Molecular Biology of the Cell
Vol. 9, 2803–2817, October 1998
A Late Mitotic Regulatory Network Controlling Cyclin
Destruction in Saccharomyces cerevisiae
Sue L. Jaspersen, Julia F. Charles, Rachel L. Tinker-Kulberg, and
David O. Morgan*
Department of Physiology, University of California, San Francisco, California 94143-0444
Submitted May 7, 1998; Accepted July 13, 1998
Monitoring Editor: Marc W. Kirschner
Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin
complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is
regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the
budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants,
including cdc15, cdc5, cdc14, dbf2, and tem1, arrest in anaphase with a phenotype similar
to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the
possibility that the products of these genes are components of a regulatory network that
governs cyclin proteolysis. We identified a complex array of genetic interactions among
these mutants and found that the growth defect in most of the mutants is suppressed by
overexpression of SPO12, YAK1, and SIC1 and is exacerbated by overproduction of the
mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in
cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the
anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We
conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC
in late mitosis but are not required for maintenance of that activity in G1.
Progression through the eukaryotic cell division cycle
is governed by oscillations in the activities of cyclin-
dependent kinases (CDKs). Entry into mitosis is initi-
ated by mitotic CDK–cyclin complexes, including the
Cdc2–cyclin B complex in vertebrates and the Cdc28–
Clb complex of Saccharomyces cerevisiae (King et al.,
1994; Nasmyth, 1996; Morgan, 1997). Exit from mitosis
requires CDK inactivation, which is accomplished pri-
marily by ubiquitin-dependent destruction of the cy-
clin subunit (Murray, 1995; King et al., 1996; Hoyt,
1997). The importance of cyclin destruction for exit
from mitosis is underscored by the observation in a
wide range of eukaryotes that overexpression of non-
destructible forms of mitotic cyclin causes cells to
arrest in anaphase (Murray et al., 1989; Gallant and
Nigg, 1992; Holloway et al., 1993; Surana et al., 1993;
Rimmington et al., 1994; Sigrist et al., 1995; Yamano et
al., 1996). Under some conditions, however, additional
CDK inactivation mechanisms allow mitotic exit in the
absence of complete cyclin destruction (Minshull et al.,
1996; Toyn et al., 1996; Schwab et al., 1997; Visintin et
al., 1997; Jin et al., 1998).
Mitotic cyclin destruction requires the covalent at-
tachment of a chain of ubiquitin molecules to a region
near the amino terminus of the cyclin protein (Glotzer
et al., 1991). Ubiquitination of cyclin, like that of other
proteins, begins with the transfer of ubiquitin from the
ubiquitin-activating enzyme (E1) to a ubiquitin-conju-
gating enzyme (E2) (Hershko et al., 1994; King et al.,
1995; Hochstrasser, 1996). The E2, together with a
ubiquitin ligase (E3), transfers the ubiquitin onto the
cyclin substrate. The E3 required for cyclin ubiquiti-
nation is a multisubunit protein complex known as the
anaphase-promoting complex (APC) or cyclosome
(Irniger et al., 1995; King et al., 1995; Sudakin et al.,
1995; Peters et al., 1996; Zachariae et al., 1996, 1998;
Hwang and Murray, 1997; Kramer et al., 1998; Yu et al.,
1998). Several lines of evidence suggest that the APC
mediates the key regulatory step in cyclin destruction
(Hershko et al., 1994; King et al., 1995; Sudakin et al.,
* Corresponding author. E-mail address: email@example.com.
© 1998 by The American Society for Cell Biology2803
In addition to being required for the ubiquitination
of mitotic cyclins, the APC also catalyzes the ubiquiti-
nation of other mitotic regulatory proteins. APC-de-
pendent degradation of the Pds1 protein of S. cerevisiae
(or Cut2 of Schizosaccharomyces pombe) is required for
progression from metaphase to anaphase (Cohen-Fix
et al., 1996; Funabiki et al., 1996); thus, mutation or
inhibition of the APC causes a metaphase arrest and
not the anaphase arrest that results from overexpres-
sion of nondegradable cyclin (Holloway et al., 1993;
Irniger et al., 1995; Cohen-Fix et al., 1996; Zachariae et
al., 1998). Other APC substrates have also been iden-
tified in S. cerevisiae, including the microtubule-asso-
ciated protein Ase1, whose destruction is necessary for
efficient disassembly of the mitotic spindle (Juang et
al., 1997). The APC is also required for the destruction
of the WD40 repeat protein Cdc20 and the Polo-re-
lated protein kinase Cdc5 (Charles et al., 1998; Prinz et
al., 1998; Shirayama et al., 1998).
Studies of APC regulation have focused almost ex-
clusively on its cyclin–ubiquitin ligase activity, which
increases in metaphase or anaphase and remains high
throughout G1 (Amon et al., 1994; King et al., 1995;
Lahav-Baratz et al., 1995; Sudakin et al., 1995; Brandeis
and Hunt, 1996; Zachariae and Nasmyth, 1996;
Charles et al., 1998). In higher eukaryotes, activation of
the APC toward cyclin substrates is initiated by Cdc2–
cyclin B (Felix et al., 1990; Lahav-Baratz et al., 1995;
Sudakin et al., 1995), whereas in budding yeast there is
evidence that Cdc28-associated kinase activity inhibits
cyclin ubiquitination by the APC (Amon, 1997). Re-
cent studies have also implicated other protein kinases
in APC regulation: Polo-related kinases (Plk1 in mam-
mals, Plx1 in Xenopus, and Cdc5 in budding yeast)
promote APC activation, whereas in mammals and
fission yeast protein kinase A (PKA) appears to inhibit
cyclin-directed APC activity (Yamashita et al., 1996;
Charles et al., 1998; Descombes and Nigg, 1998; Kotani
et al., 1998; Shirayama et al., 1998). Little is known
about how these various regulatory influences are
integrated to provide the correct timing of cyclin de-
To ensure the proper order of mitotic events, the
APC may also be regulated at the level of substrate
specificity. APC-dependent ubiquitination of proteins
involved in sister chromatid cohesion (Pds1) occurs at
the metaphase-to-anaphase transition, whereas mi-
totic cyclins (e.g., Clb2), Cdc20, and Ase1 remain sta-
ble until the end of anaphase (Pellman et al., 1995;
Cohen-Fix et al., 1996; Zachariae et al., 1996; Shirayama
et al., 1998). Recent work suggests that this additional
level of regulation may be conferred in S. cerevisiae by
Cdc20 and Hct1/Cdh1 (Schwab et al., 1997; Visintin et
al., 1997; Lim et al., 1998; Shirayama et al., 1998). Over-
expression of CDC20 results in APC-dependent desta-
bilization of Pds1 but has little effect on the destruction
of Ase1 and Clb2; cdc20 mutants arrest in metaphase
with stable Pds1 (Sethi et al., 1991; Visintin et al., 1997;
Shirayama et al., 1998). Similar evidence suggests that
HCT1 promotes the destruction of Clb2 and Ase1 but
not that of Pds1 (Schwab et al., 1997; Visintin et al.,
1997). The regulation of these putative specificity fac-
tors is not well understood, although recent studies
suggest that Cdc20 may be regulated by multiple
mechanisms: its levels increase during mitosis, and its
function may be negatively regulated in response to
spindle damage (Hwang et al., 1998; Kim et al., 1998;
Prinz et al., 1998; Shirayama et al., 1998).
In S. cerevisiae, various genetic screens have led to
the identification of a group of mutants that arrest in
late anaphase with large buds, an elongated spindle,
and separated DNA (Hartwell et al., 1973; Johnston
and Thomas, 1982; Johnston et al., 1990; Molero et al.,
1993; Shirayama et al., 1994a,b; Luca and Winey, 1998).
This arrest phenotype is similar to that observed in
yeast overexpressing a nondegradable form of Clb2,
raising the possibility that the late mitotic gene prod-
ucts are required for the inactivation of Cdc28–Clb
complexes (Surana et al., 1993). Interestingly, the late
mitotic mutants all encode potential regulatory pro-
teins, including the protein kinases Cdc15 and Dbf2,
the Polo-like kinase Cdc5, the protein phosphatase
Cdc14, and the Ras-like GTPase Tem1 (Johnston et al.,
1990; Schweitzer and Philippsen, 1991; Wan et al.,
1992; Kitada et al., 1993; Shirayama et al., 1994b). Re-
cent studies suggest that Cdc5 promotes mitotic exit
by stimulating APC activity toward cyclins (Charles et
al., 1998; Shirayama et al., 1998), and it seems likely
that the other late mitotic proteins also contribute to
the control of cyclin destruction.
In the present work, we address the hypothesis that
the proteins encoded by the late mitotic gene family
form a regulatory network governing Cdc28 inactiva-
tion in late mitosis. In support of this hypothesis, we
find that several late mitotic mutants display an ex-
tensive array of genetic interactions. These mutants
arrest with elevated levels of Clb2, decreased amounts
of Pds1, and negligible cyclin-specific APC activity.
We therefore conclude that the proteins encoded by
the late mitotic genes promote mitotic exit by activat-
ing the cyclin–ubiquitin ligase activity of the APC.
MATERIALS AND METHODS
Yeast Strains and Plasmids
All strains (Table 1) were derivatives of W303 (MATa ade2-1 trp1-1
leu2-3, 112 his3-11, 15 ura3-1 can1-100). Strains were made cogenic by
backcrossing at least four times to AFS34 and were made bar1 by a
subsequent cross to AFS92 (a gift from A. Straight, University of
California, San Francisco, CA) or were constructed in AFS92 using
a pop-in, pop-out strategy (Guthrie and Fink, 1991).
Multicopy plasmids carrying the genes encoded by the late mi-
totic mutants were cloned as follows. pSJ107 (pRS426-CDC15HA)
was made by cloning the hemagglutinin (HA) epitope into a PstI site
generated by oligonucleotide mutagenesis at the stop codon of a
S.L. Jaspersen et al.
Molecular Biology of the Cell2804
4-kb genomic CDC15 fragment. pJC29 (pRS426-HACDC5) was cre-
ated by inserting the HA epitope into an NcoI–EcoRI site generated
at the start codon of CDC5. The construct contains 300 bp of 5?
sequence and 500 bp of 3? sequence in addition to the CDC5 open
reading frame. pPD.2 (pRS426-CDC14HA3) contains 564 bp of pro-
moter sequence and the open reading frame of CDC14 ligated in
frame to a triple HA (HA3) tag in pRS426. To construct pSJ57
(pRS426-HA3DBF2), the DBF2 open reading frame and 380 bp of 3?
sequence were ligated in frame into a 2? plasmid containing the
DBF2 promoter sequence and a triple HA tag. Finally, pSJ56
(pRS426-TEM1HA3) was generated by fusing the 3? end of the
TEM1 open reading frame (accompanied by 300 bp of 5? sequence)
to an HA3 tag in pRS426. All of these constructs were shown to
complement the appropriate temperature-sensitive mutant in single
copy and on the multicopy plasmid.
Strains containing GAL-CLB2-URA3 were obtained from crosses
to ADR58 (a gift from A. Rudner, University of California, San
Francisco, CA; Hwang and Murray, 1997). Wild-type and mutant
strains containing PDS1HA-URA3 were obtained from crosses to
ADR1002, a wild-type strain containing PDS1HA-URA3 integrated
at the PDS1 locus (a gift from D. Koshland, Carnegie Institution of
Washington, Baltimore, MD; Cohen-Fix et al., 1996). To construct
pSJ50 (GAL-CLB2HA) and pRTK-C1 (GAL-PDS1HA), the open read-
ing frames of CLB2 and PDS1 were amplified from genomic DNA
by PCR and cloned into a pRS304-based plasmid (Sikorski and
Hieter, 1989) containing the GAL1/10 promoter and a single C-
terminal HA tag. Strains containing GAL-CLB2HA or GAL-PDS1HA
were made by digesting pSJ50 and pRTK-C1 with Bsu36I for inte-
gration at TRP1.
All CDC15 constructs were derived from a 4-kb PvuII genomic
fragment containing the CDC15 gene (a gift from A. Rudner;
Schweitzer and Philippsen, 1991). To create SLJ23, CDC15 was
tagged at the carboxyl terminus with an HA3 tag and integrated
into AFS92 at the CDC15 locus using a pop-in, pop-out strategy
(Guthrie and Fink, 1991). pSJ103 (pRS426-CDC15HA3) was made by
subcloning the CDC15HA3 genomic fragment into pRS426. A ki-
nase-deficient mutant CDC15 (pSJ59) was generated by site-directed
mutagenesis of pSJ103 using the following oligonucleotide to
change lysine 54 to a leucine (K54L): 5?-GTACACGACCTCTA-
GAATTGCCACGAC-3?. The wild-type HA3-tagged CDC15 con-
structs fully complement the growth defects of cdc15-2 and cdc15?.
The K54L mutant does not complement either strain (our unpub-
Standard protocols were used for yeast transformation, genetic
analysis, and cell propagation (Guthrie and Fink, 1991). To arrest
temperature-sensitive strains, cells were grown at 23°C to midlog
phase and arrested with 1 ?g/ml ?-factor or 15 ?g/ml nocodazole
at 23°C for 3.5 h or by shifting cells to 37°C for 3.5 h. During the last
30 min of the arrests, ?-factor- and nocodazole-arrested cultures
were shifted to 37°C in the continued presence of the arresting
agent. To measure the turnover of Pds1 and Clb2, cells were grown
in YP/2% raffinose to an OD600of 0.3 and arrested. Expression from
the GAL promoter was induced by the addition of galactose to 2%
for 30 min. Transcription and translation were then repressed with
2% dextrose and 10 ?g/ml cycloheximide, and cells were harvested
at the indicated times. Arrest and release from ?-factor were done
by growing cells at 30°C to an OD600of 0.3. ?-Factor (1 ?g/ml) was
added for 3 h, cells were pelleted, washed three times in fresh
media, and released in fresh media at 30°C.
High-Copy Suppressor Screen
To screen for high-copy suppressors of cdc15-2, SLJ02 was trans-
formed with a URA3-marked GAL-cDNA library (a gift from Aaron
Straight, University of California; Liu et al., 1992). Transformants
were selected on SC-ura/dextrose plates at 23°C. Cells were washed
off the plates and resuspended in SC-ura/galactose-raffinose media
and allowed to grow for 6 h at 23°C. The culture was then diluted
and plated onto YP/galactose-raffinose plates at 37°C to select for
suppressors. From ?25,000 SC-ura/dextrose transformants, 312 col-
onies formed on YP/galactose-raffinose at 37°C. The putative sup-
Table 1. Yeast strains
MATa ade2-1 can1-100 ura3-1 leu2-3,112, his3-11,15, trp1-1
MATa bar1 cdc16-1
MATa bar1 cdc15-2
MATa bar1 cdc5-1
MATa bar1 cdc14-1
MATa bar1 dbf2-2
MATa bar1 tem1-3
MATa bar1 ura3?GAL-CLB2-URA3 (pDK27)
MATa bar1 pds1?PDS1HA-URA3
MATa bar1 cdc16-1 pds1?PDS1HA-URA3
MATa bar1 cdc15-2 pds1?PDS1HA-URA3
MATa bar1 cdc5-1 pds1?PDS1HA-URA3 trp1?LacO-TRP1
MATa bar1 cdc14-1 pds1?PDS1HA-URA3
MATa bar1 dbf2-2 pds1?PDS1HA-URA3
MATa bar1 tem1-3 pds1?PDS1HA-URA3
MATa bar1 cdc15-2 trp1?GAL-CLB2HA-TRP1 (pSJ50)
MATa bar1 cdc15-2 trp1?GAL-PDS1HA-TRP1 (pRTK-C1)
MATa bar1 cdc15?CDC15HA3
aAll strains are in a W303 background.
bConstructed in AFS92 as described (Shirayama et al., 1994b).
Regulation of Cyclin Destruction
Vol. 9, October 19982805
pressors were retested for growth at 37°C. Growth at 37°C was then
shown to be plasmid and galactose dependent for 189 of the sup-
Ninety-two suppressors were chosen for further analysis. Restric-
tion mapping and sequence analysis of 12 cDNAs revealed that
SPO12 or SIC1 were responsible for suppression. To allow rapid
analysis of the remaining suppressors, whole-colony PCR was done
using a primer complementary to the GAL promoter and a primer in
the SPO12 gene or the SIC1 gene. In two independent PCR analyses,
71 of the suppressors were found to be SPO12, and 15 of the
suppressors were SIC1. Sequencing of plasmids rescued from the six
remaining suppressors revealed that three were an identical fusion
with the kinase domain of YAK1 (594 bp downstream of the start
codon), one was CDC15, and two were YGR230W, an open reading
frame with homology to SPO12 on chromosome VII. All of these
plasmids retested in their ability to restore growth to cdc15-2 at
Lysate Preparation and Immunoblotting
Yeast lysates were prepared by resuspending cells in 3–5 pellet vol
of ice cold LLB (50 mM HEPES-NaOH, pH 7.4, 75 mM KCl, 50 mM
NaF, 50 mM ?-glycerophosphate, 1 mM EGTA, 0.1% NP40, 1 mM
dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride, 2 ?g/ml
aprotinin, 1 ?g/ml leupeptin, and 1 ?g/ml pepstatin) and lysing by
mechanical disruption in a Beadbeater (Biospec Products, Bartles-
ville, OK). Lysates were clarified by centrifugation at 14,000 ? g for
10 min at 4°C. Protein concentrations of extracts were determined
with the Bio-Rad (Hercules, CA) protein assay, using BSA as a
For immunoblots, equal amounts of total protein were loaded on
SDS-PAGE gels, and proteins were electrophoretically transferred
to nitrocellulose. Clb2 and Cdc28 proteins were detected with af-
finity-purified polyclonal antibodies as described (Gerber et al.,
1995; Charles et al., 1998). For detection of HA-tagged proteins, the
mouse monoclonal antibody 12CA5 was used as previously de-
scribed (Gerber et al., 1995). Sic1 immunoblots were performed with
a 1:1000 dilution of ?-Sic1 polyclonal antibodies (a gift from M.
Tyers, University of Toronto, Toronto, Canada; Skowyra et al., 1997).
To measure Cdc15-associated kinase activity, cell lysates (250 ?g–1
mg) were incubated with 3 ?g of 12CA5 and protein A-Sepharose
(Sigma, St. Louis, MO) for 2 h at 4°C. Immune complexes were
washed three times in LLB and once in kinase buffer (50 mM
HEPES-NaOH, pH 7.4, 5 mM MgCl2, 2.5 mM MnCl2, 5 mM ?-glyc-
erophosphate, and 1 mM DTT) and incubated for 10 min at 23°C in
a 20-?l reaction mixture containing 20 ?M ATP, 2 ?g of myelin basic
protein (MBP), and 5 ?Ci of [?-32P]ATP (3000 mCi/mmol) in kinase
buffer. Reaction products were analyzed on 12% SDS-PAGE gels
followed by autoradiography. Clb2-associated kinase activity was
measured as described (Gerber et al., 1995) by immunoprecipitating
Clb2 from 100 ?g of yeast lysate with 0.3 ?g of affinity-purified
anti-Clb2 antibody and protein A-Sepharose for 2 h at 4°C.
In Vitro Ubiquitination Assay
Ubiquitin ligase activity of the APC was measured as described
(Charles et al., 1998). Briefly, the APC was immunoprecipitated with
12CA5 monoclonal antibodies from 500 ?g of yeast lysate (contain-
ing Cdc27HA, a gift from P. Hieter, University of British Columbia,
Vancouver, Canada; Lamb et al., 1994). Immune complexes were
washed three times in LLB, once in high-salt QA (20 mM Tris-HCl,
pH 7.6, 250 mM KCl, 1 mM MgCl2, and 1 mM DTT), and twice in
buffer QA (20 mM Tris-HCl, pH 7.6, 100 mM KCl, 1 mM MgCl2, and
1 mM DTT) and were then incubated for 15 min at 23°C in a 15-?l
reaction containing 3.5 pmol of Uba1, 47 pmol of Ubc4, 1 mM ATP,
20 ?g of bovine ubiquitin (Sigma), and 0.25 ?l of125I-labeled sea
urchin (13–91) cyclin B1 in buffer QA. Reaction products were
electrophoresed on 7.5–15% gradient gels and analyzed for ubiq-
uitin conjugates by autoradiography with the Bio-MaxMS system
(Eastman Kodak, Rochester, NY).
Genetic Interactions among Late Mitotic Mutants
The similar anaphase arrest phenotype of cdc15-2,
cdc5-1, cdc14-1, dbf2-2, and tem1-3 mutants suggests
that the proteins encoded by these genes may have
overlapping functions in the control of mitotic exit.
Consistent with this possibility, a variety of previous
studies have revealed that overexpression of some late
mitotic genes results in growth of other late mitotic
mutants at the nonpermissive temperature (Kitada et
Table 2. Multicopy suppression of late mitotic mutants
Effect of multicopy plasmid on colony growth at 37°Ca
VectorCDC15HA HACDC5CDC14HA3 HA3DBF2 TEM1HA3
aWild-type and mutant strains were transformed with the indicated gene on a 2? plasmid carrying a URA3 marker (pRS426; Sikorski and
Hieter, 1989) and transformants were selected on SC-ura plates at 23°C. Suppression of each mutant arrest was analyzed by streaking for
single colonies on SC-ura plates at 37°C. ?, Robust growth on SC-ura plates at 37°C; ?/?, weak growth on SC-ura plates at 37°C; ?, no
growth on SC-ura plates at 37°C. In some cases, similar results have been obtained in previous studies, as indicated by the superscripts.
bKitada et al. (1993).
cResult differs from that previously reported; previous work used the cdc15-1 and dbf2-1 alleles in a different strain background.
dShirayama et al. (1996).
eShirayama et al. (1994b).
S.L. Jaspersen et al.
Molecular Biology of the Cell 2806
al., 1993; Shirayama et al., 1994b, 1996). We extended
these studies by carrying out a systematic high-copy
suppression analysis of the major late mitotic mutants
in a common strain background. Multicopy plasmids
carrying CDC15, CDC5, CDC14, DBF2, and TEM1
were each sufficient to rescue the temperature-sensi-
tive growth defects of many of the late mitotic mutants
(Table 2). The tem1-3 mutant was suppressed by all of
the late mitotic genes except DBF2, whereas cdc14-1
and dbf2-2 grew only when their wild-type genes were
supplied. Interestingly, CDC14 was unique in its abil-
ity to restore growth to the majority of mutants at
Further evidence that the late mitotic mutants are
functionally linked is that many double mutants are
inviable (Table 3). In addition, most of the viable dou-
ble mutants exhibited growth defects and reduced
viability at semipermissive temperatures (Table 3). In
particular, the cdc5-1 and tem1-3 mutants exhibited
synthetic interactions with all other late mitotic family
members examined. In contrast, the cdc14-1 mutant
had no obvious synthetic interaction with cdc15-2 and
dbf2-2 mutants and only minor interactions with cdc5-1
and tem1-3. These genetic interactions suggest that the
proteins encoded by the late mitotic mutants work
together to coordinate exit from mitosis.
High-Copy Suppressors of cdc15-2
To identify additional genes involved in control of exit
from mitosis, we performed a screen for GAL-driven
cDNAs that allowed growth of a cdc15-2 strain at 37°C
sors of cdc15-2. Four of the
cdc15-2 high copy suppressors
(GAL-CDC15, GAL-SIC1, GAL-
cdc15-2 strain (SLJ02), grown in
YP/raffinose to midlog phase,
serially diluted fivefold, and
spotted onto YP/dextrose or
Plates were incubated at 23 or
37°C for 2.5 d.
Table 3. Synthetic interactions between late mitotic mutants
Maximum permissive temperature of double mutants (°C)
cdc15-2 cdc5-1 cdc14-1dbf2-2tem1-3
Heterozygous diploids were generated by crossing single haploid mutant strains. Diploids were sporulated for 4 d at 23°C. For each cross,
20–40 tetrads were dissected and analyzed. Dead indicates that no viable double-mutant spores were obtained from nonparental ditype
tetrads at 23°C. The maximum permissive temperature for growth of viable double mutants was determined by comparing growth with that
of the single-mutant parents at 23, 30, 33, and 37°C. The data are presented twice for clarity.
Regulation of Cyclin Destruction
Vol. 9, October 19982807
(Figure 1). Other than GAL-CDC15, the most robust
suppressor of cdc15-2 was GAL-SPO12, which also
suppressed the growth arrest of a complete deletion of
CDC15 (our unpublished data) and has previously
been shown to suppress the growth defect in dbf2 and
dominant CDC15 mutants (Parkes and Johnston, 1992;
Shirayama et al., 1996). The SPO12 locus encodes a
protein of unknown function; mutation or deletion of
this gene causes diploid cells to skip a meiotic division
and produce dyad spores (Klapholz and Esposito,
1980; Malavasic and Elder, 1990). Disruption of SPO12
has minor effects on progression through mitosis (Ma-
lavasic and Elder, 1990; Parkes and Johnston, 1992).
We also found that growth of cdc15-2 was restored at
37°C upon overexpression of a putative open reading
frame, YGR230W, that encodes a protein with homol-
ogy to Spo12. The function of this protein is unknown.
A fourth suppressor contained a 3? fragment of the
YAK1 gene. YAK1 encodes a nonessential protein with
homology to protein kinases (Garrett and Broach,
1989), and our suppressor encoded an amino-termi-
nally truncated version of Yak1 that is initiated at
methionine 233, several residues before the beginning
of the kinase domain. Mutants in YAK1 were origi-
nally identified as extragenic suppressors of ras2 mu-
tants (Garrett and Broach, 1989). RAS2 encodes a
GTPase involved in activating adenylate cyclase, the
enzyme responsible for cAMP production in yeast
(Toda et al., 1985). Subsequent genetic studies sug-
gested that Yak1 antagonizes the effects of the cAMP-
dependent kinase PKA (Garrett et al., 1991; Hartley et
al., 1994; Ward and Garrett, 1994). Its ability to sup-
press cdc15-2 is therefore consistent with previous
studies showing that the anaphase arrest in cdc15 mu-
tants is accompanied by high levels of cAMP, and
decreasing cAMP levels alleviates the cdc15-2 defect at
37°C (Spevak et al., 1993).
Finally, growth of cdc15-2 cells was partially re-
stored at 37°C by GAL-driven overexpression of SIC1
(Figure 1), which encodes an inhibitor of Cdc28–Clb
kinases and has previously been reported to suppress
cdc15 mutants when overexpressed (Mendenhall,
1993; Schwob et al., 1994; Toyn et al., 1996). Growth of
cdc15-2 cells was rescued even more effectively by
SIC1 on a 2? plasmid (our unpublished data). The
ability of SIC1 to suppress the growth defect in the
cdc15 mutant is of particular interest, because it sug-
gests that the primary defect in this mutant is an
inability to inactivate Cdc28.
Overexpression of SIC1, SPO12, and Truncated
YAK1 Allows Growth of Late Mitotic Mutants
If Cdc15 cooperates with the other late mitotic pro-
teins to regulate exit from mitosis, then high-copy
suppressors of cdc15-2 should also allow growth of the
other mutants at 37°C. Indeed, overexpression of SIC1
partially restored growth to all of the late mitotic
mutants at 37°C (Table 4) (Donovan et al., 1994; Toyn
et al., 1996; Charles et al., 1998). SPO12 overexpression
resulted in robust growth of cdc15-2, cdc5-1, dbf2-2,
and tem1-3 at 37°C but did not restore growth to
cdc14-1 cells (Table 4) (Parkes and Johnston, 1992;
Toyn and Johnston, 1993; Shirayama et al., 1996). Sim-
ilarly, overproduction of truncated Yak1 partially res-
cued the temperature-sensitive growth defect of all
late mitotic mutants except cdc14-1 (Table 4).
Overexpression of CLB2 Is Toxic in Late Mitotic
Mutants defective in cyclin destruction should be sensi-
tive to increased production of cyclin protein. Overpro-
duction of Clb2 is known to be toxic in cdc5-1 and tem1-3
mutants at the permissive temperature but has no effect
Table 4. Suppression of late mitotic mutants by GAL-cDNAs
Effect of GAL-cDNA on colony growth at 37°Ca
CDC15 SIC1SPO12 YAK1?Nb
aWild-type and mutant strains were transformed with the indicated GAL-cDNA plasmids, and transformants were selected on SC-ura/
dextrose at 23°C. To analyze the ability of the GAL-cDNA to restore growth of the mutant at 37°C, cultures were grown overnight to OD600
? 1.0 and spotted onto YP/galactose-raffinose plates in a series of six fivefold dilutions. After 3 d at 37°C, growth on the plates was scored
as follows: ???, robust growth (at the fourth dilution and beyond); ??, growth at the third dilution; ?, growth at the first and second
dilution; ?/?, growth of only the first dilution; ?, no growth above background.
bThe YAK1 clone contains only the 3? end of the gene (see MATERIALS AND METHODS for details).
cGrowth was very poor, mainly microcolonies.
S.L. Jaspersen et al.
Molecular Biology of the Cell 2808
on growth of wild-type strains (Shirayama et al., 1994b;
Charles et al., 1998). In the present work, we found that
overexpression of CLB2 also prevents growth of cdc14-1
strain overexpressing CLB2 was able to grow at the
permissive temperature, the excess CLB2 was lethal in
this mutant at a semipermissive temperature (Figure 2).
These synthetic interactions are consistent with the pos-
sibility that the late mitotic proteins act as positive reg-
ulators of cyclin destruction.
Clb2 Destruction Is Reduced in Late Mitotic
The late mitotic mutants arrest in anaphase with
separated chromosomes, suggesting that mutants in
these genes may be defective in the destruction of
cyclins but not that of Pds1 (Hartwell et al., 1973;
Kitada et al., 1993; Surana et al., 1993; Shirayama et
al., 1994b; Toyn and Johnston, 1994). We therefore
compared Clb2 and Pds1 protein levels in the late
mitotic mutants at their arrest point. As previously
reported, cdc15-2 and cdc5-1 mutants arrest with
high Clb2 levels, whereas only a small fraction of
the Pds1 protein remains (Figure 3A) (Cohen-Fix et
al., 1996; Charles et al., 1998; Shirayama et al., 1998).
Similarly, cdc14-1, dbf2-2, and tem1-3 mutants all
arrest with mitotic levels of Clb2 and low levels of
Pds1 (Figure 3A), supporting the notion that the late
mitotic mutants are defective specifically in the de-
struction of mitotic cyclins.
To directly measure the stability of Clb2 and Pds1,
we constructed cdc15-2 mutant strains containing an
integrated copy of CLB2 or PDS1 under the control
of the GAL promoter. Each protein was fused to a
single copy of an HA epitope tag at its carboxyl
terminus. The half-lives of both proteins at various
enhances the growth defect in
tem1-3 mutants. Mutant strains
containing GAL-CLB2 were gen-
erated by crossing to ADR58.
Progeny from tetratype spores
were grown to midlog phase in
YP/raffinose at 23°C, serially di-
luted fivefold, and spotted onto
YP/dextrose or YP/galactose-
raffinose plates. Plates were in-
cubated for 2 d at 30°C or 3 d at
Regulation of Cyclin Destruction
Vol. 9, October 1998 2809
points in the cell cycle were determined by inducing
their expression with galactose and then repressing
transcription and translation with dextrose and cy-
cloheximide, respectively. Clb2HA and Pds1HA
were competent for destruction, as both were highly
unstable in a G1 arrest (Figure 3B). The rapid deg-
radation of both proteins in G1 was dependent on
APC function (our unpublished data). In cdc15-2
cells arrested in metaphase with the microtubule-
depolymerizing drug nocodazole, Pds1 and Clb2
proteins were both stable (Figure 3B). In cdc15-2
cells arrested in late anaphase, Clb2 was greatly
stabilized relative to G1 cells (Figure 3B). In con-
trast, the majority of the Pds1 protein was rapidly
degraded at the mutant arrest point (Figure 3B),
although a significant fraction of the protein re-
mained stable. This pool of stable Pds1 was larger
than that observed in our studies of endogenous
Pds1 (Figure 3A), suggesting that it represents an
artifact of Pds1 overproduction in late mitotic cells.
Cyclin Ubiquitination by the APC Is Defective in
Late Mitotic Mutants
To determine whether decreased Clb2 destruction in the
late mitotic mutants is due to a defect in the cyclin-
specific proteolysis machinery, we measured the cyclin–
ubiquitin ligase activity of the APC in vitro. We used a
recently described assay (Charles et al., 1998) in which
the APC is immunoprecipitated from yeast extracts with
antibodies against an epitope-tagged APC subunit, in
this case Cdc27HA expressed on a plasmid under the
control of its own promoter (Lamb et al., 1994). The
immunoprecipitated APC is incubated with purified
yeast E1 (Uba1), E2 (Ubc4), bovine ubiquitin, ATP, and
125I-labeled amino terminus of sea urchin cyclin B1
(Glotzer et al., 1991; Holloway et al., 1993). The conjuga-
tion of ubiquitin to the cyclin amino terminus is assessed
by PAGE of reaction products.
Mutant strains were arrested in late anaphase by
shifting asynchronous cultures to 37°C until 80–95%
stabilized in late mitotic mutants.
(A) Wild-type or mutant strains
in which the endogenous copy of
Pds1 was replaced with Pds1HA
(SLJ423–429) were grown in YPD
to midlog phase at 23°C. Cultures
were divided and either grown as
asynchronous cultures or arrested
as indicated for 3.5 h. During the
last 30 min of the nocodazole and
?-factor arrests, these cultures
were also shifted to 37°C. Cell ly-
sates were subjected to immuno-
blotting with the anti-HA anti-
(which normally migrates as a
doublet; left panels) or with anti-
Clb2 antibodies (right panels). (B)
GAL-PDS1HA or GAL-CLB2HA
was integrated at the TRP1 locus
of cdc15-2 to create SLJ272 and
phase YP/raffinose cultures were
arrested in 1 ?g/ml ?-factor, in 15
?g/ml nocodazole, or at 37°C for
3.5 h. During the last 30 min,
?-factor- and nocodazole-arrested
cultures were shifted to 37°C. Ga-
lactose was added to a final con-
centration of 2% to induce expres-
sion of Pds1HA or Clb2HA. After
30 min of induction, transcription
and translation were repressed by
addition at time zero of 2% dex-
trose and 10 ?g/ml cyclohexi-
mide, and cells were harvested at
the indicated times. Cell lysates
were subjected to Western blot-
ting with 12CA5 antibodies.
Clb2, but not Pds1, is
S.L. Jaspersen et al.
Molecular Biology of the Cell 2810
of the cells were arrested as large budded cells. The
APC isolated from the late mitotic mutants at 37°C
had negligible cyclin–ubiquitin ligase activity, except
in the case of cdc14-1 mutants, which reproducibly
contained a small amount of activity (Figure 4A). The
level of APC activity measured in vitro was reflective
of the amount of Clb2 protein and Clb2-associated
kinase activity (Figure 4, B and C, respectively). Thus,
the late mitotic proteins are required for activation of
the APC toward mitotic cyclins.
When mutant cells were arrested in G1 with ?-factor
and then shifted to the restrictive temperature in the
continued presence of ?-factor, the APC activity from
cdc15-2, cdc14-1, dbf2-2, and tem1-3 cells was equivalent
to that of wild-type cells arrested in ?-factor (Figure 4A).
cdc5-1 cells displayed low APC activity in G1, probably
because this mutation results in a severe defect in APC
activation even at the permissive temperature (Charles et
al., 1998). We conclude that the late mitotic gene prod-
ucts are required for initiation but not maintenance of
APC activity toward cyclin.
All of the late mitotic mutants arrest with negligible
levels of the Cdk inhibitor Sic1 (Figure 4D). This is
consistent with previous evidence that Cdc28-depen-
dent kinase activity inhibits Swi5-dependent SIC1
transcription and also inhibits Sic1 stabilization (Moll
et al., 1991; Donovan et al., 1994; Toyn et al., 1996;
Verma et al., 1997).
CDC15 Encodes a Protein Kinase Whose Activity Is
Not Regulated in the Cell Cycle
If the products of the late mitotic genes are activators
of the APC, their activity might be expected to in-
arrest with low APC activity toward
cyclin. (A) Wild-type and mutant
strains were transformed with a
plasmid carrying CDC27HA under
of cultures was arrested for 3.5 h at
shifted to 37°C in the presence of
?-factor for an additional hour. The
Cdc27HA subunit of the APC was
immunoprecipitated from 500 ?g of
cell lysate with 12CA5, and conjuga-
tion of ubiquitin to the125I-labeled
amino terminus of sea urchin cyclin
TERIALS AND METHODS. Ubiq-
uitin conjugates were observed at
gated cyclin B1 fragment. The aster-
isk indicates a nonspecific back-
ground band observed
presence of cyclin substrate alone
(far left lane). Note that APC activity
in wild-type asynchronous cells is
normally lower than that in G1-ar-
rested cells (Charles et al., 1998); in
this experiment, the high activity in
asynchronous cells is due to the rel-
atively high protein levels in these
samples (see anti-Cdc28 Western
blot in B). (B) Lysates (?35 ?g) from
the experiment in panel (A) were
subjected to Western blotting with
polyclonal antibodies against Clb2
(top) and Cdc28 (bottom). (C) His-
tone H1 kinase activity was mea-
sured in anti-Clb2 immunoprecipi-
tates from 100 ?g of cell lysate. (D)
Cell lysates (100 ?g) were subjected
ified polyclonal antibodies against
The late mitotic mutants
Regulation of Cyclin Destruction
Vol. 9, October 19982811
crease in mitosis. Indeed, the expression of CDC5,
CDC14, and DBF2 is known to peak during mitosis
(Johnston et al., 1990; Wan et al., 1992; Kitada et al.,
1993); in addition, the levels and kinase activities of
the Cdc5 and Dbf2 proteins rise during mitosis and
decline as cells enter G1 (Toyn and Johnston, 1994;
Hardy and Pautz, 1996; Charles et al., 1998; Shirayama
et al., 1998). Studies of Cdc15 protein levels or activity
during the cell cycle have not been reported.
CDC15 is predicted to encode a 110-kDa protein
kinase (Schweitzer and Philippsen, 1991). To verify
this prediction, we constructed a version of Cdc15
with three copies of the HA epitope tag at its carboxyl
terminus and either expressed the gene from its own
promoter on a 2? plasmid or replaced the endogenous
gene with the epitope-tagged copy. Cells expressing
Cdc15HA3 but not those expressing untagged Cdc15
contained a 110-kDa protein that was recognized by
the anti-HA monoclonal antibody 12CA5 (Figure 5A).
Immunoprecipitates from cells expressing Cdc15HA3
contained an associated kinase activity that phosphor-
ylated MBP in vitro (Figure 5B). Kinase activity was
abolished by a point mutation at a conserved lysine in
the ATP binding site of the Cdc15 kinase domain
(K54L; Figure 5B). In addition to phosphorylating
MBP, Cdc15HA3 also phosphorylated itself (Figure 5B
and our unpublished data).
To analyze Cdc15 protein levels across the cell cycle,
cells in which the endogenous CDC15 was replaced
with CDC15HA3 were arrested in G1 with mating
pheromone and then released. Whereas Clb2 protein
levels oscillated as cells progressed through the cell
cycle, levels of Cdc15 protein remained constant (Fig-
ure 6A). We also measured Cdc15-associated kinase
activity across the cell cycle, using a strain expressing
Cdc15HA3 from its own promoter on a multicopy
plasmid. As before, Cdc15 protein levels did not fluc-
tuate as cells were released from a G1 arrest and
allowed to proceed through the cell cycle (Figure 6B).
Furthermore, neither Cdc15 autophosphorylation nor
Cdc15-associated MBP kinase activity appeared to
change across the cell cycle (Figure 6B).
Several lines of genetic evidence, presented here and
in previous work, reveal extensive overlaps in the
functions of Cdc15, Cdc5, Cdc14, Dbf2, and Tem1
(Kitada et al., 1993; Donovan et al., 1994; Shirayama et
al., 1994b, 1996). First, mutants in these genes arrest at
the restrictive temperature with remarkably similar
phenotypes, including large buds, extended spindles,
separated DNA masses, high levels of Clb2, low levels
of Pds1 and Sic1, and low cyclin-directed APC activ-
ity. Second, the temperature-sensitive growth defect
in many late mitotic mutants can be suppressed by
overexpression of other genes in the family. Third, the
growth defect in all of the mutants is enhanced by
CLB2 overexpression and suppressed in all but one
mutant by overexpression of SIC1, SPO12, and trun-
cated YAK1. Finally, we have found an extensive array
of synthetic lethal interactions in strains bearing two
late mitotic mutations. These results are all consistent
with the possibility that the late mitotic genes promote
overlapping functions required for the exit from mi-
The functions of the late mitotic genes appear to
converge on the cyclin destruction machinery. All five
of the genes we studied are required for the activation
of cyclin–ubiquitin ligase activity of the APC in late
carrying a carboxyl-terminal triple HA tag was used to replace the
endogenous CDC15 gene (SLJ23; lane 2) or was cloned onto a 2?
plasmid (pSJ103; lane 4). Lysates from the indicated asynchronous
cultures (120 ?g in lanes 1 and 2, 35 ?g in lanes 3–5) were subjected
to Western blotting with 12CA5 antibodies. (B) 12CA5 immunopre-
cipitates from 1 mg (lanes 1 and 2) or 250 ?g (lanes 3–5) of cell
extract were tested for their ability to phosphorylate MBP in a
standard kinase reaction. A protein the size of Cdc15HA3 was also
labeled in these immunoprecipitates. In other experiments with
singly tagged Cdc15HA, this band migrates slightly faster, indicat-
ing that it represents the Cdc15 protein itself (our unpublished
data). In lane 5, the kinase reaction was performed with a version of
Cdc15 (pSJ59) carrying a point mutation (K54L) that is predicted to
abolish kinase activity.
CDC15 encodes a protein kinase. (A) A version of CDC15
S.L. Jaspersen et al.
Molecular Biology of the Cell 2812
mitosis, whereas none is required for the maintenance
of that activity in G1. We suspect that the products of
the late mitotic genes directly promote cyclin-specific
APC activation, rather than controlling it indirectly by
promoting an essential mitotic process whose comple-
tion is required to allow cyclin destruction. The latter
possibility does not seem consistent with the ability of
SIC1 overexpression to suppress the growth defects in
these mutants. Good evidence for a direct regulatory
role exists for Cdc5, whose overproduction at any cell
cycle stage triggers APC activation (Charles et al.,
1998; Shirayama et al., 1998); in addition, the mamma-
lian homologue of Cdc5, Plk1, is able to directly phos-
phorylate and activate the APC (Kotani et al., 1998).
Previous work showed that overexpression of genes
that antagonize the cAMP pathway suppresses the
growth defect in the cdc15-2 mutant (Spevak et al.,
1993). Similarly, we found that many of the late mi-
totic mutants are suppressed by overexpression of
truncated YAK1, which may, like full-length YAK1,
oppose the actions of PKA (Garrett and Broach, 1989;
Garrett et al., 1991; Hartley et al., 1994; Ward and
Garrett, 1994). Considering recent evidence that PKA
acts as an inhibitor of the APC in vitro (Kotani et al.,
1998), it might be predicted that inhibition of the PKA
pathway by YAK1 could increase APC activity and
thereby allow late mitotic mutants to exit mitosis.
The late mitotic mutants are defective primarily in
the degradation of cyclin and not that of Pds1, sug-
gesting that these genes activate the Hct1-dependent
pathway that is thought to specify the ubiquitination
of late mitotic substrates such as Clb2, Ase1, and Cdc5
(Schwab et al., 1997; Visintin et al., 1997; Charles et al.,
1998; Shirayama et al., 1998). The destruction of the
majority of Pds1 in cdc15-2, cdc5-1, cdc14-1, dbf2-2, and
tem1-3 is consistent with the fact that these mutants
complete chromosome segregation. Interestingly, late
mitotic mutants arrested in anaphase still contain a
small amount of stable Pds1 protein, which may rep-
resent an inactive pool of the protein whose destruc-
tion is not required for chromosome segregation.
The products of the late mitotic genes may also
contribute to Cdc28 inactivation by mechanisms other
than cyclin destruction. Recent studies suggest that
cyclin destruction is not essential for mitotic exit un-
der some conditions (Minshull et al., 1996; Toyn et al.,
and kinase activity are constant
was arrested for 3 h at 30°C with 1
?g/ml ?-factor, released from the
arrest, and allowed to grow at
30°C. Cells were harvested at the
indicated times, and lysates (100
?g) were analyzed by Western
blotting with 12CA5 (top) or anti-
Clb2 antibodies (bottom). (B) Wild-
type cells carrying CDC15HA3 on a
2? plasmid were arrested for 3 h at
30°C with 1 ?g/ml ?-factor, re-
leased from the arrest, and allowed
to progress through the cell cycle at
30°C. Cells were harvested at the
indicated times, and lysates (35 ?g)
were analyzed by Western blotting
with 12CA5 (top) or anti-Clb2 anti-
Cdc15HA3 was immunoprecipi-
tated from 250 ?g of lysate and
tested for its ability to phosphory-
late itself (third from top) or MBP
Cdc15 protein levels
Regulation of Cyclin Destruction
Vol. 9, October 1998 2813
1996; Schwab et al., 1997; Visintin et al., 1997; Jin et al.,
1998). Cells lacking HCT1 are able to exit mitosis de-
spite a severe defect in cyclin destruction, possibly
because Cdc28 is inactivated in these cells by the in-
hibitor Sic1 (Schwab et al., 1997; Visintin et al., 1997).
The fact that the late mitotic genes are essential for
mitotic exit implies that they may have functions in
addition to the activation of cyclin destruction. For
example, they may stimulate the synthesis or stabili-
zation of Sic1 (Figure 7).
In light of previous evidence that APC-dependent
proteolysis is inhibited by Cdc28 activity (Amon,
1997), it is conceivable that late mitotic gene products
act entirely through the up-regulation of Sic1, which
would lead indirectly to APC activation. This seems
unlikely, however, given the fact that the late mitotic
genes are essential for viability and SIC1 is not, and
given the biochemical evidence that at least one late
mitotic gene product, Cdc5, acts directly on the APC
(Kotani et al., 1998).
The reversal of Cdc28 action in late mitosis cannot
be accomplished solely by Cdc28 inactivation: de-
phosphorylation of its substrates is presumably re-
quired. Thus, defects in the dephosphorylation of
Cdc28 substrates would also be expected to result in
a late mitotic arrest. Interestingly, Cdc14 is homol-
ogous to protein phosphatases and possesses phos-
phatase activity in vitro (Wan et al., 1992; Taylor et
al., 1997), raising the possibility that it is responsible
for dephosphorylating Cdc28 substrates. Interest-
ingly, the cdc14-1 mutant displayed unique behav-
iors in our experiments that are consistent with this
possibility: the cdc14-1 mutant defect was not res-
cued effectively by any of the suppressors, and over-
expressed CDC14 was the most effective suppressor
of the other mutants.
To understand how the products of the late mitotic
genes fit into the complex pathways that trigger Cdc28
inactivation after chromosome segregation, we will
need a better understanding of the regulation of these
proteins. Production of three of the late mitotic gene
products (Cdc5, Cdc14, and Dbf2) is increased during
mitosis at the time when APC activation occurs, but
the mechanisms underlying this regulation remain ob-
scure (Johnston et al., 1990; Wan et al., 1992; Kitada et
al., 1993; Toyn and Johnston, 1994; Hardy and Pautz,
1996; Charles et al., 1998; Shirayama et al., 1998). We
found that bulk Cdc15 protein levels and activity do
not appear to be regulated during the cell cycle, but
this does not exclude cell cycle-dependent changes in
Cdc15 localization or accessibility of Cdc15 substrates.
through a regulated component of the pathway (such
as Cdc5) to specifically activate cyclin proteolysis at
the end of mitosis.
The five genes studied in the present work are
members of a growing family of genes with over-
lapping functions in the completion of mitosis. Ad-
ditional genes in this family include LTE1, which
interacts genetically with CDC15 and TEM1 and
encodes a putative guanine nucleotide exchange
factor (Shirayama et al., 1994a,b, 1996). MOB1 en-
codes a protein that physically associates with Dbf2
and is required for the completion of anaphase;
mob1 mutants display genetic interactions with
DBF2, CDC15, CDC5, and LTE1 (Komarnitsky et al.,
1998; Luca and Winey, 1998). Dbf2 also interacts
physically with the CCR4 transcription complex and
might thereby exert effects on gene expression in
late mitosis (Liu et al., 1997). The existence of this
complex network of late mitotic regulatory proteins
implies that progression from anaphase to G1 is a
key regulatory transition in the cell cycle. It seems
likely that the late mitotic regulators serve as com-
ponents in signaling pathways that monitor mitotic
events and promote Cdc28 inactivation and mitotic
exit only upon successful completion of anaphase
and preparation for cytokinesis.
in late mitosis. Late mitotic gene products stimulate mitotic cyclin
destruction and may also induce increased levels of Sic1 (see DIS-
CUSSION). This model also accommodates evidence that Cdc28
inhibits APC activity (Amon, 1997) and also inhibits SIC1 transcrip-
tion and Sic1 stability (Moll et al., 1991; Toyn et al., 1996; Verma et al.,
1997), resulting in a feedback system that triggers rapid and com-
plete Cdc28 inactivation when Cdc28 activity is reduced to some
threshold. For simplicity, this diagram does not include an addi-
tional feedback loop suggested by the observation that Cdc28–Clb
complexes stimulate CLB transcription (Amon et al., 1993).
Model of regulatory pathways governing Cdc28 activity
S.L. Jaspersen et al.
Molecular Biology of the Cell 2814
We thank Aaron Straight, Lena Hwang, Alex Szidon, Adam Rud-
ner, Phil Heiter, Doug Koshland, and Mike Tyers for reagents, Paul
DiGregorio and Simon Chan for their initial work on CDC14, Cathe-
rine Takizawa and Sue Biggins for comments on the manuscript,
and Megan Grether, Andrew Murray, and members of the Morgan
and Murray laboratories for valuable discussions. This work was
supported by funding from the National Institute of General Med-
ical Sciences (to D.O.M.), a Howard Hughes Medical Institute Pre-
doctoral Fellowship (to S.L.J.), and a Damon Runyon–Walter
Winchell postdoctoral fellowship (to R.T.K.).
Amon, A. (1997). Regulation of B-type cyclin proteolysis by Cdc28-
associated kinases in budding yeast. EMBO J. 16, 2693–2702.
Amon, A., Irniger, S., and Nasmyth, K. (1994). Closing the cell cycle
circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until
the activation of G1 cyclins in the next cycle. Cell 77, 1037–1050.
Amon, A., Tyers, M., Futcher, B., and Nasmyth, K. (1993). Mecha-
nisms that help the yeast cell cycle clock tick—G2 cyclins transcrip-
tionally activate G2 cyclins and repress G1 cyclins. Cell 74, 993–
Brandeis, M., and Hunt, T. (1996). The proteolysis of mitotic cyclins
in mammalian cells persists from the end of mitosis until the onset
of S phase. EMBO J. 15, 5280–5289.
Charles, J.F., Jaspersen, S.L., Tinker-Kulberg, R.L., Hwang, L., Szi-
don, A., and Morgan, D.O. (1998). The Polo-related kinase Cdc5
activates and is destroyed by the mitotic cyclin destruction machin-
ery in S. cerevisiae. Curr. Biol. 8, 497–507.
Cohen-Fix, O., Peters, J.-M., Kirschner, M.W., and Koshland, D.
(1996). Anaphase initiation in Saccharomyces cerevisiae is controlled
by the APC-dependent degradation of the anaphase inhibitor
Pds1p. Genes Dev. 10, 3081–3093.
Descombes, P., and Nigg, E.A. (1998). The polo-like kinase Plx1 is
required for M phase exit and destruction of mitotic regulators in
Xenopus egg extracts. EMBO J. 17, 1328–1335.
Donovan, J.D., Toyn, J.H., Johnson, A.L., and Johnston, L.H. (1994).
P40SDB25, a putative CDK inhibitor, has a role in the M/G1 transi-
tion in Saccharomyces cerevisiae. Genes Dev. 8, 1640–1653.
Felix, M.-A., Labbe, J.-C., Doree, M., Hunt, T., and Karsenti, E.
(1990). Triggering of cyclin degradation in interphase extracts of
amphibian eggs by cdc2 kinase. Nature 346, 379–382.
Funabiki, H., Yamano, H., Kumada, K., Nagao, K., Hunt, T., and
Yanagida, M. (1996). Cut2 proteolysis required for sister-chromatid
separation in fission yeast. Nature 381, 438–441.
Gallant, P., and Nigg, E.A. (1992). Cyclin B2 undergoes cell cycle-
dependent nuclear translocation and, when expressed as a non-
destructible mutant, causes mitotic arrest in HeLa cells. J. Cell Biol.
Garrett, S., and Broach, J. (1989). Loss of Ras activity in Saccharomy-
ces cerevisiae is suppressed by disruptions of a new kinase gene,
YAK1, whose product may act downstream of the cAMP-dependent
protein kinase. Genes Dev. 3, 1336–1348.
Garrett, S., Menold, M.M., and Broach, J.R. (1991). The Saccharomyces
cerevisiae YAK1 gene encodes a protein kinase that is induced by
arrest early in the cell cycle. Mol. Cell. Biol. 11, 4045–4052.
Gerber, M.R., Farrell, A., Deshaies, R., Herskowitz, I., and Morgan,
D.O. (1995). Cdc37 is required for association of the protein kinase
Cdc28 with G1 and mitotic cyclins. Proc. Natl. Acad. Sci. USA 92,
Glotzer, M., Murray, A.W., and Kirschner, M.W. (1991). Cyclin is
degraded by the ubiquitin pathway. Nature 349, 132–138.
Guthrie, C., and Fink, G.R., ed. (1991). Guide to Yeast Genetics and
Molecular Biology. Methods in Enzymology, San Diego, Academic
Hardy, C.F.J., and Pautz, A. (1996). A novel role for Cdc5p in DNA
replication. Mol. Cell. Biol. 16, 6775–6782.
Hartley, A.D., Ward, M.P., and Garrett, S. (1994). The Yak1 protein
kinase of Saccharomyces cerevisiae moderates thermotolerance and
inhibits growth by an Sch9 protein kinase-independent mechanism.
Genetics 136, 465–474.
Hartwell, L.H., Mortimer, R.K., Culotti, J., and Culotti, M. (1973).
Genetic control of the cell division cycle in yeast: V. Genetic analysis
of cdc mutants. Genetics 74, 267–286.
Hershko, A., Ganoth, D., Sudakin, V., Dahan, A., Cohen, L.H., Luca,
F.C., Ruderman, J.V., and Eytan, E. (1994). Components of a system
that ligates cyclin to ubiquitin and their regulation by the protein
kinase cdc2. J. Biol. Chem. 269, 4940–4946.
Hochstrasser, M. (1996). Ubiquitin-dependent protein degradation.
Annu. Rev. Genet. 30, 405–439.
Holloway, S.L., Glotzer, M., King, R.W., and Murray, A.W. (1993).
Anaphase is initiated by proteolysis rather than by the inactivation
of maturation-promoting factor. Cell 73, 1393–1402.
Hoyt, M.A. (1997). Eliminating all obstacles: regulated proteolysis in
the eukaryotic cell cycle. Cell 91, 149–151.
Hwang, L.H., Lau, L.F., Smith, D.L., Mistrot, C.A., Hardwick, K.G.,
Hwang, E.S., Amon, A., and Murray, A.W. (1998). Budding yeast
Cdc20: a target of the spindle checkpoint. Science 279, 1041–1044.
Hwang, L.H., and Murray, A.W. (1997). A novel yeast screen for
mitotic arrest mutants identifies DOC1, a new gene involved in
cyclin proteolysis. Mol. Biol. Cell 8, 1877–1887.
Irniger, S., Piatti, S., Michaelis, C., and Nasmyth, K. (1995). Genes
involved in sister chromatid separation are needed for B-type cyclin
proteolysis in budding yeast. Cell 81, 269–277.
Jin, P., Hardy, S., and Morgan, D.O. (1998). Nuclear localization of
cyclin B1 controls mitotic entry after DNA damage. J. Cell Biol. 141,
Johnston, L.H., Eberly, S.L., Chapman, J.W., Araki, H., and Sugino,
A. (1990). The product of the Saccharomyces cerevisiae cell cycle gene
DBF2 has homolgy with protein kinases and is periodically ex-
pressed in the cell cycle. Mol. Cell. Biol. 10, 1358–1366.
Johnston, L.H., and Thomas, A.P.M. (1982). The isolation of new
DNA synthesis mutants in the yeast Saccharomyces cerevisiae. Mol.
Gen. Genet. 186, 439–444.
Juang, Y.-L., Huang, J., Peters, J.-M., McLaughlin, M.E., Tai, C.-Y.,
and Pellman, D. (1997). APC-mediated proteolysis of Ase1 and the
morphogenesis of the mitotic spindle. Science 275, 1311–1314.
Kim, S.H., Lin, D.P., Matsumoto, S., Kitazono, A., and Matsumoto,
T. (1998). Fission yeast Slp1: an effector of the Mad2-dependent
spindle checkpoint. Science 279, 1045–1047.
King, R.W., Deshaies, R.J., Peters, J.-M., and Kirschner, M.W. (1996).
How proteolysis drives the cell cycle. Science 274, 1652–1659.
King, R.W., Jackson, P.K., and Kirschner, M.W. (1994). Mitosis in
transition. Cell 79, 563–571.
King, R.W., Peters, J.-M., Tugendreich, S., Rolfe, M., Hieter, P., and
Kirschner, M.W. (1995). A 20S complex containing CDC27 and
CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to
cyclin B. Cell 81, 279–288.
Kitada, K., Johnson, A.L., Johnston, L.H., and Sugino, A. (1993). A
multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell
Regulation of Cyclin Destruction
Vol. 9, October 19982815
cycle mutant gene dbf4 encodes a protein kinase and is identified as
CDC5. Mol. Cell. Biol. 13, 4445–4457.
Klapholz, S., and Esposito, R.E. (1980). Isolation of SPO12-1 and
SPO13-1 from a natural variant of yeast that undergoes a single
meiotic division. Genetics 96, 567–588.
Komarnitsky, S.I., Chiang, Y., Luca, F.C., Chen, J., Toyn, J.H., Winey,
M., Johnston, L.H., and Denis, C.L. (1998). DBF2 protein kinase
binds to and acts through the cell cycle-regulated MOB1 protein.
Mol. Cell. Biol. 18, 2100–2107.
Kotani, S., Tugendreich, S., Fujii, M., Jorgensen, P., Watanabe, N.,
Hoog, C., Hieter, P., and Todokoro, K. (1998). PKA and MPF-
activated Polo-like kinase regulate anaphase-promoting complex
activity and mitosis progression. Mol. Cell 1, 371–380.
Kramer, K.M., Fesquet, D., Johnson, A.L., and Johnston, L.H. (1998).
Budding yeast RSI1/APC2, a novel gene necessary for initiation of
anaphase, encodes an APC subunit. EMBO J. 17, 498–505.
Lahav-Baratz, S., Sudakin, V., Ruderman, J.V., and Hershko, A.
(1995). Reversible phosphorylation controls the activity of cyclo-
some-associated cyclin-ubiquitin ligase. Proc. Natl. Acad. Sci. USA
Lamb, J.R., Michaud, W.A., Sikorski, R.S., and Hieter, P.A. (1994).
Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis.
EMBO J. 13, 4321–4328.
Lim, H.H., Goh, P.-Y., and Surana, U. (1998). Cdc20 is essential for
cyclosome-mediated proteolysis of both Pds1 and Clb2 during M
phase in budding yeast. Curr. Biol. 8, 231–234.
Liu, H., Krizek, J., and Bretscher, A. (1992). Construction of a GAL1-
regulated yeast cDNA expression library and its application to the
identification of genes whose overexpression causes lethality in
yeast. Genetics 132, 665–673.
Liu, H., Toyn, J.H., Chiang, Y., Draper, M.P., Johnston, L.H., and
Denis, C.L. (1997). DBF2, a cell cycle-regulated protein kinase, is
physically and functionally associated with the CCR4 transcrip-
tional regulatory complex. EMBO J. 16, 5289–5298.
Luca, F.C., and Winey, M. (1998). MOB1, an essential yeast gene
required for completion of mitosis and maintenance of ploidy. Mol.
Biol. Cell 9, 29–46.
Malavasic, M.J., and Elder, R.T. (1990). Complementary transcripts
from two genes necessary for normal meiosis in the yeast Saccharo-
myces cerevisiae. Mol. Cell. Biol. 10, 2809–2819.
Mendenhall, M.D. (1993). An inhibitor of p34CDC28protein kinase
activity from Saccharomyces cerevisiae. Science 259, 216–219.
Minshull, J., Straight, A., Rudner, A.D., Dernburg, A.F., Belmont, A.,
and Murray, A.W. (1996). Protein phosphatase 2A regulates MPF
activity and sister chromatid cohesion in budding yeast. Curr. Biol.
Molero, G., Yuste-Rojas, M., Montesi, A., Vazquez, A., Nombela, C.,
and Sanchez, M. (1993). A cdc-like autolytic Saccharomyces cerevisiae
mutant altered in budding site selection is complemented by SPO12,
a sporulation gene. J. Bacteriol. 175, 6562–6570.
Moll, T., Tebb, G., Surana, U., Robitsch, H., and Nasmyth, K. (1991).
The role of phosphorylation and the CDC28 protein kinase in the
cell cycle-regulated nuclear import of the S. cerevisiae transcription
factor SWI5. Cell 66, 743–758.
Morgan, D.O. (1997). Cyclin-dependent kinases: engines, clocks,
and microprocessors. Annu. Rev. Cell Dev. Biol. 13, 261–291.
Murray, A.W. (1995). Cyclin ubiquitination: the destructive end of
mitosis. Cell 81, 149–152.
Murray, A.W., Solomon, M., and Kirschner, M. (1989). The role of
cyclin synthesis and degradation in the control of maturation pro-
moting factor activity. Nature 339, 280–286.
Nasmyth, K. (1996). At the heart of the budding yeast cell cycle.
Trends Genet. 12, 405–412.
Parkes, V., and Johnston, L.H. (1992). SPO12 and SIT4 suppress
mutations in DBF2, which encodes a cell cycle protein kinase that is
periodically expressed. Nucleic Acids Res. 20, 5617–5623.
Pellman, D., Bagget, M., Tu, H., and Fink, G.R. (1995). Two micro-
tubule-associated proteins required for anaphase spindle movement
in Saccharomyces cerevisiae. J. Cell Biol. 130, 1373–1385.
Peters, J.-M., King, R.W., Hoog, C., and Kirschner, M.W. (1996).
Identification of BIME as a subunit of the anaphase-promoting
complex. Science 274, 1199–1201.
Prinz, S., Hwang, E.S., Visintin, R., and Amon, A. (1998). The
regulation of Cdc20 proteolysis reveals a role for the APC compo-
nents Cdc23 and Cdc27 during S phase and early mitosis. Curr. Biol.
Rimmington, G., Dalby, B., and Glover, D.M. (1994). Expression of
N-terminally truncated cyclin B in the Drosophila larval brain leads
to mitotic delay at late anaphase. J. Cell Sci. 107, 2729–2738.
Schwab, M., Lutum, A.S., and Seufert, W. (1997). Yeast Hct1 is a
regulator of Clb2 cyclin proteolysis. Cell 90, 683–693.
Schweitzer, B., and Philippsen, P. (1991). CDC15, an essential cell
cycle gene in Saccharomyces cerevisiae, encodes a protein kinase do-
main. Yeast 7, 265–273.
Schwob, E., Bohm, T., Mendenhall, M.D., and Nasmyth, K. (1994).
The B-type cyclin kinase inhibitor p40SIC1controls the G1 to S
transition in S. cerevisiae. Cell 79, 233–244.
Sethi, N., Monteagudo, M.C., Koshland, D., Hogan, E., and Burke,
D.J. (1991). The CDC20 gene product of Saccharomyces cerevisiae, a
?-transducin homolog, is required for a subset of microtubule-
dependent cellular processes. Mol. Cell. Biol. 11, 5592–5602.
Shirayama, M., Matsui, Y., Tanaka, K., and Toh-e, A. (1994a). Isola-
tion of a CDC25 family gene, MSI2/LTE1, as a multicopy suppressor
of ira1. Yeast 10, 451–461.
Shirayama, M., Matsui, Y., and Toh-e, A. (1994b). The yeast TEM1
gene, which encodes a GTP-binding protein, is involved in termi-
nation of M phase. Mol. Cell. Biol. 14, 7476–7482.
Shirayama, M., Matsui, Y., and Toh-e, A. (1996). Dominant mutant
alleles of yeast protein kinase gene CDC15 suppress the lte1 defect
in termination of M phase and genetically interact with CDC14. Mol.
Gen. Genet. 251, 176–185.
Shirayama, M., Zachariae, W., Ciosk, R., and Nasmyth, K. (1998).
The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/
fizzy are regulators and substrates of the anaphase promoting com-
plex in Saccharomyces cerevisiae. EMBO J. 17, 1336–1349.
Sigrist, S., Jacobs, J., Stratmann, R., and Lehner, C.F. (1995). Exit
from mitosis is regulated by Drosophila fizzy and the sequential
destruction of cyclins A, B, and B3. EMBO J. 14, 4827–4838.
Sikorski, R.S., and Hieter, P. (1989). A system of shuttle vectors and
yeast host strains designed for efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics 122, 19–27.
Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J., and Harper, J.W.
(1997). F-box proteins are receptors that recruit phosphorylated
substrates to the SCF ubiquitin-ligase complex. Cell 91, 209–219.
Spevak, W., Keiper, B.D., Stratowa, C., and Castanon, M.J. (1993).
Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in
anaphase are rescued by Xenopus cDNAs encoding N-ras or a pro-
tein with ?-transducin repeats. Mol. Cell. Biol. 13, 4953–4966.
Sudakin, V., Ganoth, D., Dahan, A., Heller, H., Hershko, J., Luca,
F.C., Ruderman, J.V., and Hershko, A. (1995). The cyclosome, a large
complex containing cyclin-selective ubiquitin-ligase activity, targets
S.L. Jaspersen et al.
Molecular Biology of the Cell2816
cyclins for destruction at the end of mitosis. Mol. Biol. Cell 6,
Surana, U., Amon, A., Dowzer, C., McGrew, J., Byers, B., and
Nasmyth, K. (1993). Destruction of the CDC28/CLB mitotic kinase
is not required for the metaphase-to-anaphase transition in budding
yeast. EMBO J. 12, 1969–1978.
Taylor, G.S., Liu, Y., Baskerville, C., and Charbonneau, H. (1997).
The activity of Cdc14p, an oligomeric dual specificity protein phos-
phatase from Saccharomyces cerevisiae, is required for cell cycle pro-
gression. J. Biol. Chem. 272, 24054–24063.
Toda, T., Uno, I., Ishikawa, T., Powers, S., Kataoka, T., Broek, D.,
Cameron, S., Broach, J., Matsumoto, K., and Wigler, M. (1985). In
yeast, RAS proteins are controlling elements of adenylate cyclase.
Cell 40, 27–36.
Toyn, J.H., Johnson, A.L., Donovan, J.D., Toone, W.M., and
Johnston, L.H. (1996). The Swi5 transcription factor of Saccharomyces
cerevisiae has a role in exit from mitosis through induction of the
Cdk-inhibitor Sic1 in telophase. Genetics 145, 85–96.
Toyn, J.H., and Johnston, L.H. (1993). Spo12 is a limiting factor that
interacts with the cell cycle protein kinases Dbf2 and Dbf20, which
are involved in mitotic chromatid disjunction. Genetics 135, 963–
Toyn, J.H., and Johnston, L.H. (1994). The Dbf2 and Dbf20 protein
kinases of budding yeast are activated after the metaphase to an-
aphase cell cycle transition. EMBO J. 13, 1103–1113.
Verma, R., Annan, R.S., Huddleston, M.J., Carr, S.A., Reynard, G.,
and Deshaies, R.J. (1997). Phosphorylation of Sic1p by G1 Cdk
required for its degradation and entry into S phase. Science 278,
Visintin, R., Prinz, S., and Amon, A. (1997). CDC20 and CDH1: a
family of substrate-specific activators of APC-dependent proteoly-
sis. Science 278, 460–463.
Wan, J., Xu, H., and Grunstein, M. (1992). CDC14 of Saccharomyces
cerevisiae. J. Biol. Chem. 267, 11274–11280.
Ward, M.P., and Garrett, S. (1994). Suppression of a yeast cyclic
AMP-dependent protein kinase defect by overexpression of SOK1, a
yeast gene exhibiting sequence similarity to a developmentally reg-
ulated mouse gene. Mol. Cell. Biol. 14, 5619–5627.
Yamano, H., Gannon, J., and Hunt, T. (1996). The role of proteolysis
in cell cycle progression in Schizosaccharomyces pombe. EMBO J. 15,
Yamashita, Y.M., Nakeseko, Y., Samejima, I., Kumada, K., Yamada,
H., Michaelson, D., and Yanagida, M. (1996). 20S cyclosome com-
plex formation and proteolytic activity inhibited by the cAMP/PKA
pathway. Nature 384, 276–279.
Yu, H., Peters, J., King, R.W., Page, A.M., Hieter, P., and Kirschner,
M.W. (1998). Identification of a cullin homology region in a subunit
of the anaphase-promoting complex. Science 279, 1219–1222.
Zachariae, W., and Nasmyth, K. (1996). TPR proteins required for
anaphase progression mediate ubiquitination of mitotic B-type cy-
clins in yeast. Mol. Biol. Cell 7, 791–801.
Zachariae, W., Shevchenko, A., Andrews, P.D., Ciosk, R., Galova,
M., Strak, M.J.R., Mann, M., and Nasmyth, K. (1998). Mass spectro-
metric analysis of the anaphase-promoting complex from yeast:
identification of a subunit related to cullins. Science 279, 1216–1219.
Zachariae, W., Shin, T.H., Galova, M., Obermaier, B., and Nasmyth,
K. (1996). Identification of subunits of the anaphase-promoting com-
plex of Saccharomyces cerevisiae. Science 274, 1201–1204.
Regulation of Cyclin Destruction
Vol. 9, October 1998 2817