Volume 22 February 1, 2011
Ama1p-activated anaphase-promoting complex
regulates the destruction of Cdc20p during
Grace S. Tan a,b , Jennifer Magurno c , and Katrina F. Cooper a, c
a Department of Biochemistry and Molecular Biology, Drexel Medical School, Philadelphia, PA 19102; cDepartment
of Molecular Biology, School of Osteopathic Medicine, University of Medicine and Dentistry of New Jersey, Stratford,
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E10-04-0360) on November 30, 2010.
b Present address: Division of Rheumatology, University of Pennsylvania,
Philadelphia, PA 19104.
Address correspondence to: Katrina F. Cooper ( Cooperka@umdnj.edu ).
Abbreviations used: APC/C, anaphase-promoting complex/cyclosome; CB, C-
box; DAPI, 4′,6-diamino-2-phenylindole; Db1, destruction box degron 1; FC, fi nal
concentration; GST, glutathione S -transferase; GxEN, destruction degron; HA,
hemagglutinin; mAb, monoclonal antibody; MAPK, mitogen-activated protein
kinase; MI, meiosis I; MII, meiosis II; SPM, sporulation medium; WT, wild type.
© 2011 Tan et al. This article is distributed by The American Society for Cell
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able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
ABSTRACT The execution of meiotic divisions in Saccharomyces cerevisiae is regulated by
anaphase-promoting complex/cyclosome (APC/C)–mediated protein degradation. During mei-
osis, the APC/C is activated by association with Cdc20p or the meiosis-specifi c activator Ama1p.
We present evidence that, as cells exit from meiosis II, APC/C Ama1 mediates Cdc20p destruc-
tion. APC/C Ama1 recognizes two degrons on Cdc20p, the destruction box and destruction de-
gron, with either domain being suffi cient to mediate Cdc20p destruction. Cdc20p does not
need to associate with the APC/C to bind Ama1p or be destroyed. Coimmunoprecipitation
analyses showed that the diverged amino-terminal region of Ama1p recognizes both Cdc20p
and Clb1p, a previously identifi ed substrate of APC/C Ama1 . Domain swap experiments revealed
that the C-terminal WD region of Cdh1p, when fused to the N-terminal region of Ama1p, could
direct most of Ama1p functions, although at a reduced level. In addition, this fusion protein
cannot complement the spore wall defect in ama1Δ strains, indicating that substrate specifi city
is also derived from the WD repeat domain. These fi ndings provide a mechanism to tempo-
rally down-regulate APC/C Cdc20 activity as the cells complete meiosis II and form spores.
The anaphase-promoting complex/cyclosome (APC/C) is a highly
conserved ubiquitin ligase that directs the destruction of key regula-
tory proteins necessary for proper mitotic and meiotic progression
(reviewed in Yu, 2007 ). During mitotic cell division in budding yeast,
APC/C activation and substrate specifi city are directed by two highly
conserved Trp-Asp (WD40) repeat proteins: Cdc20p and Cdh1p
( Dawson et al. , 1995 ; Schwab et al. , 1997 ; Sigrist and Lehner, 1997 ;
Visintin et al. , 1997 ). Ama1p is the meiosis-specifi c APC/C activator
( Chu et al. , 1998 ; Cooper et al. , 2000 ) that directs the ubiquitylation
of the B-type cyclin Clb1p ( Cooper et al. , 2000 ) plus other unknown
substrates ( Rabitsch et al. , 2001 ). APC/C Ama1 activates Smk1p, the
meiotic mitogen-activated protein kinase (MAPK) required for spore
wall morphogenesis through an unknown mechanism ( McDonald
et al. , 2005 ). Ama1p also coordinates exit from meiosis II and is re-
quired for the early stages of spore wall assembly ( Rabitsch et al. ,
2001 ; Coluccio et al. , 2004 ; Diamond et al. , 2009 ).
To associate with the APC/C, activators contain two short motifs
called the C-box (CB) and IR motifs ( Schwab et al. , 2001 ; Passmore
et al. , 2003 ; Vodermaier et al. , 2003 ). Although the precise mecha-
nism has not been elucidated, it has been proposed that the IR
motif targets the activators to the APC/C, while the CB motif
promotes an activating change in APC/C conformation that is inde-
pendent of the activators’ substrate-recruiting function ( Vodermaier
et al. , 2003 ; Dube et al. , 2005 ; Kimata et al. , 2008 ). Interestingly,
the IR box of Cdc20p is not required for function in Saccharomyces
cerevisiae but contributes to APC/C-dependent turnover ( Thornton
et al. , 2006 ).
The recognition of substrates by the APC/C is more complex.
Initially, it was proposed that the APC/C activators played an equiv-
alent role to F-box proteins in the SCF ubiquitin ligase, serving as a
Received: Apr 27 , 2010
Revised: Nov 15 , 2010
Accepted: Nov 17 , 2010
MBoC | ARTICLE
316 | G. S. Tan et al. Molecular Biology of the Cell
studies and coimmunoprecipitation assays identifi ed two con-
served degrons on Cdc20p (destruction box, GxEN) that are rec-
ognized by the divergent amino-terminus of Ama1p. Interestingly,
replacing the C-terminal WD40 of Ama1p with that of Cdh1p pro-
vided partial APC/C Ama1 function. These fi ndings indicate that
unique information is provided by both the N-terminal and car-
boxyl terminal domains of Ama1p.
Ama1p is required for Cdc20p destruction during meiosis
CDC20 mRNA levels decline as cells exit the mitotic cell cycle, fol-
lowed by a transient induction during the fi rst meiotic prophase,
which peaked at the meiotic divisions ( Chu et al. , 1998 ). To investi-
gate Cdc20p regulation during meiosis, a strain harboring a chro-
mosomally amino-terminal tagged CDC20 allele ( CDC20- 18myc)
was induced to enter meiosis, and time points were taken before
the shift (0 h) until the completion of spore formation. Similar to its
mRNA profi le, Western blot analysis of protein samples prepared
from each time point revealed a transient accumulation of Cdc20p
peaking during the meiotic nuclear divisions ( Figure 1A , top). In
vegetative cells, Cdc20p is subjected to proteolysis in G1 mediated
by APC/C Cdh1 as well as an APC/C-independent mechanism ( Prinz
et al. , 1998 ; Fang et al. , 1998a ; Shirayama et al. , 1998 ; Huang et al. ,
2001 ; Morris et al. , 2003 ). To determine whether Ama1p or Cdh1p
bridge between the APC/C catalytic domain and the substrate
(Deshaies, 1999 ). In support of this model, APC/C activators were
shown to recognize specifi c degrons (destruction box, KEN box,
A-box, and the destruction degron [GxEN]) on their target protein
(Burton and Solomon, 2001 ; Burton et al. , 2005 ; Hilioti et al. , 2001 ;
Pfl eger et al. , 2001a ; Littlepage and Ruderman, 2002 ; Meyn et al. ,
2002 ; Castro et al. , 2003 ; Kraft et al. , 2003 ). However, more recent
studies have reported that the core APC/C also recognizes sub-
strates, although the presence of activators is almost always required
for this binding (Carroll and Morgan, 2002 ; Carroll et al. , 2005 ;
Passmore et al. , 2003 ; Passmore and Barford, 2005 ; Yamano et al. ,
2004 ; Eytan et al. , 2006 ). Taken together, a current model proposes
that substrate selection is aided by direct binding to the core APC/C
itself. Although the exact mechanism for how this promotes sub-
strate ubiquitylation remains unknown, many models have been
proposed (reviewed in Yu, 2007 ). Recent evidence has favored a
“multivalency” model in which activator binding to the APC/C
(through its CB and IR motifs) creates a bipartite binding site for the
substrate through the activator and the APC/C core (Matyskiela and
Morgan, 2009 ). Activator binding in turn may also create a confor-
mational change to the APC/C, promoting ubiquitylation of the sub-
In vegetative cells, Cdc20p is transcribed from S phase through
G2 phase and subjected to proteolysis in G1 ( Fang et al. , 1998a ;
Prinz et al. , 1998 ; Shirayama et al. , 1998 ;
Huang et al. , 2001 ; Morris et al. , 2003 ). This
proteolysis is mediated by APC/C Cdh1
in late mitosis/early G1 and a Cdh1p-
independent mechanism during G1/S ( Goh
et al. , 2000 ; Robbins and Cross, 2010 ).
In addition, APC/C Cdc20 is negatively
regulated by the binding of the spindle
checkpoint protein Mad2p (reviewed in
Bharadwaj and Yu, 2004 ) or by protein
kinase A phosphorylation ( Searle et al. ,
2004 ; Mallory et al. , 2007 ). In contrast,
Cdh1p is controlled by posttranslational
mechanisms including cell cycle–dependent
nuclear export ( Jaquenoud et al. , 2002 ),
G1 cyclin–Cdc28p phosphorylation
( Zachariae et al. , 1998 ; Jaspersen et al. ,
1999 ), and specifi c inhibitors ( Martinez
et al. , 2006 ; Dial et al. , 2007 ; Choi et al. ,
2008 ; Crasta et al. , 2008 ; Enquist-Newman
et al. , 2008 ; Hall et al. , 2008 ; Ostapenko
et al. , 2008 ). Cdh1p is activated at ana-
phase when the Cdc28p-antagonizing
phosphatase Cdc14p is released from the
nucleolus ( Anghileri et al. , 1999 ). The mei-
osis-specifi c activator Ama1p is controlled
at the level of transcription ( Chu et al. ,
1998 ) and splicing ( Cooper et al. , 2000 ;
Spingola and Ares, 2000 ). APC/C Ama1 activ-
ity is inhibited by the APC/C subunit Mnd2p
and B cyclin–Cdc28p kinase activity until
late in the meiotic program ( Oelschlaegel
et al. , 2005 ; Penkner et al. , 2005 ; Carlile
and Amon, 2008 ).
In this study, we demonstrate that
APC/C Ama1 , and not APC/C Cdh1 , mediates
the degradation of Cdc20p at the end of
the second meiotic division. Genetic
FIGURE 1: APC/C Ama1 is required for of Cdc20p destruction during meiosis. (A) Wild-type
(RSY695), cdh1Δ (RSY1210), and ama1Δ (RSY853) strains harboring integrated Cdc20p-18myc
were induced to enter meiosis and time points taken as indicated. Cdc20p-18myc levels were
monitored by Western blot analysis. (B) The percent population of tetranucleated cells in the
strains described in (A) are plotted. (C) Quantitation of the Cdc20p-18myc signal from (A) is
plotted from the 9-h time point. The results shown are the averages from three separate
experiments with error bars included. (D) Wild-type (RSY695) or mnd2Δ (KCY440) strains
harboring integrated Cdc20p-18myc were induced to enter meiosis and time points taken as
indicated. Western blot analysis of protein extracts was conducted to detect Cdc20p. The
mnd2Δ strain remained mononucleated as previously described ( Penkner et al. , 2005 ). The blots
were stripped and reprobed for Tub1p as a loading control. MI and MII indicate the approximate
times of meiosis I (MI) and meiosis II (MII) as determined by DAPI analysis.
Volume 22 February 1, 2011 Ama1p-activated anaphase-promoting complex | 317
APC/C Ama1 activity is dependent on the conserved
To further confi rm a role for Ama1p in the meiotic destruction of
Cdc20p, we examined the degradation of Cdc20p in nonfunctional
ama1 mutants in which the conserved APC/C interaction motifs (CB
and IR motifs) were deleted. Deletion of both of these motifs inacti-
vated Ama1p, as indicated by the absence of viable spores
( Figure 3A ). Interestingly, the strain expressing the CBΔ mutant ex-
hibited a sixfold reduction in spore formation with unevenly sepa-
rated nuclei whereas the IRΔ mutant displayed a more modest de-
fect in spore formation and nuclear spacing. Similar results were
obtained when the ability of these mutants to activate the meiosis-
specifi c MAPK Smk1p was tested ( Figure 3B ). As previously reported
( McDonald et al. , 2005 ), the ratio of active (phosphorylated) to inac-
tive (unphosphorylated) forms of Smk1p is reduced in the ama1Δ
background (compare lanes 1 and 2, Figure 3B ).
We next tested the requirement of the CB and IR motifs for
Cdc20p degradation. Compared with the wild type, cultures ex-
pressing Ama1p CBΔ or Ama1p IRΔ displayed a modest defect in
plays a role in Cdc20p proteolysis during meiosis, the meiotic time
course described above was repeated in homozygous diploid cells
deleted for these genes. Despite progressing though meiosis with
the same kinetics as wild type ( Figure 1B ), Cdc20p levels are at the
limits of detection in the cdh1 Δ strain during the meiotic divisions
( Figure 1A , middle). Longer exposure of this blot revealed that
Cdc20p is present but, unlike wild-type cells, does not seem to be
up-regulated as the cells execute meiosis I and II. In addition, CDC20
mRNA is expressed normally in cdh1Δ strains (Supplemental
Figure S1A). However, in ama1Δ cells, Cdc20p was not down-
regulated as the cells exited from meiosis II (monitored by the ap-
pearance of tetra-nucleated cells; Figure 1B , quantitated in
Figure 1C ). These results suggest that APC/C Ama1 is required for
Cdc20p proteolysis as cells exit from meiosis II. They also suggest
that, different from mitotic cell divisions, this degradation is not
assisted by an APC/C-independent mechanism.
Loss of Ama1p or Cdc16p function leads to defects in meiotic
progression ( Cooper et al. , 2000 ). Therefore one possible explana-
tion is that the maintenance of Cdc20p levels in these mutants is an
indirect consequence of this meiotic arrest. However, we and others
( Diamond et al. , 2009 ) have shown that the abundance of proteins
that are normally degraded at the completion of meiosis II ( Cooper
et al. , 2000 ; Carlile and Amon, 2008 ) are comparable in wild-type
and ama1Δ strains. This suggests that the failure to destroy Cdc20p
at the end of meiosis II in the ama1Δ mutant is directly due to lack of
APC/C Ama1 activity.
Mnd2p prevents premature meiotic Cdc20p
The above results suggest a model in which Ama1p regulates Cdc20
degradation as the cells exit from meiosis II. Although Ama1p is
synthesized early in the meiotic program ( Cooper et al. , 2000 ), it is
kept inactive until Mnd2p is degraded ( Oelschlaegel et al. , 2005 ).
Therefore it would be predicted that Cdc20p would be prematurely
degraded during meiosis in mnd2Δ cells. To test this possibility,
Cdc20p-18myc levels were followed in wild-type and mnd2Δ cul-
tures during meiosis. The results show that Cdc20p fails to accumu-
late in mnd2Δ cells compared with the wild type ( Figure 1D ). North-
ern analysis confi rmed that CDC20 mRNA was expressed in mnd2Δ
cells (Supplemental Figure S1B). This result is consistent with the
model that APC/C Ama1 regulates Cdc20p degradation as cells exit
from the meiotic divisions.
Ama1p is required for normal meiotic nuclear divisions
We had previously reported that ama1Δ cells arrest before meiosis
I ( Cooper et al. , 2000 ). However, other studies using the SK1 back-
ground reported that Ama1p is dispensable for meiosis I or
meiosis II ( Coluccio et al. , 2004 ; Oelschlaegel et al. , 2005 ; Penkner
et al. , 2005 ; Diamond et al. , 2009 ). Reexamination of our strain
revealed that it does duplicate its spindle pole bodies ( Figure 2A )
indicative of nuclear divisions taking place. This is consistent with
our previous fi nding that early and middle genes are expressed in
ama1Δ cells but not loci required for spore wall formation ( Cooper
et al. , 2000 ). However, 70% of the ama1Δ mutants exhibited
uneven nuclear divisions ( Figure 2A and “other” category in
Figure 2B ). Closer examination revealed that cells with aberrant
nuclear divisions also possessed defective spindle formation ( Fig-
ure 2C ). To conclude, we concur with other published reports that
ama1 mutants can execute meiotic divisions. However, the data
presented here show that ∼20% ama1Δ mutants arrest before mei-
osis I and that the segregation of nuclei in cells that do undergo
nuclear divisions is abnormal.
FIGURE 2: Ama1p is required for normal meiotic divisions.
(A) Wild-type (WT) (KCY224) and ama1Δ mutant (KCY225) harboring
integrated Tub4p-GFP were induced to enter meiosis. Samples were
fi xed and analyzed by fl uorescence microscopy at the time points
indicated. Nuclear, spindle, and spindle pole body morphologies were
determined by DAPI staining (top), indirect immunofl uorescence of
tubulin, and direct immunofl uorescence of Tub4p-GFP, respectively.
Magnifi cation is 1000×. (B) Terminal meiotic arrest phenotype of
ama1Δ mutant. WT and ama1Δ strains were induced to enter meiosis,
and samples were taken after 24 h. Cells were scored as described in
Materials and Methods . “Other” represents cells exhibiting irregular
DAPI and tubulin staining. For all morphology quantitations
presented, the SDs were ≤6% for all values. (C) Indirect
immunofl uorescence of tubulin and DAPI analysis of WT (RSY335) and
ama1Δ (RSY562) cells after 24 h in sporulation medium. 1200×
318 | G. S. Tan et al. Molecular Biology of the Cell
meiosis. To verify that the CB and IR motifs
direct Cdc20p binding to the APC/C during
meiosis, coimmunoprecipitation experi-
ments were performed 9 h after the cells
entered meiosis. These studies indicate that
Cdc20p CBΔ/IRΔ is defective in APC/C binding
( Figure 4B ). Coimmunoprecipitation studies
with the single CB or IR motif mutants re-
vealed a subtle reduction in binding effi -
ciency. These fi ndings indicate that, as
expected, the CB and IR motifs are re-
quired for Cdc20p-APC/C association during
We next examined the expression profi le
of Cdc20p CBΔ/IRΔ during meiosis. No differ-
ences were observed in the accumulation or
degradation of Cdc20p CBΔ/IRΔ compared with
the wild type ( Figure 4C ). These results indi-
cate that Cdc20p destruction occurs inde-
pendently of activator-binding motifs. In ad-
dition, no defect in meiotic progression, as
measured by the appearance of bi- and tet-
ranucleated cells, was noted, indicating that
Cdc20p CBΔ/IRΔ did not exhibit a dominant-
negative effect (unpublished data). These
observations are consistent with a model in
which Ama1p recognizes Cdc20p as a
substrate, recruiting it to the APC/C for
degradation as cells exit from meiosis II.
Two destruction motifs are utilized for
APC/C Ama1 -mediated destruction of
Cdc20p contains four motifs (two destruc-
tion boxes, one KEN box, and GxEN; see
Figure 5A ) previously implicated in APC/C-
directed ubiquitylation ( Glotzer et al. , 1991 ;
Pfl eger and Kirschner, 2000 ; Littlepage and Ruderman, 2002 ). De-
struction box 1 (Db1) mediates partial Cdc20p degradation during
mitotic divisions ( Prinz et al. , 1998 ; Goh et al. , 2000 ). To address
which of these motifs are required for Cdc20p proteolysis during
meiotic progression, the mutations described in Figure 5A were in-
troduced individually into CDC20- 18myc (pMsc7). Wild-type cells
expressing these various mutant proteins were then induced to en-
ter meiosis and their degradation profi les monitored by Western
analysis. The results show that individually mutating these elements
did not alter Cdc20p-18myc degradation profi les ( Figure 5B ; Sup-
plemental Figure S2, A and B; and quantitated in Supplemental
Figure S2C), indicating that none of these motifs are solely respon-
sible for Cdc20p degradation.
A subset of APC/C substrates (e.g., Clb2p, Hsl1p) can require
more than one degron for their effi cient destruction (Burton and
Solomon, 2001 ; Hendrickson et al. , 2001 ). To determine whether this
is also the case for meiotic Cdc20p degradation, wild-type cultures
harboring plasmids bearing mutations in different combinations were
examined as described above. The results show that combining the
Db1, Db2, and KEN degrons does not signifi cantly affect the rate of
Cdc20p degradation compared with the wild type ( Figure 5C and
quantitated in Supplemental Figure S2D). However, combining the
GxEN and Db1 mutations stabilized Cdc20p similarly to that observed
in ama1Δ cells (compare to Figure 1A ). These results indicate that ei-
ther the Db1 or GxEN degron can mediate the meiotic destruction of
Cdc20p destruction ( Figure 3C ). However, Cdc20p was substantially
protected from degradation in the Ama1p CBΔ/IRΔ double mutant
( Figure 3C , bottom), suggesting that the phenotypes of these two
mutations are additive. These results indicate that Ama1p activity is
dependent on its conserved APC/C-binding motifs. In addition, our
fi ndings suggest that these domains are performing separate roles
in mediating APC/C Ama1 activity.
Cdc20p association with the APC/C is not required for its
Cdc20p is both an activator and substrate of the APC/C. If these
processes were separate, we would predict that APC/C association
through its CB or IR domain would not be required for Cdc20p de-
struction. To test this model, both the CB and IR domains were mu-
tated in a Cdc20p derivative and introduced into a cdc20-1 strain
(RSY809). This culture, along with wild-type and vector controls,
were induced to enter meiosis at the permissive temperature (23°C).
After 4.5 h, and before the fi rst meiotic division, the cells were
switched to the restrictive temperature (34.5°C). Cells harboring the
wild-type plasmid progressed through the meiotic program nor-
mally and produced viable spores ( Figure 4A ). Cells harboring either
the vector or Cdc20p CBΔ/IRΔ -expressing plasmids arrested before the
fi rst meiotic division. As cdc20 mutants arrest before meiosis I with
high Pds1p levels (Salah and Nasmyth, 2000 ), this result indicates
that the CB and IR motifs are required for APC/C Cdc20 activity during
FIGURE 3: The C-box (CB) and IR motifs are required for normal meiotic nuclear divisions.
(A) An ama1Δ culture (RSY562) harboring the AMA1 expression plasmids indicated above each
panel was transferred to sporulation medium for 18 h and then analyzed by DAPI staining
(bottom) or Nomarski imaging (top) to monitor nuclear segregation and spore formation,
respectively. The percentage of sporulation was determined as described in Materials and
Methods . The relative viability was scored with the wild-type (WT) value set at 100%. (B) The CB
domain is required for Smk1p activation. SK1 diploid CMY15 ( ama1Δ with an integrated
SMK1 -HA allele) harboring plasmids expressing either no Ama1p (vector), the WT T7-tagged
Ama1p (pKC3036), mutant Ama1p CBΔ (pKC3045), Ama1p IRΔ (pKC3087), or Ama1p CBΔ/IRΔ
(pKC3048) was induced to enter meiosis. After 10 h, samples were analyzed by Western blot
using an HA antibody. The upper and lower immunoreactive species (arrows) correspond to the
hyperphosphorylated (active) and hypophosphorylated (inactive) forms of Smk1p, respectively.
(C) CB and IR motifs are required for Cdc20p destruction. Yeast strain RSY853 ( ama1Δ , CDC20-
18myc) harboring the same plasmids as (B) was induced to enter meiosis and time points were
taken as indicated. Western blot analysis of protein extracts was conducted to detect Cdc20p.
The blots were stripped and reprobed for Tub1p, which served as a loading control.
Volume 22 February 1, 2011 Ama1p-activated anaphase-promoting complex | 319
Cdc20p. As before, meiotic progression was assessed by the appear-
ance of bi- and tetranucleated cells. Again, no difference in the rate of
meiotic progression was noted in cultures expressing the Db1/GxEN
double mutant (unpublished data), suggesting that Cdc20p destruc-
tion is not essential for meiotic progression (discussed below).
GxEN does not mediate mitotic Cdc20p degradation
Db1 was previously shown to mediate partial destruction of Cdc20p
in vegetative cells ( Prinz et al. , 1998 ). To investigate whether GxEN
is also involved in the mitotic destruction of Cdc20p, we examined
the stability of Cdc20p GxEN following release from α-factor–induced
G1 arrest in wild-type vegetative cultures ( Prinz et al. , 1998 ). Before
release, wild-type strains expressing galactose-inducible CDC20 al-
leles ( CDC20 , CDC20 Db1 , CDC20 GxEN , CDC20 Db1/GxEN ) were treated
with glucose and cycloheximide to stop transcription and translation
of these genes, respectively (see Materials and Methods for details).
Samples were taken and Western blot analysis was used to monitor
Cdc20p-24myc levels. As previously reported ( Prinz et al. , 1998 ;
Goh et al. , 2000 ), partial stabilization of Cdc20p was observed in the
Db1Δ strain ( Figure 6A ). However, mutating GxEN did not affect
Cdc20p destruction compared with the wild type. Mutating both
Db1 and GxEN did not appear to stabilize Cdc20p more than the
single Db1Δ mutation. Interestingly, all the galactose-inducible
CDC20 constructs are functional as determined by their ability to
complement the temperature-sensitive growth defect associated
with a cdc20-1 mutation ( Shirayama et al. , 1999 and Figure 6B ).
However, strains expressing the Db1/GxEN double mutant exhib-
ited a reduced plating effi ciency compared with strains expressing
the wild type or any single mutant allele ( Figure 6B ), suggesting that
overexpression of the double mutant is deleterious to mitotic cell
division. To conclude, these experiments show that Db1 is required
for normal Cdc20p destruction during mitotic cell division, but both
Db1 and GxEN are active in meiotic cells.
Cdc20p destruction is not required for meiotic progression
Our results suggest that Cdc20p is destroyed by APC/C Ama1 -
mediated degradation upon exit from the second meiotic division.
As described in Figure 2 , Ama1p is required for normal meiotic nu-
clear divisions and spore morphogenesis. To determine whether
these phenotypes are the result of the failure to destroy Cdc20p, we
placed wild-type CDC20 (pKC5069) and the Db1/GxEN double mu-
tant (pKC5070) under the control of AMA1 promotor on single-copy
plasmids. The AMA1 promotor was chosen as AMA1 and CDC20
are transcribed at similar times during meiosis. Moreover, placing
CDC20 under the control of a meiotic promotor alleviates potential
meiotic effects due to the vegetative growth defects associated with
Cdc20p Db1ΔGxENΔ expression (see Figure 6B ). This mutant CDC20 al-
lele was able to complement the cdc20-1 meiotic phenotype, allow-
ing the cells to form tetrads at wild-type rates at both permissive
and restrictive temperatures (unpublished data). In vegetative cells,
stabilizing Cdc20p impacts cell division only when combined with
signifi cant overexpression ( Shirayama et al. , 1999 ; Robbins and
Cross, 2010 ). Thus we repeated these experiments using high-copy
versions of the same plasmids (see Supplemental Table S3). In addi-
tion to examining the effi ciency of spore formation, meiotic progres-
sion was also monitored by 4′,6-diamino-2-phenylindole (DAPI)
analysis. There was no change in the appearance of tetranucleated
cells ( Figure 6C ) or spore formation/viability ( Figure 6D ). This result
was not due to plasmid loss as the vector was still present in dis-
sected spore colonies (unpublished data). Taken together, these
data indicate that Cdc20p destruction is not required for exit from
meiosis II or spore formation.
FIGURE 4: Cdc20p binding to the APC/C is not required for its
destruction. (A) The CB and IR regions are required for Cdc20p
function. A cdc20-1 strain (RSY801) harboring plasmids expressing
wild-type (WT) CDC20 or the CDC20 CBΔ/IRΔ alleles were induced to
enter meiosis and then shifted to the restrictive temperature to
inactivate Cdc20-1p. After 24 h, samples were taken for DAPI analysis.
(B) The CB and IR regions are required for Ama1p binding to the
APC/C. The Cdc16-TAP–expressing strains (RSY1055) harboring either
vector, WT (pMSC8), or mutant versions of Cdc20p (CBΔ/IRΔ =
pKC5022, CBΔ = pKC5020, IRΔ = pKC5021) were induced to enter
meiosis and cells harvested for analysis at 9 h, when both CDC16 and
CDC20 are expressed ( Cooper et al. , 2000 ). Western blot analysis was
conducted to detect the presence of Cdc16p-TAP in Cdc20p-18myc
immunoprecipitates. The top and middle panels control for protein
expression. (C) WT (RSY1055) strains harboring either Cdc20p-18myc
(pMSC7) or Cdc20 CBΔ/IRΔ -18myc (pKC5065) centromere plasmids were
induced to enter meiosis and time points taken as indicated. Western
blot analysis of protein extracts was conducted to detect Cdc20p.
The blots were stripped and reprobed for Tub1p, which served as a
320 | G. S. Tan et al. Molecular Biology of the Cell
coimmunoprecipitation studies were conduced in a strain harboring
the nonfunctional allele of Ama1p (Ama1p CBΔ/IRΔ ). Samples were
taken following 9 h in sporulation medium (SPM), when both Ama1p
and Cdc20p are expressed. The results show that Cdc20p can coim-
munoprecipitate with Ama1p ( Figure 7A, lanes 2 and 5 ), suggesting
that these two proteins interact in vivo. This interaction was not sim-
ply a result of the protein extraction procedure as coimmunopre-
cipitation between Ama1p CBΔ/IRΔ and Cdc20p was not observed
when these proteins were isolated from separate extracts and then
mixed (Supplemental Figure S3). An alternative scenario is that
Cdc20p and Ama1p are simply binding two independent sites on
the APC/C. To rule out this explanation, the experiment was re-
peated with the Cdc20p CBΔ/IRΔ , which cannot associate with the
APC/C ( Figure 4B ). These results also indicate that Ama1p can
FIGURE 5: Identifi cation of Cdc20p degrons. (A) Location of
destruction boxes, KEN and GxEN motifs, in Cdc20p. Amino acid
substitutions corresponding to the mutations generated are shown
below the sequence. (B) Cdc20p deleted for either Db1 or GxEN is
destroyed during meiosis with the same kinetics as wild type.
Wild-type cells (RSY335) harboring the Cdc20p-18myc centromere
plasmids indicated (see Supplemental Table S3 for details) were
induced to enter meiosis and samples taken at the time points shown
for Western analysis. The blots were stripped and reprobed for Tub1p
as a loading control. (C) Cdc20p utilizes two degrons (Db1 and GxEN)
for its destruction during meiosis. Cells were treated as described
above except that Cdc20p harbored the mutations indicated.
The Db1 and GxEN degrons of Cdc20p are recognized by
Ama1p during meiosis
If Cdc20p is a substrate of APC/C Ama1 , it should interact with Ama1p
in vivo. As association with Ama1p could lead to Cdc20p destruction,
FIGURE 6: The GxEN degron does not function in mitotic cells.
(A) Wild-type cells (RSY10) harboring either WT (pUS995), Db1
(pKC5006), GxEN (pKC5009), or Db1/GxEN (pKC5016) galactose-
inducible Cdc20p-24myc plasmids were grown in raffi nose to mid-log
phase, arrested in G1, and then induced by the addition 2%
galactose. Degradation of Cdc20p was monitored by Western analysis
at the time points indicated following G1 release. The blots were
stripped and reprobed for Tub1p as loading control. (B) The Cdc20p
Db1, KEN, GxEN, and Db1/GxEN mutants are functional. Mid-log
phase cdc20-1 cultures (RSY809) containing either vector (pRS426) or
galactose-inducible Cdc20p constructs (derived from functional
Cdc20p-24myc CEN plasmid [pUS995] using the oligonucleotides
described in Supplemental Table S2) were grown in raffi nose, serial
diluted (1:10), and spotted onto 2% galactose medium selecting for
plasmid maintenance at 23°C (permissive) and the restrictive
temperatures (34.5°C). Images were collected following 72 h
incubation. (C) Meiotic progression was monitored in cdc20-1 cultures
(RSY809) expressing either WT CDC20 (pKC5071) or the DB1/GxEN
mutant (pKC5072) under the control of AMA1 promotor. The cells
were induced to enter meiosis at the permissive temperature (23°C)
and switched to the restrictive temperature after 15 h in SPM. The
cells were analyzed by DAPI staining to monitor the appearance of
tetranucleated cells. (D) Nomarski imaging (top) and DAPI staining
(bottom) of cells described in (C) after 24 h in SPM. The percentage of
mono-, bi-, and tetranucleated cells in each culture was determined by
DAPI analysis. The relative spore viability was scored with the WT
value set at 100%.
Volume 22 February 1, 2011 Ama1p-activated anaphase-promoting complex | 321
2005 ; Kimata et al. , 2008 ). To determine
whether the Ama1p WD40 region also rec-
ognizes Cdc20p, coimmunoprecipitation
experiments were repeated with the
Ama1p 201–596 deletion mutant. Extracts were
prepared as just described and immunopre-
cipitated with the myc monoclonal antibody
(mAb). Western blot analysis of these immu-
noprecipitates did not detect Ama1p 201–596
although Ama1p CBΔ/IRΔ was observed as be-
fore ( Figure 8B , bottom, lanes 2 and 4). No
difference in the expression levels of
Ama1p 201–596 and Ama1p CBΔ/IRΔ was de-
tected (compare lanes 6 and 7 with lane 10),
suggesting that the WD region did not as-
sociate directly with Cdc20p. To verify this
conclusion, these experiments were re-
peated except that the Ama1p derivative
immunoprecipitates were probed for the
presence of Cdc20p-18myc. As expected, a
strong Cdc20-18 myc signal was observed
in Ama1p CBΔ/IRΔ -containing extracts. How-
ever, the Ama1p 201–596 signal was not above
the no-antibody control (compare lanes 7
and 8). These negative results do not ex-
clude the possibility that the WD40 region
interacts with Cdc20p. However, they do
show that if an interaction exists, it is below the limits of detection
using coimmunoprecipitation analysis of protein extracts.
Previously we have shown that APC/C Ama1 mediates the destruc-
tion of the B-type cyclin Clb1p as cells complete the meiotic nuclear
divisions ( Cooper et al. , 2000 ). To determine whether the amino-
terminal domain of Ama1p also binds Clb1p, the coimmunopre-
cipitation experiments described in Figure 8A were repeated using
Clb1p-3HA as the substrate. The results ( Figure 8C ) show that
Clb1p does coimmunoprecipitate with GST-Ama1p 1–200 above the
background level with GST alone. Taken together, these data indi-
cate that the amino terminus of Ama1p is suffi cient to bind Cdc20p
and Clb1p. However, GST-Ama1 1–200 is unable to complement the
sporulation defect of an ama1Δ strain (unpublished data), indicat-
ing that the WD40 region is also necessary for Ama1p function
Ama1-Cdh1 hybrid protein promotes APC/C Ama1 -specifi c
To further investigate the role of the conserved WD40 domain in
Ama1p activity, a domain swap experiment was performed. A fusion
protein was made that contained the amino-terminal domain of
Ama1p fused to the WD40-containing region of Cdh1p (Ama1p hybrid ;
see Figure 9A ). As a control, hemagglutinin (HA) epitope–tagged
CDH1 was placed under the control of the Ama1p promoter (Cdh1p-
HA). Both of these constructs produced full-length protein at the
same time during meiosis ( Figure 9B ). These constructs, plus plas-
mids expressing wild-type Ama1p, Ama1p CBΔ/IRΔ , or vector, were in-
troduced into an ama1Δ mutant host, and three APC/C Ama1 -specifi c
activities were assayed. The results show that the hybrid protein was
able to direct Cdc20p-18myc destruction with kinetics similar to the
wild type ( Figure 9C and quantitated in Figure 9D ). Next, the ability
of the hybrid protein to activate Smk1p was determined. Ama1p hybrid
expression was able to stimulate Smk1p activation as determined
by the increased ratio of phosphorylated to unphosphorylated spe-
cies of the MAP kinase above vector control levels ( Figure 9E ) but
not to the extent observed in the wild type. We also examined the
interact with Cdc20p CBΔ/IRΔ ( Figure 7A, lane 3 ), further supporting
the model that Cdc20p is a substrate of APC/C Ama1 .
Our previous results indicate both Db1 and GxEN motifs on
Cdc20p can mediate its meiotic destruction. To address whether
Ama1p recognizes these degrons, the coimmunoprecipitation ex-
periments described above were repeated with either wild-type
Cdc20p or Cdc20p Db1/GxEN and Ama1 CBΔ/IRΔ . The results ( Figure 7B )
show that the interaction between Ama1p and Cdc20p Db1/GxEN is
diminished compared with wild-type Cdc20p (compare lanes 3
and 4, bottom). Given the nonspecifi c interaction observed between
Cdc20p-18myc and protein G beads in these experiments (also see
Figure 8B ), it is diffi cult to assign any signifi cance to the apparent
association between Cdc20p Db1/GxEN and Ama1 CBΔ/IRΔ . However, the
genetic results presented in Figure 5 are consistent with a model
that a functional interaction with Ama1p is mediated through the
Db1 and GxEN degrons on Cdc20p.
The N-terminal domain of Ama1p directs Cdc20p
To identify the Ama1p domain(s) suffi cient for Cdc20p interaction,
expression constructs were generated containing either the di-
verged amino third of Ama1p (codons 1–200) or the more con-
served carboxyl region containing WD40 repeats (codons 201–
596). As the 1–200 portion of Ama1p is unstable when expressed
independently (unpublished data), this region was fused to gluta-
thione S -transferase (GST) under the control of the ADH1 pro-
moter. These plasmids, and the appropriate controls, were trans-
formed into a wild-type strain carrying an integrated copy of
CDC20- 18myc. After 9 h in SPM, samples were collected and pro-
tein extracts prepared for coimmunoprecipitation studies. These
experiments revealed that GST-Ama1p 1–200 associated with
Cdc20p-18myc, whereas GST could not ( Figure 8A ). These results
indicate that the amino-terminal region of Ama1p is suffi cient for
Previous studies have indicated that the Cdh1p WD40 domain
plays a role in degron recognition in Xenopus extracts ( Kraft et al. ,
FIGURE 7: The Db1 and GxEN degrons direct Ama1p association. (A) An ama1Δ mutant
(RSY562) harboring different Ama1p and Cdc20p expression plasmids (Ama1p CBΔ/IRΔ , pKC3048;
Cdc20p, pMSC8; Ama1p CBΔ/IRΔ , pKC3048; Cdc20p CBΔ/IRΔ , pKC5022; vector, pRS424) as indicated
was induced to enter meiosis and cells were harvested for analysis at 9 h, when both AMA1 and
CDC20 are expressed ( Cooper et al. , 2000 ). Immunoprecipitation and Western blot analyses
were conducted to detect the presence of both proteins (top and middle) or
coimmunoprecipitation (bottom). (B) Coimmunoprecipitation experiments performed as
described in (A) were repeated except with the inclusion of the Cdc20p DB1/GxEN -18myc
(pKC5023) expression plasmid. The no-antibody mock immunoprecipitation is represented by [ ].
322 | G. S. Tan et al. Molecular Biology of the Cell
from the Ama1p promoter, Cdh1p was unable to induce Cdc20p
destruction ( Figure 9C ), activate Smk1p ( Figure 9E ), or promote
meiotic nuclear divisions ( Figure 9F ). Likewise, Cdh1p under the
control of its own promotor also could not complement the ama1Δ
null defect (unpublished data). These fi ndings suggest that the
WD40 domain is interchangeable with respect to Cdc20p destruc-
tion, Smk1p activation, and nuclear divisions although the effi ciency
of function is reduced (see Discussion ). However, the hybrid protein
was unable to promote detectable spore wall formation. These re-
sults suggest that specifi c information included in the Ama1p WD40
region is required for function.
In this study, we demonstrate that the meiosis-specifi c APC/C Ama1
activity destroys Cdc20p at the end of meiosis II. Moreover, unlike
mitotic cell division, APC/C-dependent ubiquitylation is the primary
pathway leading to Cdc20p degradation. We show that Ama1p ac-
tivity is mediated through its conserved APC/C-binding CB and IR
motifs, although individually neither domain is essential for Ama1p
function. Using a combination of genetic and biochemical assays,
we demonstrate that Ama1p recognizes two degrons, Db1 and
GxEN, to dock with Cdc20p. The N-terminal domain of Ama1p me-
diates this activator/substrate interaction as well as interaction with
another APC/C Ama1 substrate, Clb1p. Finally, domain swap experi-
ments reveal that specifi c information is contained within the WD
region of Ama1p that is required for spore wall formation.
Model for substrate recognition by APC/C activators
It has been proposed that APC/C activators may have two function-
ally distinct roles: APC/C activation and substrate recognition
( Kimata et al. , 2008 ). This protein family is structurally similar in that
they contain a divergent amino third of the protein, with the remain-
ing two-thirds containing the conserved WD repeats. Currently
there is no consensus as to which region orchestrates substrate
binding ( Pfl eger et al. , 2001a ; Sorensen et al. , 2001 ; Burton et al. ,
2005 ). The results from several studies support a model that the N-
terminal region dictates target specifi city while the C-terminal do-
main is more involved in interacting with APC/C components (Zhang
and Lees, 2001 ; Burton et al. , 2005 ). Consistent with this possibility,
our data demonstrate that the amino-terminal region of Ama1p
binds Cdc20p through the Db1 and GxEN degrons. However, in
vitro cross-linking studies provided evidence that the carboxyl-
terminal WD40 region directly interacts with degrons (reviewed in
Thornton et al. , 2006 ; Benanti and Toczyski, 2008 ). However, we
could not detect an interaction between Cdc20p and the C-terminal
region of Ama1p ( Figure 8C ). One explanation is that the interaction
strength between the WD40 repeats and the degrons is below the
limits of detection using this assay. Alternatively, the different meth-
ods used to detect protein–protein interactions may be the source
of this discrepancy. Interestingly, domain swap experiments in which
we combined the amino-terminal domain of Ama1p with the Cdh1p
WD40 region provided most, but not all, of APC/C Ama1 function in
three different assays. These fi ndings would suggest that the WD40
region plays a more general role in providing APC/C activator func-
tion. However, expression of the hybrid protein was not able to
complement the spore wall assembly defect found in ama1Δ mu-
tants. This fi nding strongly argues against the notion that the WD40
domains are completely interchangeable. Rather, it supports the no-
tion that the WD40 domain of Ama1p also plays a specifi c role in
substrate recognition. Therefore the precise role the C-terminal
WD40 region plays with respect to Ama1p activity remains unde-
fi ned. One possibility is that this region could be required for
ability of the hybrid protein to promote normal meiotic nuclear divi-
sions and spore wall assembly. Expression of Ama1p hybrid was able
to stimulate meiosis I and meiosis II well above levels observed in
the vector control (31% vs. <0.5%, respectively). However, this level
was still below the wild-type value (75%; see Figure 3A ), suggesting
again that the hybrid protein was able to provide partial Ama1p
function. However, the hybrid protein did not allow spore wall as-
sembly ( Figure 9F ), as scored by the appearance of natural fl uores-
cence observed when yeast spore walls are exposed to UV light
(unpublished data). These data suggest that either the hybrid pro-
tein cannot suffi ciently activate Smk1p to promote spore wall as-
sembly or that another, unknown function of Ama1p is required that
is not provided by the hybrid protein. Finally, to assess the contribu-
tion of the Cdh1p WD40 domain for Ama1p-specifi c activities, we
tested the functionality of Cdh1p in these assays. When expressed
FIGURE 8: The N-terminus of Ama1p binds to Cdc20p. (A) Wild-type
cultures (RSY335) containing the indicated expression plasmids
(CDC20-18myc, pMsc8; GST-Ama1p1–200, pKC3070; GST, pQYAC-GST)
were induced to enter meiosis. After 9 h, the cells were harvested for
coimmunoprecipitation and Western blot analysis as indicated. The
top and middle panels control for protein expression. The bottom
panel assays coimmunoprecipitation. (B) The coimmunoprecipitation
experiments described in (A) were repeated with ama1Δ (RSY562)
cells harboring the indicated expression plasmids (vector, pRS426;
Ama1201–596p-T7, pKC3084; Cdc20–18myc, pMsc8; Ama1pCBΔ/IRΔp-T7,
pKC3048). (C) Clb1p, a previously identifi ed substrate of Ama1p
(Cooper et al., 2000), also associates with the amino-terminus of
Ama1p. As (A) except that cells were harvested after 12 h and
GST-Ama1p1–200 was coimmunoprecipitated with Clb1p-3HA (pKC430)
after 12 h. In all panels, [ ] represents the no-antibody mock
Volume 22 February 1, 2011 Ama1p-activated anaphase-promoting complex | 323
of this idea is the observation that ACM1
has two Ndt80p consensus sites, suggesting
that it is up-regulated after meiotic recombi-
nation is executed ( Chu et al. , 1998 ). An-
other intriguing observation resulting from
our studies is that progression through the
meiotic program is similar in wild-type and
cdh1 strains (as assessed by DAPI and
mRNA profi les). Despite this, Cdc20p levels
are signifi cantly down-regulated earlier in
meiotic cdh1Δ cells. If APC/C Ama1 triggers
this event, this would suggest that, in cdh1Δ
cells, APC/C Ama1 is precociously activated
similar to what we observe in an mnd2Δ mu-
tant. A possible mechanism for this could be
that APC/C Cdh1 regulates a protein whose
activity regulates Mnd2p. In this scenario,
Mnd2p is precociously inactivated in cdh1Δ
cells, thus allowing APC/C Ama1 to destroy
Cdc20p before the meiotic divisions are
completed. Alternatively, APC/C Cdh1 could
regulate a protein that directly activates
Ama1p. Thus, in the absence of APC/C Cdh1
activity, Ama1p is precociously activated to
destroy Cdc20p. However, it has been pro-
posed that both the removal of Mnd2p and
the inactivation of Cdk1 are required for
APC/C Ama1 activation as cells exit meiosis
( Diamond et al. , 2009 ). So do both these
events have to occur for APC/C Ama1 to be
precociously active? The answers to this
question may be addressed by studying the
role Cdh1p plays during meiotic divisions in
The APC/C and exit from meiosis
APC/C regulation is critical for proper exe-
cution of the meiotic divisions as unsched-
uled APC/C activity can lead to missegre-
gametes. Recently it was suggested that
APC/C inactivation at the end meiosis is
critical for embryonic development in
Drosophila (Pesin and Orr-Weaver, 2007 ).
Here the meiosis-specifi c APC/C activator
CORT (also known as CORTEX; Chu et al. ,
2001 ) is destroyed by APC/C FZY (Cdc20p
homologue) at the completion of meiosis
in the early embryo (Pesin and Orr-Weaver, 2007 ). Here we provide
a mechanism to inactivate APC/C Cdc20 as cells exit from meiosis II
in S. cerevisiae. In this model system, Cdc20p is destroyed by
APC/C Ama1 -mediated degradation, utilizing either the destruction
box and/or GxEN motifs as degrons. However, the destruction of
this activator is not essential, as introducing stabilized alleles of
CDC20 did not affect spore viability. Intriguingly, FZY levels are not
reduced in the early embryo (Pesin and Orr-Weaver, 2007 ). If inhib-
iting APC/C is important for normal gametogenesis, then these
results suggest that APC/C Cdc20 can be inactivated by alternative
mechanisms. For example, dephosphorylation of core APC/C sub-
units, possibly by PP1 or PPa2, decreases APC/C activity, although
the mechanism of this inhibition is not known (reviewed in Harper
et al. , 2002 ). Alternatively, or in addition, the association of APC/C
binding to the chaperonin-containing TCP1 ( Valpuesta et al. , 2002 ;
Camasses et al. , 2003 ), an event that is essential in activating Cdc20p
and Cdh1p during mitotic cell divisions ( Camasses et al. , 2003 ).
Relationship between Cdh1p and Ama1p during meiosis
Our results show that APC/C Ama1 , and not APC/C Cdh1 , down-regulates
Cdc20p as cells complete meiosis II. However, Cdh1p is expressed
during meiosis, with increased amounts of the activator being
present as cells execute the meiotic divisions (our unpublished data
and Chu et al. , 1998 ). How APC/C Cdh1 activity is regulated during
meiosis remains unknown. One possibility is that Acm1p, an
inhibitor of Cdh1p ( Martinez et al. , 2006 ; Choi et al. , 2008 ; Enquist-
Newman et al. , 2008 ; Ostapenko et al. , 2008 ) whose mRNA is also
expressed during meiosis, plays a role ( Chu et al. , 1998 ). In support
FIGURE 9: The Ama1-Cdh1p fusion protein partially complements ama1Δ phenotypes.
(A) Diagram depicting the proteins tested. N represents the amino-terminal divergent region,
C the carboxyl terminus–conserved WD40 repeat domain. All constructs are under the control
of the AMA1 promotor and terminator. (B) The expression of the AMA1 -hybrid and AMA1 pro -
CDH1 constructs were verifi ed in meiotic cultures. Extracts prepared from samples harvested
9 h following transfer to SPM were immunoprecipitated, and then Western blots were
performed with the indicated antibodies. (C) Cdc20p levels were followed in meiotic RSY853
( ama1Δ , Cdc20-18myc) harboring either the Ama1-hybrid (pKC3077), Cdh1p-HA (pKC3078),
Ama1p-T7 (pKC3056), or Ama1 CboxΔ/IRΔ p-T7 (pKC3057) expression plasmids or vector control.
(D) The Cdc20p signals obtained in (C) were quantitated and graphed. All Cdc20p values were
compared with Tub1p levels to control for protein quantitation. (E) Smk1p activation assays were
performed on CMY15 containing either an empty vector, AMA1 -T7, AMA1-hybrid , or AMA1 pro -
CDH1 –expressing plasmids as described in Figure 3B . (F) An ama1Δ culture (RSY562) harboring
plasmids expressing either Cdh1p-HA or Ama1p-hybrid was induced to enter meiosis and
harvested for analysis after 12 h in SPM. Nuclei and spindle microtubules were visualized by
DAPI (bottom) and indirect immunofl uorescence (top), respectively. The number of cells seen at
each stage of meiosis was scored as a percentage of the total cells counted. 1000× fi nal
324 | G. S. Tan et al. Molecular Biology of the Cell
Pst I restriction site into pVZCDH1 (W. Seufert) at amino acid 234
using oligonucleotides KCO230 and KCO231 to make pKC6000.
The 900–base pairs fragment encoding Cdh1p 234–566 was then in-
serted into the Pst I site of pKC3062 to make pKC3064 (called
Ama1p hybrid -T7). To place CDH1 under the control of AMA1 pro-
moter, the Spe I/ Sac I fragment from pWS216 (W. Seufert) contain-
ing functional CDH1- 3HA was placed downstream of the AMA1
promoter in pRS424 ( Christianson et al. , 1992 ) to form pKC3067
Protein extract preparation, coimmunoprecipitation, and
Protein extracts for coimmunoprecipitation and Western blot
analyses were prepared as described (Cooper and Strich, 1999 ),
except recombinant Protein G agarose beads (Invitrogen) were
used instead of Protein A resin. Western blot analysis and coim-
munoprecipitation experiments were conducted with 100 µg and
1 mg soluble protein, respectively. Epitope-tagged derivatives
were visualized as follows: Myc and HA by using mouse mAb
(Roche) at a fi nal concentration (FC) of 2 µg/ml; T7 with mouse
mAb (Novagen) at an FC of 0.1 ng/ml; TAP with rabbit polyclonal
antibody (Open Biosystems) at an FC of 0.4 µg/ml; and Tub1p
(tubulin) using rabbit polyclonal antibody (V. Guacci) at an FC of
0.1 µl/ml. Signals were quantitated using a Kodak Image Station
Quantitation of meiosis I and II execution was achieved by analyz-
ing DAPI-stained cells as described ( Akamatsu et al. , 1998 ;
Cooper and Strich, 2002 ). At least 200 cells were counted per
time point. For determining percent sporulation of ama1Δ cells
harboring various mutant plasmids, at least three independent
isolates were sporulated as described above and the results pre-
sented as the mean with standard deviation (SD). Spindle mor-
phology was determined using indirect immunofl uorescence as
described previously ( Cooper et al. , 2000 ) on meiotic cells fi xed
in 4% paraformaldehyde and 3.4% sucrose for 15 min. Cells were
scored as follows: prophase I, spindle smaller than nucleus; meta-
phase I, spindle the same width as nucleus; pseudo-anaphase,
spindle masses separate with elongated, but not separated, nu-
clei; and fragmented nuclei, more than four DAPI-staining bod-
ies, irregular-sized DAPI staining bodies, and/or misoriented
(more than two poles) spindle formation. The results presented
are the mean of the three strains with SD. Zymolyase was pur-
chased from US Biological (Z1004), and the rat α-tubulin mAb
and Alexa Fluor 594 anti-rat immunoglobulin G secondary anti-
body were obtained from Harlan Sera-Lab (MAS 078) and
Molecular Probes (A11007), respectively. An Olympus PROVIS
AX70 fl uorescence microscope was used for all experiments at a
fi nal magnifi cation of 1000×. Northern blots were performed as
described previously ( Cooper et al. , 2009 ).
inhibitors may play a role in down-regulating this ubiquitin
ligase. Taken together, these results suggest a model in which
inhibiting APC/C Cdc20 activity at the end of meiosis is important
for normal development and that this task is accomplished using
multiple mechanisms including Ama1p-dependent destruction
in yeast. Interestingly, Ama1p protein levels also decrease as
cells exit from meiosis II ( Cooper et al. , 2000 ). It will be of inter-
est to address whether this down-regulation is also APC/C
MATERIALS AND METHODS
Yeast strains and culture conditions
The strains used in this study (Supplemental Table S1) are isogenic
to RSY335 ( Cooper et al. , 1999 ) except RSY10, a W303 derivative
(see Figure 6, A and B) ( Strich et al. , 1989 ; Cooper et al. , 1997 ), and
CMY15, an SK1 strain used for Smk1 assays (E. Winter) ( McDonald
et al. , 2005 ). Our wild-type parent strain (RSY335) is derived from
the SK1 and W303 backgrounds. Strains harboring epitope-tagged
CDC20- 18myc were constructed using the integrating plasmid from
W. Zachariae or derivatives thereof. RSY1210 ( cdh1 :: LEU2 ) was
made using pWS176 (W. Seufert). All other deletion strains were
made by the method outlined by Longtine et al. ( 1998 ). Details are
available upon request. Meiotic time course and mitotic culture con-
ditions experiments were conducted as previously described
( Cooper et al. , 1997 ). To permit cdc20-1 cultures to exit mitosis and
enter meiosis, the cells were maintained at the permissive tempera-
ture (23°C) and then shifted to the restrictive temperature (34.5°C)
for the times indicated in the text. Cdc20p stability experiments in
G1-arrested cultures were performed as described ( Prinz et al. ,
Supplemental Tables S2 and S3 list the oligonucleotides and plas-
mids described in this study, respectively. Details of plasmid con-
structions are available on request. In brief, the Cdc20-myc18 plas-
mids pMSC7 (CEN) and pMSC8 (2µ) were derived from
pCDC20-myc18 (W. Zachariae). From these plasmids, mutations
were introduced using the QuikChange Site-directed Mutagenesis
(SDM) Kit (Stratagene) according to the manufacturer’s protocol. All
introduced mutations were verifi ed by DNA sequencing (MWG/
Operon). For the galactose induction experiments ( Figure 6 ), muta-
tions were introduced into pUS995 (galactose-inducible Cdc20p-
24myc) (U. Surana). The constitutive GST-Ama1 1–200 fusion construct
( ADH1 promotor; Figure 8A ) was made by inserting the PCR-
amplifi ed AMA1 1–600 open reading frame into pQYAC (Quattromed)
(oligonucleotides KCO171 and KCO173) to make pKC3070. The
ADH1 GST vector (pQYAC-GST) was made by inserting the Bam HI
fragment from pKC3070, containing just the GST open reading
frame, into pQYAC. Galactose-inducible GST-Ama1 fusion con-
structs were made by introducing AMA1 into pEG[KT], which con-
tains GST under the control of the galactose promotor (a gift from
The epitope-tagged derivative of AMA1 (pKC3036) was con-
structed by inserting a single 10–amino acid T7 epitope tag at the
amino terminus by SDM. The T7-tagged AMA1 - CDH1 hybrid
gene was constructed by introducing a Pst I site into pKC3036 at
amino acid 200 using oligonucleotides KCO204 and KCO205.
Next, the Pst I restriction site in the promoter of AMA1 was de-
leted using oligonucleotides KCO217 and KCO218 to form
pKC3061. The carboxyl terminus of AMA1 was then removed by
excising the internal Pst I fragment to form pKC3062. The carboxyl
terminus of CDH1 was inserted into pKC3062 by introducing a
We thank David Bardford, Wolfgang Zachariae, Uttam Surana,
Wolfgang Seufert, Mark Solomon, Edward Winter, and Andrew
Murray for the plasmids and Vincent Guacci for Tub1p antibodies.
We thank Michael Mallory for technical assistance. We thank Randy
Strich and Michael Law for helpful discussions and comments on the
manuscript and Edward Winter for his help with the Smk1p assays.
This work was supported by American Cancer Society grant
CCG106162 to K.F.C.
Volume 22 February 1, 2011 Ama1p-activated anaphase-promoting complex | 325
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Diamond AE, Park JS, Inoue I, Tachikawa H, Neiman AM ( 2009). The
anaphase promoting complex targeting subunit Ama1 links meiotic exit
to cytokinesis during sporulation in Saccharomyces cerevisiae . Mol Biol
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