
10B, lanes 1 and 2 and data not shown).

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FIG. 10.
Comparison of myc-tagged Cln3 proteins. (A) Indirect
immunolocalization of NLS-Cln3- 22mycp. Wild-type
(1255-5C) cells were transformed with plasmids pMM99
(Cln3mycp), pMM98 (Cln3- 22mycp), and pMM104
(NLS-Cln3- 22mycp). All plasmids are episomal CEN (low
copy number) and express the myc-tagged Cln3 protein from the
CLN3 promoter. Transformants were assayed by indirect
immunofluorescence as described in Materials and Methods. The first row
shows DIC images (cells), the second row shows indirect
immunofluorescence with monoclonal anti-myc antibody 9E10 ( -myc),
and the third row shows DAPI staining of DNA. The vector control from
this experiment (data not shown) gave no detectable signal. (B)
Wild-type strain (1255-5c) transformed with plasmids pMM99
(Cln3myc, set 1), pMM98 (Cln3- 22myc, set 2),
pMM104 (NLS-Cln3- 22myc, set 3), pMM105
(mnls-Cln3- 22myc, set 4), pMM106
(NES-Cln3- 22myc, set5), and pMM107
(mnes-Cln3- 22myc, set 6). All plasmids are episomal CEN
(low copy number) and express the myc-tagged Cln protein from the
CLN3 promoter. Cellular lysates of two independent
transformants for each clone were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (10% polyacrylamide) and
analyzed by Western blot analysis as described in Materials and
Methods. (C) Coimmunoprecipitation assays were carried out as described
in Materials and Methods. Strain 1559-3B-7 expressing HA-epitope-tagged
Cdc28p was transformed with plasmids pRS414 (vector), pMM99
(CLN3myc), and pMM98
(cln3- 22myc). The
top panel shows the anti-HA immunoblot of immunoprecipitated
Cdc28hap. The middle panel shows the anti-myc immunoblot of
Cln3myc protein present in the immunoprecipitation. The
bottom panel shows the anti-myc immunoblot of Cln3myc
protein present in the total cellular lysate used for the
immunoprecipitation.
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The NLS could overlap with functional PEST sequences in the Cln3p
C-terminal third (13, 81); alternatively, altering Cln3p localization could directly affect Cln3p stability. To test this, exogenous localization signals (SV40 NLS or PKI NES) were added to
Cln3-
22mycp to accentuate or correct the
localization defect. The NLS resulted in a partial accumulation of
Cln3-
22mycp in the nucleus (Fig. 10A). In
repeated immunofluorescence experiments, the PKI NES appeared to result
in a tighter exclusion of Cln3-
22mycp from the
nucleus (data not shown), although the effect is subtle, since the bulk
of Cln3-
22mycp is already cytoplasmic even
without the NES. The mnls and mnes mutant sequences (containing
single-amino-acid changes that disrupt the localization activity of
the sequences) did not significantly alter the localization of
Cln3-
22mycp (data not shown). No
significant difference was observed between the steady-state levels of
the NLS-Cln3-
22mycp (shifted to the nucleus
[Fig. 10]) versus mnls-Cln3-
22mycp
(cytoplasmic [data not shown]); therefore, mislocalization does not
appear to cause the increase in steady-state levels of
Cln3-
22mycp (Fig. 10B). The increase in
steady-state expression of Cln3-
22mycp may be
due to stabilization, since part of the final Cln3p PEST region is
missing in this deletion (81). The effect of this mutation
on Cln3mycp accumulation is significantly less
than the effect of complete deletion of the C-terminal 177 amino acids
of Cln3p (45), consistent with the presence of additional
PEST sequences in Cln3p (81).
Differences in subcellular localization of Cln3p verses Cln3-
22p may
influence Clnp-Cdc28p complex formation. To test this idea, a strain
expressing HA-tagged Cdc28p was transformed with plasmids that express
9× myc-tagged Cln3p or Cln3-
22p. Immunoprecipitations of the
HA-epitope-tagged Cdc28p results in coimmunoprecipitation of the
myc-epitope-tagged Cln3p. We find that levels of Cln3p able to
coimmunoprecipitate with Cdc28p correspond to the expression levels of
these proteins found in total cellular lysates (Fig. 10C). These data
indicate that the deletion of the last 22 amino acids of Cln3p does not
significantly alter the ability of Cln3p to associate with Cdc28p.
Functional consequences of Cln3p mislocalization.
The last 22 amino acids of Cln3p encode an NLS that is important for the nuclear
localization of Cln3p. Functional analysis was carried out to determine
if this NLS was significant for Cln3p function in vivo. Previously, we
demonstrated that shifts in the localization patterns of Cln3p
correlate with changes in the functional profile of Cln3p. This study
was carried out with the Cln3-1p mutant, which lacks the
carboxy-terminal 177 amino acids of Cln3p. In addition to missing the
C-terminal 22 amino acids (NLS characterized above), this mutant lacks
several PEST domains and demonstrates a dramatic stabilization
phenotype (13, 45, 81). In contrast, the Cln3-
22p
mutant is a much smaller deletion, missing only the NLS and parts of a
single PEST domain and causes significantly less stabilization of the
protein than the deletion of the C-terminal 177 amino acids. In
addition to determining if the C-terminal NLS defined in this study is
a functionally significant NLS in vivo, we were also interested in
whether the changes in the functional profile previously observed for
the mislocalized Cln3-1p mutant (45) required hyperstabilization.
A cln1 cln2 cln3 GAL1::CLN1 strain is
inviable on glucose medium (which turns off the
GAL1::CLN1 cassette, resulting in cln1 cln2
cln3 inviability). We introduced plasmids encoding Cln3-
22p with or without mislocalization signals into this strain and
compared the ability of these plasmid-bearing strains to form colonies on glucose-containing medium. The strain expressing NES-Cln3-
22p was
inviable, while the strains expressing NLS-, mnls-, or
mnes-Cln3-
22p and Cln3-
22p were viable (Fig.
11A). This result is different from that obtained with the Cln3-1p mutant and
reveals an essential nuclear requirement for Cln3p not previously
observed. The ability of NES-Cln3-1p to support viability in this
strain may result from its overexpression. These data indicate that
nuclear localization of Cln3p is required for efficient rescue of the
triple cln deletion. The ability of Cln3-
22p to rescue
the cln1 cln2 cln3 strain may reflect residual nuclear
accumulation of Cln3-
22p, which may be eliminated by NES (but not
mnes) addition.

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FIG. 11.
Functional assays for Cln3- 22p. Mutant
strains, each containing a
GAL1::CLN for viability, were
transformed with CLN plasmids. For each transformant,
10-fold serial dilutions were prepared from independent pools of
transformants (5 to 10 colonies), and 3 µl of each dilution was
plated onto both YPD (dextrose) and YPG (galactose) plates. Plates were
incubated at 30°C, except where 38°C is indicated. The
CLN plasmids used are pMM82
(CLN2myc), pMM99
(CLN3myc), pMM98
(cln3- 22myc), pMM104
(NLS-cln3- 22myc),
pMM105 (mnls-cln3- 22myc),
pMM106 (NES-cln3- 22myc),
pMM107
(mnes-cln3- 22myc),
pMM60 (NLS-CLN2myc), pMM61
(mnls-CLN2myc), pMM92
(NES-CLN2myc), and pMM93
(mnes-CLN2myc). All plasmids are episomal
CEN (low copy number) and express the myc-tagged Cln protein from the
CLN3 promoter. Strains are listed in Table 1. Genotypes
are as follows (lowercase denotes deletion; uppercase denotes wild
type, and GAL1:: CLN gene not indicated):
cln1 cln2 cln3 and cln1 cln2 cln3 bck2
(A); cln1 cln2 cln3 pcl1 pcl2 and cln1 cln2 CLN3
pcl1 pcl2 (B); cln1 cln2 CLN3 swi4 and
cln1 cln2 cln3 swi4 (C). (cl1 clz cl3 swi4
data were reproduced for comparison to the cln1 cln2 CLN3
swi4 strain from reference 45.)
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The genetic requirement for Cln3p is increased in the absence of the
BCK2 gene product, since Bck2p works in parallel with Cln3p
to trigger transcription of SCF- and MBF-regulated genes in late
G1 (see Introduction). Cln3-
22p has a reduced
ability to rescue the bck2 cln1 cln2 cln3 strain, at least
at elevated temperatures, compared to wild-type Cln3p (Fig. 11A). This
defect was rescued by addition of the NLS to Cln3-
22p, consistent
with the idea that this defect is specifically due to the largely
cytoplasmic localization of Cln3-
22p. Addition of the mnls to
Cln3-
22p was significantly less effective at restoring its activity
(Fig. 9A). These data indicate that the C-terminal NLS of Cln3p is
functionally significant, consistent with the idea that nuclear
localization of Cln3p is important for function (45).
These data also reveal an essential role of nuclear Cln3p in the
cln strain that was not previously detected with Cln3-1p.
Deletion of pcl1 and pcl2 results in the
inability of CLN3, but not CLN1 or
CLN2, to provide the essential CLN function for cell cycle initiation (see Introduction). Thus a cln1 cln2 cln3 pcl1 pcl2 GAL1::CLN1 strain is rescued on
glucose medium by CLN2, but not by CLN3 on
plasmids. As observed for the CLN3-1 mutant (45), CLN3-
22 exhibits a weak
ability to rescue this strain (Fig. 11B). This rescue was
abolished by addition of the NLS (but not the mnls) to Cln3-
22p,
suggesting that significant cytoplasmic localization of Cln3-
22p was
required for rescue. As expected from the inability of NES-Cln3-
22p
to rescue the cln1 cln2 cln3 strain (Fig. 11A), it was also
unable to rescue the cln1 cln2 cln3 pcl1 pcl2 strain (data
not shown). Taken together, these results could imply that rescue of
the cln1 cln2 cln3 pcl1 pcl2 strain by Cln3-
22p required
both cytoplasmic Cln3-
22p and some residual nuclear Cln3-
22p. If
this were correct, then we would predict that the combination of
wild-type nuclear Cln3p and the strongly cytoplasmic
NES-Cln3-
22p might complement each other for rescue of a
cln1 cln2 cln3 pcl1 pcl2 strain. Indeed, this was observed (Fig. 11B): provision of wild-type CLN3 in the chromosome
and NES-CLN3-
22 on a plasmid resulted in
significant viability in the absence of pcl1,
pcl2, cln1, and cln2, while neither
one alone was sufficient (Fig. 11B) (data not shown). Essentially
similar results were obtained with a swi4 cln1 cln2 CLN3
GAL1::CLN1 deletion strain (data not shown).
Therefore, as with pcl1 pcl2, deletion of
swi4 appears to uncover a strong requirement for both
cytoplasmic and nuclear CLN activity.
Consistent with this, we found previously that addition of an NES (but
not an mnes) to Cln2p strongly reduced its ability to rescue a
cln1 cln2 cln3 swi4 strain (45). According to
the hypothesis that both nuclear and cytoplasmic CLN
activities may be required in this context, NES-Cln2p should complement
Cln3p for rescue of a cln1 cln2 cln3 swi4 strain, since
Cln3p should provide the nuclear CLN function and NES-Cln2p
should provide the cytoplasmic CLN function. This was
observed (Fig. 11C): provision of wild-type CLN3 in the
chromosome and NES-CLN2 on a plasmid resulted in significant
viability in the absence of pcl1, pcl2, cln1, and cln2, while CLN3 alone was
insufficient and NES-CLN2 alone was very inefficient (Fig.
11C).
Thus, wild-type Cln3p requires nuclear localization to carry out its
normal roles, probably including activation of
G1/S transcription (see Introduction), so that
its mislocalization to the cytoplasm blocks its ability to rescue the
cln1 cln2 cln3 strain. In contrast, mislocalization of Cln3p
to the cytoplasm allows it to carry out some biological roles normally
restricted to the cytoplasmic Cln2p. These results confirm and extend
our previous results with mislocalized Cln3-1p, with much less
experimental confounding of localization and protein abundance effects.
Localization-dependent cell size control by Cln3p.
We also
assayed for CLN activity by monitoring cell volume.
Initiation of the cell cycle requires CLN activity for bud
emergence and activation of Clbp-Cdc28p complexes. Cells with reduced
CLN activity will have a delay in these events resulting in
a population of relatively large cells due to a longer period of cell
growth after cell division and before cell cycle initiation.
Conversely, high CLN activity will result in a population of
relatively small cells (11, 48, 53). Links between cell
cycle progression and cellular growth may also exist in higher
eukaryotic cells, suggesting that this process could be conserved (as
reviewed in reference 10). Plasmids encoding the various
mutant Cln3 proteins were introduced into a cln2 cln3
strain, and transformants were assayed for cell volume. (A cln2
cln3 strain was used to sensitize the assay to CLN
function.) As expected, expression of Cln2mycp
and Cln3mycp confers smaller cell volume than
that of the vector control. Expression of
NES-Cln3-
22mycp (but not the mnes controls)
produces populations of cells with larger cell volumes, near the volume
of control vector transformants (Fig.
12). These data show a strong reduction
of CLN3 functional activity when Cln3p is exported out of
the nucleus and into the cytoplasm through the combination of the Cln3p
NLS deletion and addition of the PKI NES. Thus, as with the cln1
cln2 cln3 rescue assay, the cell volume assay for CLN
activity leads to the conclusion that nuclear Cln3 may be required to
efficiently drive cell cycle initiation.

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FIG. 12.
Cell size assay for CLN gene function.
CLN1 cln2 cln3 cells (1421-21D) were transformed with
plasmids containing various CLN2 and CLN3
constructs (see the legend to Fig. 9). Cell size analysis was carried
out as described in Materials and Methods. Data for two transformants
are shown on each graph.
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Although we expected the shift of Cln3-
22p to the cytoplasm (even
without the NES) to decrease its activity in this assay, we find only a
slight shift and widening of the peak distribution of cells expressing
Cln3-
22p compared to wild-type Cln3p. This slight, but reproducible
change is relieved by addition of the functional SV40 NLS, but not the
mnls control (Fig. 12). These data are relevant to models describing
the regulation of cell size in response to Clnp dosage resulting from
Cln3p compartmentalization in the nucleus. Assuming that the nucleus
maintains a constant volume through G1, as cells
grow, the concentration of nuclear Cln3p could increase until some
critical threshold is reached allowing cell cycle progression (as
proposed in reference 23 and quantitatively modeled by
reference 8). If so, the prediction would be that the
redistribution of a significant amount of Cln3p to the cytoplasm (via
deletion of the Cln3 NLS) should reduce the amount of Cln3p in the
nucleus, thus delaying the ability of the nuclear Cln3p to reach this
critical threshold. In this case, Cln3-
22p should give a delay in
cell cycle progression compared to that of the wild type and a
commensurate increase in cell size. This prediction is complicated in
our experiment by the fact that Cln3-
22p is present at an elevated
level compared to wild-type Cln3 (Fig. 10). Increased Cln3p protein
levels are known to accelerate cell cycle progression (11,
53), consistent with the quantitative model (8).
Hence, the lack of phenotype observed with Cln3-
22p may reflect a
balance between accelerated cell cycle progression due to higher
steady-state levels of Cln3p (at least some of which is likely to be
nuclear) and slowed cell cycle progression due to the reduction of
nuclear Cln3p. However, comparison of the NLS-Cln3-
22p and the
mnls-Cln3-
22p provides us with a situation in which a clear
difference in subcellular localization is observed by indirect
immunofluorescence (Fig. 10), with no significant changes in expression
levels (Fig. 10). In this case, we find only minor changes in cell size
distribution dependent on subcellular localization, contrary to
predictions made based on models in which nuclear compartmentalization
of Cln3p is crucial for cell size control. It is possible that there is
some compensating acceleration of cell cycle initiation by cytoplasmic
Cln3p in this case.
Taken together, these data suggest that efficient nuclear localization
of Cln3p is important for maintaining cell size, since NES-Cln3-
22p
is almost unable to function in this assay. However, the relationship
between Cln3p function, nuclear localization, and cell size control may
be somewhat complex.
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DISCUSSION |
Different mechanisms control localization of the G1
cyclins Cln2p and Cln3p.
In addition to activating Cdks through
conformational changes associated with cyclin-Cdk binding, the spatial
context in which cyclin proteins function is an important component of
Cdk regulation. In previous work, we found that the localization
pattern of the G1cyclin Cln2p is primarily
cytoplasmic and that of the G1 cyclin Cln3p is
primarily nuclear (45). Here we have investigated the role
that the Cdk Cdc28p plays in the control of distinct subcellular localization patterns of these cyclins. Using a mutant Cln2p in which
all potential Cdc28p target sites have been mutated, we find a partial
accumulation of Cln2p in the nucleus (Fig. 1). Inactivation of
CDC28 also results in a partial accumulation of Cln2p in the
nucleus as demonstrated by indirect immunofluorescence (Fig. 2). The
nuclear accumulation of hypophosphorylated Cln2p does not reflect Cln2p
stabilization, since stabilization of Cln2p via mutation of the
CDC34 gene or deletion of the GRR1 gene does not
alter Cln2p localization (Fig. 2 and data not shown). Taken together,
these data are consistent with direct Cdc28p-dependent phosphorylation
of Cln2 being required for nuclear depletion of Cln2p. Additionally,
there is a requirement of Cln2p-Cdc28p complex formation in regulated
Cln2p localization. Both nuclear depletion and nuclear accumulation are
influenced by physical association with Cdc28p, as seen with mutations
in Cln2p that are defective in binding Cdc28p and with mutations in
Cdc28p that are defective for binding Cln2p (Fig. 4).
We also find a cell cycle-dependent shift in the phosphorylation state
of Cln2p. Populations of small unbudded cells harvested by centrifugal
elutriation from a log-phase culture (presumably the youngest cells in
the population) contain largely hypophosphorylated Cln2p compared to
populations of larger unbudded cells (presumably from later in the cell
cycle). These newborn cells with hypophosphorylated Cln2p show little
or no depletion of Cln2p from the nucleus. These data are consistent
with regulated shuttling of Cln2p in and out of the nucleus, where
nonphosphorylated Cln2p is enriched in the nucleus and phosphorylated
Cln2p is enric