Research Paper 1329

Cut8, essential for anaphase, controls localization of 26S

proteasome, facilitating destruction of cyclin and Cut2
Hisashi Tatebe* and Mitsuhiro Yanagida†

Background: Anaphase-promoting complex (APC)/cyclosome and 26S Addresses: *CREST Research Project, Department
proteasome are respectively required for polyubiquitination and degradation of of Biophysics, Graduate School of Science and
†Department of Gene Mechanisms, Graduate
mitotic cyclin and anaphase inhibitor Cut2 (Pds1/securin). In fission yeast, School of Biostudies, Kyoto University,
mutant cells defective in cyclosome and proteasome fail to complete mitosis Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto
and have hypercondensed chromosomes and a short spindle. A similar 606-8502, Japan.
phenotype is seen in a temperature-sensitive strain cut8-563 at 36°C, but the
Correspondence: Mitsuhiro Yanagida
molecular basis for Cut8 function is little understood. E-mail: yanagida@kozo.biophys.kyoto-u.ac.jp

Results: At high temperature, the level of Cut8 greatly increases and it Received: 26 January 2000
becomes essential to the progression of anaphase. In cut8 mutants, Revised: 12 September 2000
Accepted: 20 September 2000
chromosome mis-segregation and aberrant spindle dynamics occur, but
cytokinesis takes place with normal timing, leading to the cut phenotype. This is Published: 13 October 2000
due to the fact that destruction of mitotic cyclin and Cut2 in the nucleus is
dramatically delayed, though polyubiquitination of Cdc13 occurs in cut8 mutant. Current Biology 2000, 10:1329–1338
Cut8 is localized chiefly to the nucleus and nuclear periphery, a distribution 0960-9822/00/$ – see front matter
highly similar to that of 26S proteasome. In cut8 mutant, however, 26S © 2000 Elsevier Science Ltd. All rights reserved.
proteasome becomes mostly cytoplasmic, showing that Cut8 is needed for its
proper localization.

Conclusion: Cut8 is a novel evolutionarily conserved heat-inducible regulator. It

facilitates anaphase-promoting proteolysis by recruiting 26S proteasome to a
functionally efficient nuclear location.

Background M.Y., unpublished results). Mitotic cyclin and Cut2, two

During anaphase, sister chromatids are physically dissoci-
ated from each other and move towards opposite poles of
the spindle. The onset of sister-chromatid separation is,
however, prevented in metaphase, the mitotic stage
immediately preceding anaphase, and occurs only after all
the chromosomes and the spindle apparatus are ready for
concerted separation [1–4]. We have used the fission yeast
Schizosaccharomyces pombe as a model system for studying
anaphase [5,6].
Ubiquitin-mediated protein degradation is essential for
promoting anaphase, and protein complexes such as 26S
proteasome [7–9] and 20S APC/cyclosome [4,10–13] are
required for the degradation and polyubiquitination,
respectively, of substrate proteins. These complexes are
evolutionarily conserved. If substrates such as mitotic
cyclin and anaphase inhibitor Cut2/Pds1/securin are not
degraded, cells fail to exit from mitosis and sister chro-
matids fail to separate [2,3]. Seven of twelve subunits in
cyclosome [14] have been identified in fission yeast
[15–19]. All mutants in cyclosome subunit genes are
arrested in metaphase, and polyubiquitination of mitotic
cyclin does not occur in mutant cells. Cyclosome subunits
are regulated in a mitosis-specific manner [16,17]. Cyclo-
some subunits are enriched in the nucleus (Y. Kimata and
known substrates of cyclosome, are enriched in the
nucleus during interphase and additionally at the spindle
pole bodies (SPBs) and the short spindle during mitosis,
and abruptly disappear in anaphase [4,20–22].
In fission yeast, conditional-lethal mutations in protea-
some subunits were isolated as mts1-5 mutants; they were
resistant to the microtubule poison methylbenzylcarbamy-
late and were also temperature-sensitive [23–26]. mts
mutant cells enter mitosis but are blocked at a metaphase-
like stage, judging from the short spindle and condensed
chromosomes present in the arrested cells. The pheno-
types of mts mutants are similar to those of cyclosome
mutants. Localization of 26S proteasome is somewhat
unexpected, as it is highly enriched in the nuclear enve-
lope as well as in the nucleus [27]. Immunogold staining
for proteasome subunit Pad1/Mts5 indicates its presence
on the inner side of the nuclear membrane. Similar local-
ization of proteasome subunits was also observed in
budding yeast [28]. In fission and budding yeasts, ubiqui-
tin-dependent proteolysis, including that mediated by
cyclosome, thus seems to be highly active in the perinu-
clear regions. Little is known about the regulation of pro-
teasome during the cell cycle, however. It is presumed to
be active throughout the cell cycle.
1330 Current Biology Vol 10 No 21
S. pombe cut8-563 is a temperature-sensitive (ts) mutant which
shows chromosome overcondensation and short-spindle
formation in the absence of sister chromatid separation at
36°C, the restrictive temperature [29]. The mutant cells
undergo cytokinesis, however, which cuts across the undi-
vided nucleus, thus producing cells with a cut (‘cell
untimely torn’) phenotype. Cut8 protein contains 262
amino acids and a database search suggests that it is evolu-
tionarily conserved and similar to budding yeast Dbf8p
[30], which is identical to Sts1p [31], and to sequences
from Candida albicans and Botrytis cinerea. DBF8/STS1 is an
essential gene, mutations in which result in delay in
mitotic progression and chromosome instability. A multi-
copy suppressor plasmid pCEK1 for the ts phenotype of
cut8-563 was obtained; Cek1 is an exceptionally large
protein kinase containing 1309 amino acids. Here, we
provide evidence that Cut8 is a novel regulator of 26S
proteasome localization. To our knowledge, this is the first
evidence that localization of proteasome is regulated by a
component other than the known proteasome subunits
(some proteasome subunits contain nuclear localization
signals [32]). Many cut8 mutant phenotypes can now be
explained by the impaired localization of proteasome.
Results
The cut8+ gene is required for normal mitosis at high
temperature
For disruption of the cut8+ gene by one-step replacement,
two plasmids were made for deletion and insertion and
used to transform a diploid strain (see Materials and
methods). Heterozygous diploids obtained were analyzed
by Southern blotting, and 4.5 and 3.6 kb bands for inser-
tion and deletion respectively were obtained (data not
shown). The diploid cells were then sporulated and
tetrads dissected (Figure 1a). Two segregant spores with
the gene deleted (del, ∆cut8) showed the ts growth pheno-
type: colonies were produced at 26–30°C, but not at
33–36°C. The identical ts phenotype was also obtained for
segregants obtained by insertion (int). Gene disruption in
the ts haploid segregants was confirmed by Southern and
northern hybridization and immunoblotting (data not
shown). Cut8 is thus not absolutely required for cell via-
bility; it is required only at 33–36°C. The cellular pheno-
types of ∆cut8 cells were observed. After the shift from
26–36°C for 2 hours, mitotic cells had hypercondensed
chromosomes accompanying the cut phenotype ([3,33],
and Figure 1b). These aberrant mitotic cells were similar
to those found in the ts mutant cut8-563 [29].
The level of Cut8 increases at 36°C
We found that the amount of Cut8 increased dramatically
in wild-type cells after the temperature shift to 36°C
(Figure 1c); the increase was observed 15 minutes after
the shift to 36°C and the level of Cut8 reached a steady
state after 60 minutes. Quantitative estimations indicated
that the increase was roughly 10-fold (α-tubulin was the
loading control). The level of cut8+ mRNA was also tested
by northern hybridization, using wis2+ (cyclophilin 40) as a
positive control (its mRNA increases with rising tempera-
ture [34]) (Figure 1d). The level of cut8 mRNA remained
identical after the temperature shift, indicating that the
Cut8 increase was due to post-transcriptional rather than
transcriptional regulation.
To confirm that the increase in Cut8 depended on protein
synthesis, the protein synthesis inhibitor cycloheximide
(CYH) was added to the wild-type culture exponentially
grown at 26°C (Figure 1e, upper panel) or after the shift to
36°C (lower panel). Cut8, which had increased after the
shift to 36°C for 30 or 60 minutes, rapidly decreased after
the addition of CYH at a similar rate between 26 and 36°C.
Cut8 was thus unstable at both 26 and 36°C (half-life
~7 minutes). This decay in the presence of CYH was
dependent on 26S proteasome because the level of Cut8
greatly increased in mts2-1, mts3-1 and pad1-1 mutant cells
(Figure 1f, and data not shown). The rate of decay did not
alter in G2-arrested cdc25-22 and M-arrested cut7-446 and
nuc2-663 mutant cells at 36°C (data not shown), indicating
that the instability of Cut8 did not alter during the cell cycle.
cut8-563 shows aberrant anaphase
To investigate the role of Cut8 at 36°C in the cell cycle,
synchronous cultures were obtained using an elutriator rotor
(see Materials and methods). Small early G2 cells of cut8-
563 grown at 26°C were collected and cultured at 36°C.
Mutant cells rapidly lost viability around 120 minutes, at a
mitotic stage corresponding to anaphase (Figure 2a, upper
panel). The histone H1 kinase activity measured for cell
extracts peaked at 80–100 minutes, corresponding to
metaphase (Figure 2a, lower panel) and decreased at
120 minutes. Note that cell viability was still high during
metaphase. The spindle index (percentage of cells contain-
ing the short and elongating spindle stained by anti-tubulin
antibody TAT-1) became maximal from 100–120 minutes.
The frequencies of the cut phenotype increased from
120 minutes (30%) and reached 80% after 160 minutes.
Cells were stained with anti-tubulin antibodies (TAT-1)
for microtubules, anti-Sad1 antibodies for SPBs (Sad1 is an
essential component of SPBs [35]) and DAPI for chromo-
somal DNA (Figure 2b). Normal mitotic cells with a short
spindle were abundant at 80 minutes (Figure 2b, left panels),
but abnormal anaphase cells displaying streaked chromo-
somes and the extended spindle were plentiful at
120 minutes (Figure 2b, right panels), consistent with the
timing of viability loss. The quantitative measurements
(Figure 2c) indicated that abnormal anaphase cells con-
taining streaked chromosomes reached 40% at 120 minutes.
These results indicate that Cut8 becomes essential during
anaphase for proper sister chromatid separation.
The destruction of Cdc13 in synchronous cultures of cut8
mutant at 36°C was monitored by immunoblot. No clear
 Research Paper Control of 26S proteasome localization Tatebe and Yanagida 1331

Figure 1

Cut8 is essential for mitosis at high

temperature. (a) The cut8+ gene was (a) Segregants Segregants (b)
(del) (int)
disrupted in two ways, by deletion (del) and 26ºC
a b c d a b c d ºC
by insertion (int). One set of tetrad YPD 26
segregants (a, b, c, d) from heterozygous 30
33
diploids of each type are shown. Ura+ 36
segregants containing the disrupted gene EMM2 w/o Ura
showed the ts growth phenotype.
(c) Min after shift up to 36ºC
(b) Cytological phenotypes of ∆cut8 (del)

180
0

0
125
150

240
cells. Cells growing at 26°C are normal. After

30

60

90
15

45

75

10

21
36ºC, 2 h

0
2 h at 36°C, cells displaying condensed Cut8
chromosomes and abnormally separating Tubulin
chromosomes with the cut phenotype are
seen. The identical phenotype was also (d) Min after shift up to 36ºC
obtained in ∆cut8 (int) cells (data not shown).

0
30

60
90
15

45

12
The scale bar represents 10 µm. (c) An 0
cut8+
immunoblot pattern of Cut8 protein in extracts
of wild-type cells after the temperature shift wis2+
from 26°C (time 0) to 36°C. Polyclonal anti-
EtBr (f) Wild type mts2-1
Cut8 antibodies were used. An approximately
10-fold increase in Cut8 protein as measured + CYH (0 min) + CYH (60 min)
min at
by densitometry was observed after 60 min. (e)

0
+ CYH (0 min)

30
60
90

30
60
90
12

12
36ºC

0
No band was obtained for ∆cut8 (del) and
0

Cut8
30
60
90

min at 26ºC
12

∆cut8 (int) (data not shown). α-Tubulin

0

Cut8 Tubulin
detected by monoclonal antibody is used as a
Tubulin
loading control. (d) Northern blot of cut8+
mRNA. Total RNAs of wild-type cells were
isolated (ethidium bromide (EtBr) stain shown + CYH (0 min) + CYH (30 min) + CYH (60 min)
as a loading control) and probed with the min at
0

0
30
60
90

30
60
90

30
60
90
12

12

12
36ºC
0

cut8+ and wis2+ genes. No detectable

change was found in the level of the cut8+
transcript. The level of wis2+ mRNA is known
to increase after temperature shift [34].
(e) Decay of Cut8 protein in the presence of
cycloheximide (CYH). CYH was added (to a
final concentration of 100 µg/ml) to the wild-
type culture grown at 26°C, and the level of
Cut8 protein was estimated from 0–120 min
by immunoblot (upper panel). CYH was
added at 0 min to the culture when it was
Cut8
Tubulin
shifted from 26 to 36°C (lower left). CYH was
added to the culture at 30 min (lower center)
and 60 min after the temperature shift (lower
right). The arrowheads indicate the time of
CYH addition, after which a rapid decay in
Cut8 was observed. α-Tubulin is the loading
Current Biology
control. (f) Wild-type and mts2 mutant cells
grown at 26°C were shifted to 36°C, and
CYH was added 60 min after the temperature
shift. Immunoblotting used polyclonal
antibodies against Cut8. Tubulin was the
loading control.

difference could be found in the degradation patterns of
Cdc13 between mutant and wild-type cells in repeated
experiments (data not shown). The anaphase degradation
of Cut2 was difficult to detect in the synchronous culture
made by elutriation (the degradation was visible only in
cdc25 block and release experiments, as its synchrony was
much higher than that by elutriation under our experi-
mental conditions; [21], and our unpublished results).
results are consistent with the idea that Cut8 is implicated
in ubiquitin-dependent proteolysis through interaction
with cyclosome and/or proteasome. In contrast to the syn-
thetic lethal interactions described above, the ts pheno-
type of cut8-563 was suppressed by an elevated dosage of
Cek1 [29]. Plasmids carrying Cek1 also suppressed ∆cut8
(data not shown). Cut8 function might thus be modulated
or substituted by Cek1.

Genetic interactions between cut8 and mutations
implicated in anaphase-promoting proteolysis
To search for mutations that might interact with cut8-563,
genetic crosses were made between cut8 and various
mutants (Table 1). Synthetic lethal combinations were
obtained by crossing with cut4-533, cut9-665 or slp1-362
mutants impaired in cyclosome function, with mts2-1 and
mts3-1 defective in 26S proteasome, and dis2-11 defective
in type 1 protein phosphatase (PP1). All these genes are
involved in anaphase-promoting proteolysis [4]. These
Decay of the Cdc13–GFP signal is delayed in cut8
We asked whether the disappearance of Cdc13, a mitotic
cyclin, during anaphase is cytologically normal in cut8-563
cells. To this end, cut8-563 was integrated with the GFP-
tagged cdc13+ gene with the native promoter, and the inte-
grated strain was observed by confocal microscopy. First,
the wild-type control cells carrying the integrated cdc13–gfp
gene were cultured at 36°C under the microscope as
described previously [36], and micrographs were taken at
30 second intervals (Figure 3a). The green fluorescence
1332 Current Biology Vol 10 No 21

Table 1

Mutations that show synthetic growth defect with cut8 mutation at 26°C or 30°C.

Gene Double mutant Phenotype Notes

mts2 mts2-1 cut8-563 Tiny colony at 26°C 26S proteasome subunit

mts3 mts3-1 cut8-563 No colony at 26°C 26S proteasome subunit
cut4 cut4-533 cut8-563 Tiny colony at 26°C APC/cyclosome subunit
cut9 cut9-665 cut8-563 Tiny colony at 26°C APC/cyclosome subunit
slp1 slp1-362 cut8-563 Tiny colony at 26°C APC/cyclosome activator
dis2 dis2-11 cut8-563 No colony at 26°C and 30°C PP1 catalytic subunit

signal of Cdc13–GFP at 36°C was enriched in the nucleus
and the single SPB in interphase (0–20 minutes), whereas in
mitotic cells (starting from 21 minutes) the spindle signal
was visible. The short-spindle signal lasted for several
minutes and very rapidly disappeared around 25 minutes.
The bulk of the signal on the spindle was destroyed in only
1–2 minutes, whereas disappearance of the nuclear signal
was slightly delayed (it could be seen till 27 minutes). Septa-
tion occurred at 49 minutes, 29 minutes after the onset of
mitosis (the position of septation is indicated by the arrow-
heads in Figure 3a,b).
In cut8-563 cells at 36°C, the timing and intensity of
Cdc13–GFP images seen in interphase (0–20 min) and
early to mid-mitotic cells (20–24 min) were basically iden-
tical to those of wild-type controls. The decay of the GFP
signal from mid-mitosis was, however, strikingly delayed
(Figure 3b). The decline of the spindle and nuclear
signals occurred slowly compared with wild type, taking
approximately five times longer (the bulk of the signal dis-
appeared within 5–10 minutes). The short-spindle signal
disappeared around 36 minutes, whereas the faint nuclear
GFP signal persisted till very late. Note, however, that
septation took place 29 minutes after the onset of mitosis,
as in wild-type cells (indicated by the arrowhead in
Figure 3b). Therefore, timing of cytokinesis was not
delayed. Ten living cut8-563 cells were monitored at 36°C,
and essentially the same results were obtained. Although

Figure 2

Anaphase is defective in cut8-563 mutant.

(a) 80 (b) 80 min 120 min The synchronous cultures of cut8-563 were
grown at 36°C. Small G2 cells of cut8-563
Percentage

60
Viability exponentially grown at 26°C were collected,
DNA

40 Spindle and then cultured at 36°C. (a) Cell viability

cut (upper panel, filled circles) decreased sharply
20 after 120 min at 36°C, whereas the H1 kinase
activity measured in extracts peaked at
0
0 60 120 180 240 100 min and decreased already at 120 min
3.0 (lower panel). The viability decrease occurred
SPB

during the inactivation period of H1 kinase.

H1 kinase activity
(arbitrary units)

The spindle index (upper panel, open circles),

2.0 as determined by anti-tubulin antibody TAT-1
went up at 100 min, corresponding to the
1.0 activation of H1 kinase, but remained relatively
high for ~1 h. Spindle destruction was
Tubulin

anomalously delayed. Cells showing the cut

0.0 phenotype (upper panel, filled squares)
0 60 120 180 240 increased after 120 min and reached 80%
Min at 36°C after 180 min (upper panel). (b) Cells taken
(c) 30 from 80–100 min at normal metaphase and
from 120 min at aberrant anaphase. They
were stained by DAPI and antibodies against
Percentage

20 Prometaphase α-tubulin (Tubulin) and Sad1 protein (SPB).

Normal anaphase
Abnormal anaphase The scale bar represents 10 µm.
10 (c) Percentages of cells showing normal
anaphase (open squares), the short
metaphase spindle (open circles) and the
0
0 60 120 180 240 elongated anaphase spindle with abnormal
Min at 36°C Current Biology chromosomes (filled squares) were estimated
from cells stained by DAPI, anti-tubulin and
Sad1 antibodies.
 Research Paper Control of 26S proteasome localization Tatebe and Yanagida 1333

Figure 3

Cdc13–GFP destruction and spindle

(a) (c)
dynamics depends on the presence of
Wild type Wild type
functional Cut8. (a,b) GFP-tagged Cdc13 20 24 28 6 12 18
expressed from the integrated gene with the 0 7 13 19
0 21 25 29
native promoter is observed in a background 2 8 14 20
of (a) wild type and (b) cut8-563 at 36°C. 10 22 26 30
3 9 15 21
Arrowheads indicate timing and position of 16 23 27 49 4 10 16 22
septation. (c,d) GFP-tagged α-tubulin
5 11 17 23
expressed from the chromosomally integrated
atb2–gfp gene is observed in a background of (b) (d)
(c) wild type and (d) cut8-563 at 36°C. Scale cut8-563 27 38 49
cut8-563 12 24 36
bars represent 10 µm. 0 13 25 37
0 28 39 50
2 14 26 38
10 29 40 51
3 15 27 39
16 30 41 52 4 16 28 40
20 31 42 53 5 17 29 41

21 32 43 54 6 18 30 42
7 19 31 43
22 33 44 64
8 20 32 44
23 34 45 74
9 21 33 45
24 35 46 10 22 34 46
84
25 36 47 11 23 35 47

26 37 48
Current Biology

the delay in Cdc13 degradation could not be detected in
extracts of synchronous cultures by immunoblotting, the
delay in the degradation of nuclear mitotic cyclin tagged
with GFP was established.
The period of mitotic spindle dynamics is prolonged in
cut8-563
Spindle extension is delayed in cut8-563 at 36°C. To observe
microtubule dynamics, the α-tubulin gene atb2+ tagged
with GFP [37] was chromosomally integrated with the
nmt1 promoter [38] in the wild type and cut8-563. In wild-
type (Figure 3c), changes in the spindle are seen from
3–15 minutes. In cut8-563 cells (Figure 3d), however, the
duration of spindle dynamics is much longer than that of
wild type. This was true in all the mutants examined. The
period between the late G2 microtubule arrays (0 min)
and formation of the short spindle (2–8 min) was somewhat
prolonged (twofold delay). The short spindle, though
gradually elongating, was seen for the following 20 minutes.
Spindle extension was incomplete, and it altered to a
curved structure around 39 minutes. The maximal spindle
length in the straight form was on average half that of the
fully extended spindle in wild-type cells. Anaphase spindle
dynamics is thus highly abnormal in cut8-563.
The decline of Cut2–GFP signal is delayed
Similar experiments were performed for cut2–gfp with the
native promoter chromosomally integrated in the wild type
and the cut8-563 mutant. As seen in the wild-type control
grown at 36°C (Figure 4a), Cut2–GFP was enriched in the
nucleus and the mitotic spindle as reported previously
[22]. The nuclear and short-spindle signals decayed very
rapidly within 1 or 2 minutes. In cut8-563 cells at 36°C
(Figure 4b), however, the decline of Cut2–GFP was greatly
delayed, in line with the delay in cyclin destruction. The
intense nuclear signal was the first to disappear, whereas
the weak spindle signal remained for a significantly long
time (~20 minutes in cut8-563 versus 4 minutes in wild
type). More than 20 living cells were observed, and essen-
tially the same delay in the time course of destruction was
obtained. Normal Cut8 is thus required for the destruction
of nuclear and spindle Cut2–GFP in anaphase.
To determine whether Cut8 has a genetic relationship
with Cut2, Cut2 was overproduced using the inducible
promoter REP81 (the weakest of three inducible nmt1
promoters available [39]) in cut8-563 at the permissive
temperature (data not shown). Mild overexpression of
Cut2 blocks colony formation of cut8-563 at 26°C (colony
formation of the wild type is not inhibited at this tempera-
ture). This result is consistent with the idea that normal
destruction of Cut2 at 36°C requires functional Cut8.
Destruction of Cdc13 and Cut2 in the G1 phase is inhibited
in cut8 mutant
Cut2 and Cdc13, both of which contain the destruction
box, an essential signal for polyubiquitination by cyclosome,
were efficiently degraded in G1-arrested cdc10 mutant
cells as they were the targets of cyclosome [21]. We inves-
tigated whether the level of these proteins changed in the
absence of Cut8 function. Cdc13 and Cut2 were already low
in cdc10-V50 mutant, even at the permissive temperature
1334 Current Biology Vol 10 No 21

Figure 4
(a) Wild type
Decay of Cut2–GFP in anaphase. (a,b) GFP-tagged Cut2 expressed
6 10 from the integrated gene with the native promoter is observed in a
background of (a) wild type and (b) cut8-563 at 36°C. Scale bars
0 7 20 represent 10 µm. (c) Single mutant cdc10-V50, double mutant cdc10-
V50 cut8-563 and double mutant cdc10-V50 cut9-665 were cultured
in EMM2 at 26°C to a cell concentration of 1 × 107/ml, followed by
4 8 30
transfer to nitrogen deficient EMM2-N medium after adjusting cell
concentration to 2 × 107/ml. The culture was then incubated at 26°C
5 9 for longer than 12 h. Resulting G1-arrested cells were then shifted to
the rich YPD medium at 36°C. Cells were collected at 1 h intervals for
(b) cut8-563 (upper panels) immunoblot analysis of Cdc13, Cut2 and Cdc2 using
11 20 specific antibodies and (lower panels) for DNA content analysis by
FACScan.

0 12 21
Cut8 is required for efficient destruction of Cdc13 and
4 13 22 Cut2 in G1-arrested cells.
5 14 23
Mitotic cyclin is polyubiquitinated in cut8 mutant cells
An important question was whether ubiquitin-mediated
6 15 24 proteolysis was generally impaired in cut8-563. We exam-
ined the level of ubiquitinated proteins in the single cut8
7 16 25 mutant and the double mutant mts2 cut8. Mouse mono-
clonal antibody against ubiquitin (1B3, MBL) was used to
8 17 26 detect the intense accumulation of ubiquitinated proteins
in cell extracts of proteasome single mutants mts2 and mts3
9 18 27 cultured at 36°C for 4 hours (Figure 5a). Accumulation
was strong in the double mutant mts2 cut8-563, but was
10 19 28 only slight in the single cut8-563 mutant after 4 hours at
(c) cdc10-V50 cdc10-V50 36°C. The accumulation, if any, of ubiquitinated proteins
cdc10-V50 cut8-563 cut9-665 in cut8 at 36°C should be only slight compared with that in
Async

Async

Async

Hours at 36ºC Hours at 36ºC Hours at 36ºC proteasome mutant cells.

0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
Cdc13 Secondly, we tested whether cyclosome-mediated polyu-
Cut2 biquitination of Cdc13 was normal in cut8-563 mutant cells.
The mutants mts2, mts2 cut8 and mts2 cut4 were used to
Cdc2 measure the level of ubiquitinated Cdc13. All carried the
multicopy plasmid encoding Cdc13-tagged with hemagglu-
cdc10-V50 cdc10-V50 cdc10-V50 tinin (HA) and histidine (His) (Cdc13–3HA–6His).
h cut8-563 h cut9-665 h
4 4 4 Extracts were prepared from cells cultured at 36°C for
3 3 3 0–6 hours. The His-tagged Cdc13 was isolated from extracts
2 2 2 by affinity chromatography and detected by anti-HA anti-
1 1 1 bodies (16B12, BAbCO). In both mts2 and mts2 cut8
0 0 0 mutants, polyubiquitinated Cdc13–HA was abundant after
1C2C 1C 2C 1C 2C 6 hours at 36°C, but could scarcely be detected in mts2 cut4,
Current Biology
in which cyclosome assembly is defective (Figure 5b).
These results strongly suggest that cyclosome-mediated
(0 hours, left-most panel in Figure 4c). If the cyclosome ubiquitination of Cdc13 occurs in cut8-563. Judging from
subunit mutation cut9-665 was combined with cdc10-V50, the band intensities obtained after 6 hours at 36°C,
the level of Cdc13 and Cut2 proteins considerably however, there might be a slight decrease in polyubiquiti-
increased after 4 hours at 36°C due to the block of polyu- nation in mts2 cut8 compared with mts2.
biquitination (right panel in Figure 4c). In the cdc10-V50
cut8-563 double mutant, destruction was also inhibited in On sucrose gradient centrifugation, Cut8 protein forms a
the G1-arrested cells after 4 hours at 36°C (middle panel broad peak around 4–15S (Figure 5c, middle panel), dis-
in Figure 4c). Evidence that cells were actually arrested in tinct from the peak of 20S cyclosome (top panel) and 26S
G1 is shown by the FACS analysis (bottom panels in proteasome (bottom panel). Nuc2 and Pad1 are the sub-
Figure 4c). These results strongly suggest that functional units of cyclosome and proteasome, respectively. Cut8
 Research Paper Control of 26S proteasome localization Tatebe and Yanagida 1335

Figure 5 Figure 6

h at
(a) (b) 0 6 (a)
36ºC Formaldehyde fix

mts2 cut8

mts2 cut4
cut8-563

mts2 cut8
mts2 cut4

mts2 cut8
mts2 cut4
DNA Cut8–GFP Mts3–Myc Merged
mts2

mts3
WT

mts2

mts2
h at
0 4 0 4 0 4 0 4 0 4 0 4 36ºC
kDa
Anti- –200
–93
Ub –66
–45
Anti- kDa
tubulin –116
(b) Methanol fix
–97 DNA Cut8–GFP Mts3–Myc Merged

–66

(c) 4.5S 16.5–19S

Anti-Nuc2
W 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Current Biology
4.5S 16.5–19S
Anti-HA Nuclear and nuclear envelope localization of Cut8. (a) The strain
(Cut8–3HA) containing integrated mts3–myc and cut8–gfp with the native
W 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
promoters was used for immunofluorescence microscopy.
4.5S 16.5–19S Formaldehyde-fixed cells were doubly stained using antibodies against
Anti-Pad1 Myc (red) and GFP (green). (b) The same strain as in (a) but fixed with
W 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
methanol. Scale bars represent 10 µm. Detailed results on Cut8
localization during the cell cycle are shown in Supplementary material.
Current Biology

Polyubiquitination occurs in cut8 mutant cells. (a) Mouse monoclonal

antibody against ubiquitin (anti-Ub; 1B3, MBL) was used to detect polyclonal anti-Sad1 antibodies [35] (for the SPBs) and
ubiquitinated proteins in cell extracts of wild type (WT), proteasome
mutants mts2 and mts3, and the double mutants mts2 cut8 and mts2
DAPI were used for counterstaining. Cells containing the
cut4 cultured at 36°C for 0–4 h. α-Tubulin is the loading control. two SPBs in the single nucleus were in early to mid-mitotic
(b) Cell extracts were prepared from three mutant strains, single mts2, stages. In methanol-fixed cells (preserving cytoskeletal
double mutants mts2 cut8 and mts2 cut4 cultured at 36°C for 0–6 h. structures), however, the nuclear envelope stain was more
These strains contained the multicopy plasmid carrying
cdc13–3ha–6his so that they could be collected by affinity
prominent (see Supplementary material). These results
chromatography (6His) and detected by immunoblotting (3HA). Mutant indicate that Cut8 is enriched in the nucleus, particularly at
cells were disrupted in the presence of guanidine-HCl to minimize the nuclear periphery.
enzymatic activities and the extracts were passed through Cobalt beads
to isolate His-tagged protein and then immunoblotted using anti-HA
To compare the localization of Cut8 and Mts3 (a 26S pro-
antibodies (16B12, BAbCO). (c) Fractions from sucrose gradient
centrifugation of wild type cells containing integrated Cut8–3HA were teasome subunit), formaldehyde- or methanol-fixed cells
immunoblotted using antibodies against Nuc2, Pad1 and HA. containing integrated cut8–gfp and mts3–myc with the
native promoter were doubly stained using antibodies
against GFP and Myc (Figure 6a,b). Their localization was
therefore does not form a stable complex with cyclosome very similar.
or 26S proteasome. However, two-hybrid interactions indi-
cated that Cut8 may form homodimeric or larger oligo- Localization of 26S proteasome was cytoplasmic in cut8
meric self-assembled structures (data not shown). mutant cells
To determine whether localization of proteasome was
Nuclear localization of Cut8 normal in cut8 mutant cells, immunofluorescence microscopy
Determination of the intracellular localization of Cut8 is was carried out using antibodies against Mts3–Myc and
important for understanding its role. The Myc-tagged cut8+ Mts4, subunits of proteasome. mts3–myc was integrated
gene was integrated into the chromosome with the native into the chromosome with the native promoter and its
promoter (see Materials and methods) and was functional, product was detected by anti-Myc antibodies (Figure 7a).
as the integrated strain grew normally at 36°C. Immunoflu- Polyclonal antibodies were used to visualize Mts4 ([25,27]
orescence micrographs of cells fixed by formaldehyde and Figure 7b). Localization of proteasome subunits to the
(which preserves nuclear structure) and stained by anti-Myc nucleus and the nuclear envelope appears to require func-
antibodies showed that Cut8 was enriched in the region tional Cut8. In cut8-563 mutant cells (Figure 7a), nuclear
corresponding to the nucleus throughout the cell cycle (see localization of the proteasome subunit was still detected
Supplementary material). To determine cell-cycle stages, at the permissive temperature (20°C) but was greatly
1336 Current Biology Vol 10 No 21

Figure 7 localization of proteasome subunits did not alter with the

temperature shift from 26–36°C.
(a) mts3–Myc mts3–Myc cut8–563
DNA Mts3–Myc DNA Mts-Myc
Discussion
We report here that fission yeast Cut8 protein is required
20ºC

for the normal rate of destruction of destruction-box con-

taining Cdc13 and Cut2 in the nucleus during mitotic
anaphase. As cut8 mutant cells are still viable at mitotic
36ºC 4 h 36ºC 2 h

metaphase but become inviable during anaphase, cut8

mutations are classified as anaphase-defective. However,
cut8 mutant displayed a more general physiological pheno-
type. Cut8 is heat inducible and is needed for normal resis-
tance to heat. The level of Cut8 protein increases roughly
10-fold after the shift to 36°C. The heat-inducible property
(b) Wild type ∆cut8 of Cut8 fits with its essential role at higher temperature.
DNA Mts4 DNA Mts4 The deletion mutant is heat sensitive: cell viability of ∆cut8
decreased after a transient heat shock. Cells of ∆cut8 were
20ºC

treated at 48°C for 0–30 minutes, followed by plating at

26°C. Viability of ∆cut8 was sharply decreased by treatment
36ºC 4 h 36ºC 2 h

at 48°C for 5–10 minutes, whereas the wild-type cells

(c) mts3–Myc
h at 36ºC mts3–Myc cut8–563
0 1 2 3 401234
Mts4
Mts3–Myc
Pad1
Tubulin Current Biology
26S proteasome is aberrantly localized in cut8 mutant cells. (a) Strains
were cultured at 20°C or 36°C for 2 and 4 h. Methanol-fixed cells
stained by DAPI were studied by immunofluorescence microscopy
using anti-Myc and anti-Mts4 antibodies. Wild-type and cut8-563
strains integrated with Myc-tagged mts3 were used, and Mts3–Myc
protein was detected by anti-Myc antibodies. (b) Wild-type and ∆cut8
strains were stained with polyclonal antibodies against Mts4 [25].
Scale bars represent 10 µm. (c) Strains as in (a) were cultured at
36°C for 0–4 h. Extracts were immunoblotted with antibodies against
Mts4, Myc (Mts3), and Pad1. Tubulin was the loading control.
diminished after 2 hours at the restrictive temperature
(36°C). In cut8 deleted cells (Figure 7b), only slight
nuclear localization was detected even at the permissive
temperature, and was no longer observed after 2 hours at
the restrictive temperature; the signals were distributed
broadly in cytoplasm. Immunoblotting indicated that the
level of proteasome subunits Mts4, Mts3–Myc, and Pad1
in cell extracts did not alter in cut8 mutant cells
(Figure 7c), indicating that Cut8 does not affect the level
of proteasome. Results identical to Mts3–Myc were
obtained for ts cut8-563 cells stained by antibodies against
Mts4. In wild-type cells, nuclear and nuclear envelope
remained viable after the same treatment. Cut8 is an unsta-
ble protein, judging from its rapid decay in the presence of
CYH. It is unstable at both low and high temperatures and
is possibly a substrate of proteasome, as it accumulates to
high levels in proteasome-mutant cells. Cut8 level appears
to be regulated post-transcriptionally, as the amount of
mRNA does not change at either high or low temperature.
Proteins similar to Cut8 are encoded in the genomes of
Saccharomyces cerevisiae (Dbf8p/Sts1p), Candida albicans
(Con4-3078) and Botrytis cinerea (CNS01A0C). Their
overall sequence identity is less than 25%, however. They
may have a similar function to Cut8, as the phenotype of
dbf8 is reminiscent of cut8 mutant [30]. Sts1p is involved in
protein destruction, as the defective phenotype of
budding yeast α-importin mutation srp1 in the ubiquitin-
mediated proteolytic pathway is suppressed by an
increased dosage of Sts1p [31]. No protein similar to Cut8
has yet been found in higher eukaryotic genomes.
A striking result presented here is that nuclear and nuclear
periphery localization of 26S proteasome is greatly dimin-
ished in cut8 mutant cells. In the ts mutant cells at 20°C, a
significant fraction of 26S proteasome is still at the nuclear
periphery, whereas at the restrictive temperature, or in the
deletion mutants, 26S proteasome is no longer enriched in
the nucleus and is dispersed in the cytoplasm. We propose
that the enrichment of 26S proteasome in the nucleus and
the nuclear periphery previously reported by Wilkinson
et al. [27,40] may be needed for efficient destruction of
polyubiquitinated Cdc13 and Cut2, and that such func-
tionally efficient intracellular positioning of 26S protea-
some requires the presence of functional Cut8.
This hypothesis explains many phenotypes of cut8 mutant
cells. Destruction of Cdc13 and Cut2, which were enriched
 Research Paper Control of 26S proteasome localization Tatebe and Yanagida
in the nucleus, was dramatically delayed at 33–36°C, prob-
ably owing to the dislocation of 26S proteasome. An
approximately four- to sixfold decrease in the rate of decay
in nuclear GFP fluorescence of Cut2 and Cdc13 was
observed during anaphase in cut8 mutant cells. Failure to
observe the delayed Cdc13 destruction in cut8 synchro-
nous culture was probably due to insufficient synchrony;
this period of delay (less than 10 min) might not be
detectable by low-resolution synchrony of liquid cultures.
The activity of 26S proteasome might not be affected,
but its preferred positioning was impaired as a result of
the lack of Cut8. Cdc13 is polyubiquitinated in cut8
mutant cells, as cyclosome is not affected. Mutant
mitotic chromosomes are hypercondensed because of
the delay in Cdc13 destruction, and anaphase chromo-
somes are not separated as normal because of the delay
in Cut2 destruction. In addition, spindle elongation in
anaphase is greatly delayed. This is probably due to the
strong opposing force generated by the non-separated
sister chromatids that result from the delay in Cut2
destruction. These pleiotropic and kinetically defective
phenotypes are unique to cut8 mutant and differ from
those of all known mutants defective in cyclosome or
proteasome. Cut8’s function may thus be to bring the
polyubiquitinated substrates of cyclosome to the site of
proteasome, resulting in a rapid destruction of these
nuclear substrates.
Consistent with apparently normal ubiquitination of
Cdc13 in cut8 mutant, the sucrose gradient centrifugation
pattern of cyclosome was normal in cut8 mutant cells (data
not shown). Wild-type Cut8 protein sediments in a broad
peak (4–15S) and is much smaller than 20S cyclosome or
26S proteasome. Immunoprecipitation experiments
showed that Cut8–HA did not co-precipitate with cyclo-
some subunits Cut4 or Cut9, nor with proteasome sub-
units Mts2 and Mts3 (data not shown). Therefore, Cut8 is
independent of and does not associate with these com-
plexes, at least in cell extracts. However, localization of
Myc-tagged Cut8 was nearly indistinguishable from that
of proteasome subunits. Because enriched nuclear local-
ization of Cut8 was maintained in mutant cells defective
in proteasome subunits (data not shown), proteasome did
not direct the localization of Cut8, but Cut8 controls the
localization of 26S proteasome. Note that Cut8 was dis-
pensable at 20°C, whereas proteasome was largely cyto-
plasmic in ∆cut8 mutant at 20°C. At low temperature, the
rates of cyclin and Cut2 destruction may be slowed to such
an extent that a much smaller than normal accumulation
of nuclear proteasome is functionally sufficient. Alterna-
tively, polyubiquitinated cyclin and Cut2 in mutant cells
at 26°C may be exported to the cytoplasm followed by
degradation by cytoplasmic proteasome; the mutant cells
are still able to progress into anaphase because mitotic
progression is slow.
1337
How Cut8 is implicated in recruiting 26S proteasome to
the nucleus and nuclear periphery remains to be deter-
mined. Cut8 possibly interacts directly with proteasome
subunits by tethering proteasome to the nuclear periph-
ery. Alternatively, Cut8 may indirectly affect localization
of proteasome through interacting with other proteins. We
are trying to identify a Cut8-binding protein(s), which
might be conserved in higher eukaryotes. One such candi-
date is Cek1 kinase, the strongest multicopy suppressor
for cut8-563 [29]. In the genome of fission yeast, there is
another sequence (SPBC725.06c) similar to Cek1. It is of
considerable interest to examine whether the double gene
knockouts are lethal, and whether Cut8 and Cek1 directly
interact. Cek1 kinase is evolutionarily conserved: RIM15
in Saccharomyces cerevisiae [41], LATS (large tumor sup-
pressor) or WTS (warts) in Drosophila, Lats1 in human and
mouse [42–45]. In budding yeast, RIM15 is a downstream
effector of protein kinase A [46]. As Cek1 kinase could
suppress the ts phenotype of cut8 null mutant, Cut8 might
be an upstream positive regulator of Cek1 kinase, which
could subsequently control localization of proteasome by
protein phosphorylation. Cut8 may be a substrate of
nuclear proteasome as it is very unstable and accumulates
in proteasome mutant cells. Cut8 may be a feedback
sensor for the amount of proteasome locating to the
nucleus. If nuclear proteasome becomes sufficient or
excess, Cut8 may be degraded. If nuclear proteasome
becomes deficient, as in mutant cells, Cut8 may accumu-
late, as it will not be degraded. In short, we report here
that Cut8 is a hitherto unknown evolutionarily conserved
regulator for anaphase-promoting proteolysis, maintaining
the localization of 26S proteasome close to polyubiquiti-
nated mitotic cyclin and Cut2 securin during mitosis.
Supplementary material
Supplementary material including full Materials and methods and details
of Cut8 localization during the cell cycle is available at http://current-
biology.com/supmat/supmatin.htm.
Acknowledgements
We thank Colin Gordon for polyclonal antibodies against Mts4. This study
was supported by the CREST Research Project of the Japan Science and
Technology Corporation (JST).
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