A role for the anaphase-promoting complex inhibitor
Emi2?XErp1, a homolog of early mitotic inhibitor 1,
in cytostatic factor arrest of Xenopus eggs
Jeffrey J. Tung*†, David V. Hansen*†, Kenneth H. Ban*†, Alexander V. Loktev*, Matthew K. Summers*,
John R. Adler III*, and Peter K. Jackson*†‡
*Department of Pathology and†Program in Cancer Biology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305
Communicated by Marc W. Kirschner, Harvard Medical School, Boston, MA, February 9, 2005 (received for review November 30, 2004)
Unfertilized vertebrate eggs are arrested in metaphase of meiosis
fertilization, exit from metaphase is blocked by an activity called
cytostatic factor (CSF), which stabilizes cyclin B by inhibiting the
anaphase-promoting complex (APC) ubiquitin ligase. The APC in-
hibitor early mitotic inhibitor 1 (Emi1) was recently found to be
required for maintenance of CSF arrest. We show here that exog-
enous Emi1 is unstable in CSF-arrested Xenopus eggs and is
destroyed by the SCF?TrCPubiquitin ligase, suggesting that endog-
enous Emi1, an apparent 44-kDa protein, requires a stabilizing
Erp1?FBXO43, a homolog of Emi1 and conserved APC inhibitor.
Emi2 is stable in CSF-arrested eggs, is sufficient to prevent CSF
release, and is rapidly degraded in a Polo-like kinase 1-dependent
manner in response to calcium-mediated egg activation. These
results identify Emi2 as a candidate CSF maintenance protein.
cyclin B ? meiosis ? maturation-promoting factor ? oocyte maturation
penetration triggers the release from metaphase arrest and the
division in the embryo. The regulatory basis for metaphase II
arrest was first characterized in frog eggs ?30 years ago and
termed cytostatic factor (CSF) (1). CSF is operationally defined
as an activity, rather than a single molecule, present in unfer-
tilized eggs that blocks cleavage of dividing blastomeres upon
injection (reviewed in ref. 2). Mos, an activator of the mitogen-
activated protein kinase?Rsk pathway, is a key component of
to block cleavage of blastomeres (3).
The anaphase-promoting complex (APC) is an E3 ubiquitin
ligase that triggers M-phase exit by directing proteasome-
dependent cyclin B destruction (4), resulting in the swift inac-
tivation of the cyclin B?Cdc2 kinase, or maturation- promoting
factor (MPF) (5, 6). A rise in intracellular calcium after fertil-
ization induces metaphase II release by relieving the APC from
repression. Early mitotic inhibitor 1 (Emi1), originally cloned
from a Xenopus oocyte cDNA library, blocks the cleavage of
injected blastomeres similar to CSF (7) and efficiently inhibits
the APC in vitro (8). Recently, Emi1 was shown to be required
for maintenance of CSF arrest in frog and mouse eggs. Immu-
nodepletion of Emi1 from Xenopus CSF egg extract causes rapid
cyclin B proteolysis and exit from metaphase arrest independent
of calcium mobilization, and ablation of Emi1 by small interfer-
ing RNA in mouse oocytes induces parthenogenesis (9, 10).
Recent work has shown that the Mos?mitogen-activated protein
kinase?Rsk pathway establishes, but is not required to maintain,
CSF arrest (11, 12). Therefore, CSF arrest is a complex process
established by the mitogen-activated protein kinase pathway and
maintained through inhibition of the APC.
o prevent parthenogenesis, unfertilized eggs from many
animals arrest in metaphase of meiosis II (MII). Sperm
Upon fertilization of Xenopus eggs, calcium signaling inactivates
CSF arrest, which requires the Xenopus Polo-like kinase 1 (Plx1).
somatic cells, MPF and human Polo-like kinase 1 (Plk1) target
Emi1 for degradation by the Skpl Cullin?F-box protein (SCF)?TrCP
ubiquitin ligase (14–17). Specifically, Plk1 phosphorylates Emi1 on
its DSGxxS sequence, creating a consensus degron recognized by
?TrCP (17). Thus, Xenopus Emi1 (xEmi1) could be a Plx1 target
levels are sustained in the CSF-arrested egg amid high MPF and
Emi1 is unstable and undetectable in Xenopus eggs (18). On the
other hand, Emi1 appears to be present in mouse eggs (10). In this
study, we want to clarify our understanding of Emi1 regulation in
to CSF arrest.
Reagents. Sera from four rabbits immunized with maltose binding
protein (MBP)-Emi1 fusion protein were affinity-purified by flow-
ing over a column of GST-Emi1 immobilized on CNBr-Sepharose
resin with acid elution. Other antibodies used were against ?-cate-
nin, cyclin B2, Plx1, Plk1 (Zymed), myc epitope, and actin (Santa
Cruz Biotechnology). xEmi2 was PCR-cloned from an oocyte
cDNA library, and a human Emi2 (hEmi2) clone was purchased
from Invitrogen. pCS2-cDNA constructs were linearized and in
kit (Ambion, Austin, TX). pCS2-cDNA constructs were in vitro-
translated (IVT) in rabbit reticulocyte lysate (TNT, Promega) and
labeled with35S-methionine. All Emi1 and Emi2 experiments used
human sequences. MBP-fusion proteins and GST-Plk1 were ex-
pressed in Escherichia coli and purified by batch binding bacterial
protein lysate to affinity resin and elution with maltose or gluta-
thione, then dialyzed into XB buffer (20 mM Hepes, pH 7.7?100
Handling of Xenopus Oocytes. Oocyteswereobtainedandprocessed
for H1 kinase activity and immunoblot as described (19). Oocytes
were injected with 30 ng of MBP-Emi1 fusion protein or 10 ng of
various mRNA in total volumes not exceeding 50 nl. Maturation
Freely available online through the PNAS open access option.
Abbreviations: APC, anaphase-promoting complex; CHX, cycloheximide; CSF, cytostatic
factor; Emi, early mitotic inhibitor; hEmi, human Emi; xEmi, Xenopus Emi; GVBD, germinal
vesicle breakdown; IVT, in vitro-translated; MI, meiosis I; MII, meiosis II; MBP, Maltose
binding protein; MPF, mitosis-promoting factor; Plk1, human Polo-like kinase 1; Plx1,
Xenopus Polo-like kinase 1; SCF, Skpl Cullin?F-box protein.
Data deposition: The sequence reported in this paper has been deposited in the GenBank
database (accession no. AY928267).
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
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was induced by treating oocytes with 10 ?g?ml progesterone. Eggs
were activated with A23187 ionophore (Sigma).
Destruction and APC Ubiquitination Assays.Eggextractwasprepared
as described (20). Destruction assays and in vitro APC ubiquitina-
tion reactions were performed as described (8).
Immunodepletion and in Vitro Phosphorylation Assays. Plx1 immu-
nodepletion, Plk1 in vitro kinase reactions, and ?TrCP binding
assays were performed as described (17).
Immunofluorescence Microscopy. Staining of Emi1 in a Xenopus
cell line (XTC) and human cell lines was performed as described
Characterization of Anti-Emi1 Antibodies. To examine Emi1 expres-
sion levels, high titer sera selected from the best four of six rabbits
immunized with recombinant MBP-Emi1 fusion protein were
purified against immobilized GST-Emi1 by affinity chromatogra-
phy. These four affinity-purified antibodies (ab1–4) vary in affinity
molecular mass of 44-kDa Emi1 in CSF extract (Fig. 1A). All four
antibodies, using crude serum or affinity-purified antibodies, im-
munoprecipitate IVT myc-Emi1 (Fig. 1B).
We tested whether the 44-kDa band in CSF extract recognized
by ab1, the most specific and highest-affinity antibody of the four
in hand, is indeed Emi1. Preincubating ab1 with increasing MBP-
Emi1 protein almost completely blocked detection of the 44-kDa
band (Fig. 1C). Incubating ab1 with MBP at twice the blocking
concentration of MBP-Emi1 did not block recognition of the
44-kDa species. Moreover, blotting ab1 and ab2 immunoprecipi-
tates from egg extract with ab1–3 showed that all three antibodies
recognize the same 44-kDa band (Fig. 1D). Using ab1, we estimate
the concentration of 44-kDa Emi1 in CSF extract to be ?50 nM
(Fig. 7, which is published as supporting information on the PNAS
web site), somewhat lower than our previous estimate of 300 nM
(9). Additional validation demonstrated that these Emi1 antibodies
detect overexpressed Emi1 and the endogenous 44-kDa protein in
oocytes, embryos, and XTC cells (Fig. 7; see also Fig. 8, which is
published as supporting information on the PNAS web site).
localization of Emi1 in XTC cells by immunofluorescence micros-
copy. hEmi1 localizes specifically to the spindle poles in a variety of
human cell lines (Fig. 1E and ref. 21). Importantly, this conserved
and specific localization of Emi1 at the spindle poles is observed by
(7). Emi1 depletion in human cell lines by small interfering RNA
abolishes the detection of Emi1 at spindle poles (data not shown).
However, we could not validate ab1 in a similar fashion because we
have found that XTC cells are refractory to small interfering RNA
whether neutralizing Emi1 in CSF extract triggers calcium-
independent metaphase release. Addition of ab1, but not control
IgG, to CSF extract triggered rapid decline of cyclin B2 levels and
Cdc2 activity and induced morphological decondensation of sperm
the effect of ab1 on meiotic progression (27). Taken together, the
above immunological evidence and conserved localization of Emi1
suggest that these antibodies most likely detect Emi1 or a highly
related protein in the oocyte, CSF-arrested egg, embryo, and XTC
Exogenous Emi1 Is Destroyed in CSF-Arrested Eggs. In mitotic egg
extract prepared by adding nondestructible ?90 cyclin B to
44-kDa protein is recognized by affinity-purified anti-
Emi1 antibodies in CSF-arrested eggs. CSF extract was
immunoblotted with affinity-purified antibodies from
four rabbits immunized against Emi1. (B) Anti-Emi1 anti-
bodies immunoprecipitate expressed Emi1. IVT myc-Emi1
purified antibodies, but not by preimmune (PI) sera. (C)
Emi1 antibody recognition of the 44-kDa species is
blocked with antigen. CSF extract was blotted with affin-
excess of MBP protein over antibody. (D) Each of the
anti-Emi1 antibodies detects the same 44-kDa protein.
Immunoprecipitates from CSF extract with two anti-Emi1
anti-Emi1 antibodies. (E) Anti-Emi1 antibody detects con-
served Emi1 localization to the spindle poles. Metaphase
chromosomes, spindles, and Emi1 were visualized in Xe-
nopus somatic XTC cells, human U2OS cells, and human
ages show DNA (blue), ?-tubulin (red), and Emi1 (green).
(Magnification: ?63.) (F) Addition of anti-Emi1 antibody
to CSF extract induces chromatin decondensation, MPF
inactivation, and cyclin B destruction without calcium ad-
and treated with anti-Emi1 antibodies or control IgG.
After 60 min, sperm chromatin was stained with Hoechst
and visualized by epifluorescence microscopy. Similar ex-
tract was incubated with anti-Emi1 antibodies or IgG and
activity and immunoblot analysis. A nonspecific band (*)
Characterization of anti-Emi1 antibodies. (A) A
Tung et al.PNAS ?
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interphase extract, IVT Emi1 requires MPF for destruction (7).
We suspected that although CSF extract contains high MPF
activity IVT Emi1 would be refractory to destruction because
Emi1 is required to maintain the CSF-arrested state. Instead, we
found that radiolabeled IVT Emi1 is degraded with similar
kinetics in mitotic and CSF extract (t1/2? 15 min; Fig. 2A). In
contrast, IVT Emi1 is stable in interphase extract for ?120 min
(data not shown). hEmi1 is also destroyed in CSF extract (Fig.
9, which is published as supporting information on the PNAS
web site). IVT Emi1 mutated in a critical serine residue (Ser-95)
of the consensus ?TrCP recognition degron is stable in CSF
extract (Fig. 2B), indicating that CSF extract contains the factors
required to ubiquitinate and destroy exogenous Emi1 through its
Emi1 levels in the egg may reflect the steady-state accumu-
lation of unstable Emi1 protein. Therefore, we examined
whether translation of myc-Emi1 mRNA in CSF would allow
Emi1 to accumulate. Translated WT Emi1 accumulates to
modest steady-state levels (Fig. 2C), whereas nondestructible
Emi1 S95N mutant accumulates to increasingly high levels,
suggesting that WT Emi1 is simultaneously translated and
destroyed. To assess the stability of translated exogenous Emi1
directly, cycloheximide (CHX) was added to the CSF extract 2 h
after transcript addition. WT Emi1 is rapidly destroyed (?15
min) in contrast to stable Emi1 S95N (Fig. 2C). Thus, newly
synthesized Emi1 appears to be dynamically accumulated by a
balance of translation and destruction, but could potentially
accumulate to higher levels if a pool of the protein was seques-
tered from destruction.
G2oocytes contain a stockpile of inactive MPF that is robustly
activated at the onset of germinal vesicle breakdown (GVBD)?MI
in response to hormonal stimulation (22). Thus, Emi1 may have
important functions in G2-MI oocytes but could be destroyed after
MPF activation after MI. We injected WT Emi1 or Emi1 S95N
G2 oocyte, although Emi1 S95N accumulates at much higher
Expression of dominant negative ?TrCP (?TrCP?F) missing the
F-box domain enables exogenous WT Emi1 protein to accumulate
to similar levels as Emi1 S95N in the MII egg (Fig. 2D). Thus,
SCF?TrCPis active in the egg and directs exogenous Emi1 for
proteolysis. The stability of both endogenous and exogenous Emi1
in the G2oocyte would be consistent with a role in stabilizing APC
substrates during G2-MI or possibly for the MI–MII transition, as
seen in mouse (12).
Endogenous Emi1 Is Stabilized in CSF-Arrested Eggs. Next, we deter-
mined whether endogenous Emi1 is stable in MII eggs with
characterized antibodies. CSF extract was treated with CHX,
incubated with MBP-Emi1 protein (300 nM final concentration),
and processed for immunoblotting at the indicated times postad-
ditions. MBP-Emi1 is destroyed with similar kinetics as IVT Emi1
(Fig. 2E). Strikingly, levels of the 44-kDa protein detected by
anti-Emi1 antibodies remain unchanged for up to 120 min in this
destruction assay. Moreover, in CHX-treated CSF extract, the
44-kDa protein is extremely stable, with no apparent degradation
in 48 h (Fig. 9B). Emi1 stability is also observed in vivo, as the
when MPF first appears in MI (Fig. 2F). Differential stability of
endogenous and exogenous forms of proteins is not uncommon.
in CSF extract by SCF?TrCP, yet endogenous ?-catenin remains
stable (Fig. 10, which is published as supporting information on the
Emi1, these results suggest that Emi1 is a stable protein in the egg
and a mechanism exists to protect it from SCF?TrCP.
Emi2 Is a Homolog of Emi1 and Crossreacts with Anti-Emi1 Antibodies.
Given that our anti-Emi1 antibodies dramatically inactivate CSF
maintenance in egg extract, we considered the possibility that a
previously unidentified Emi1 homolog could be immunologically
nonetheless, Emi1 can accumulate by de novo translation. (A) IVT Emi1 is
destroyed in mitotic extract and CSF extract. IVT, radiolabeled Emi1 was
incubated in mitotic ?90 cyclin B extract or CSF extract and processed for
CSF extract is conserved and DSGxxS sequence-dependent. IVT [35S]Met Emi1
or Emi1 S95N was added to CSF extract and processed for autoradiography at
the indicated times. (C) Accumulation of exogenous Emi1 protein translated
in CSF egg extract. myc-Emi1 mRNA (WT or S95N mutant) was added to CSF
extract with or without CHX and processed for immunoblot at the indicated
or simultaneously with myc-Emi1 and ?TrCP?F mRNA. Injected oocytes were
left at G2arrest or matured by progesterone stimulation and processed for
immunoblot. (E) Endogenous Emi1 is protected from destruction in CSF ex-
CHX and prepared for immunoblot at the indicated times after additions. (F)
Endogenous Emi1 is stable in the maturing oocyte. Stage VI oocytes were
injected with purified MBP-Emi1 protein and induced to mature by proges-
terone treatment. Emi1 was detected by immunoblotting lysates from imma-
ture oocytes, MI (GVBD) oocytes, and metaphase II eggs.
Exogenous Emi1 protein is destroyed through SCF?TrCPin CSF extract;
www.pnas.org?cgi?doi?10.1073?pnas.0501108102 Tung et al.
crossreactive. A search of TBLASTN for xEmi1 identified a homol-
ogous ORF in Xenopus Fbx26. We originally identified Fbx26 from
a Xenopus oocyte cDNA library as a highly abundant Skp1 inter-
actor (384 of 444 clones isolated) in the same yeast two-hybrid
screen that identified Emi1. However, frameshift errors in the
similarity to Emi1. The correct Fbx26 ORF is entered into the
database as Emi2?Erp1 (GenBank accession no. AY928267), a
651-aa protein that is 25% identical to Emi1 that we refer to as
Emi2. Human and mouse Emi2 orthologs are given the systematic
on xEmi2 unless noted otherwise. An alignment of Emi1 and Emi2
orthologs is shown in Fig. 3A. Residues 434–651 of Emi2 are 35%
identical to Emi1, sharing conserved F-box and IBR domains.
Furthermore, Emi2 has conserved DSG-sequence degrons.
To determine whether anti-Emi1 antibodies detect denatured
Emi2 in addition to Emi1, anti-myc immunoprecipitates from 293T
cells transfected with pCS2 myc-Emi2 or pCS2 myc-Emi1 were
processed for immunoblotting with anti-Emi1 antibodies (Fig. 3B).
Although anti-Emi1 antibodies recognize a robust Emi1 band, no
myc-Emi2 were immunoprecipitated because both were easily
detected in a blot with anti-myc antibodies. We concluded that
On this basis, the 44-kDa band detected in egg extract is most likely
not a form of Emi2.
Next, we asked whether anti-Emi1 antibodies crossreact with
native Emi2. To test this idea, lysates from 293T cells transfected
with myc-Emi1 or myc-Emi2 were immunoprecipitated with anti-
Emi1 antibodies. Blotting the anti-Emi1 immunoprecipitates with
immunoprecipitate an IVT C-terminal fragment of Emi2 (Fig. 11,
which is published as supporting information on the PNAS web
site). These results suggest that one feasible explanation for the
ability of the anti-Emi1 antibodies to cause CSF release is through
neutralization of Emi2.
Emi2 Is Destroyed upon Egg Activation. The immunological cross-
reactivity of Emi1 and Emi2 prompted us to explore whether Emi2
exhibits properties consistent with a candidate CSF maintenance
protein. One of Masui and Markert’s (1) original postulates for the
identity of CSF is that it is inactivated upon egg activation in
response to calcium signaling. To test whether Emi2 fulfills this
criterion, we incubated radiolabeled IVT myc-Emi2 in CSF extract
in the absence or presence of calcium. The autoradiogram shows
that Emi2 is stable in CSF extract but, upon calcium addition,
becomes rapidly converted to an electrophoretically retarded form
consistent with ubiquitination and is subsequently destroyed (Fig.
4A). Furthermore, Emi2 appears to be phosphorylated by an
M-phase kinase, judging by its reduced electrophoretic mobility in
CSF extract but not in interphase extract.
Emi2 has two potential sequence degrons recognized by
SCF?TrCP, one DS34GxxDS39at the N terminus and a centrally
located DS284AxxS288 sequence. We asked whether these two
sequences contributed to Emi2 destruction upon CSF release by
calcium. Whereas WT Emi2 is rapidly phosphorylated and
ubiquitinated after calcium addition, mutating the N-terminal
DSGxxDS degron (DS33AA) prevents Emi2 ubiquitination and
Emi2 is an Emi1-related protein conserved in vertebrate species. A schematic
of Emi1 and Emi2 orthologs from human (H.s.), mouse (M.m.), and frog (X.l.)
is shown. The conserved C-terminal F-box and zinc-binding ‘‘in-between-
region’’ (IBR) domains are boxed. The identified ?TrCP degrons in hEmi1 and
xEmi1 and candidate degrons (DSG?A-X2-3-S?D?E) in Emi2 orthologs are
shown. (B) Anti-xEmi1-specific antibodies can immunoprecipitate native
xEmi2, but do not recognize denatured protein. HEK 293T cells were trans-
fected with pCS2 myc-xEmi1 or pCS2 myc-xEmi2. Lysates were prepared after
48 h and either directly blotted with anti-myc antibodies or immunoprecipi-
tated with anti-myc, anti-xEmi1 (Ab1), or control antibodies and then immu-
noblotted. The band indicated by*is an unknown anti-Emi1 crossreactive
species. The band indicated by**is IgG heavy chain.
Emi2, a homolog of Emi1, is recognized by anti-Emi1 antibodies. (A)
CSF extract after calcium addition. (A) Emi2 is destroyed during CSF release.
Full-length, radiolabeled IVT Emi2 was incubated in CSF extract with or without
Ca2?addition or in interphase extract for the indicated times. (B) Emi2 is de-
in CSF extract and destruction was assayed at the indicated times after calcium
myc-Emi2 and radiolabeled IVT ?TrCP were incubated in CSF extract with pro-
teasome inhibitors, with or without calcium addition, for the indicated times.
Anti-myc immunoprecipitates were analyzed for bound ?TrCP.
Tung et al. PNAS ?
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destruction (Fig. 4B). An Emi2 mutant lacking the central
DSAxxS sequence (DS283AA) is ubiquitinated and degraded
with similar kinetics as WT Emi2 in response to calcium
addition. Consistently, the stability of Emi2 mutant lacking both
candidate degrons (2xDS-AA) is indistinguishable from the
single DS33AA mutant, indicating that DS34GxxDS39 is the
primary degron involved in Emi2 destruction during egg acti-
vation. Human IVT myc-Emi2 stability is regulated essentially
the same way with one obvious exception: hEmi2 contains two
destruction motifs that both contribute to calcium sensitivity
(Fig. 12 A and B, which is published as supporting information
?TrCP degron, we asked whether calcium triggers ?TrCP bind-
ing to Emi2. Indeed, calcium addition to CSF extract promoted
the binding of radiolabeled ?TrCP to myc-Emi2 within 5 min
(Fig. 4C), consistent with the kinetics of Emi2 ubiquitination.
This finding suggests that Emi2 requires SCF?TrCPfor
Emi2 Inhibits the APC and Is Sufficient to Prevent CSF Release.
Because Emi2 is a homolog of Emi1, a well established APC
inhibitor, we tested the likely activity of Emi2 as an APC inhibitor.
Full-length Emi2 (MBP-Emi2) or a C-terminal fragment (MBP-
Emi2 CT) blocks the ability of the APCCdc20to polyubiquitinate
radiolabeled securin substrate in vitro as effectively as Emi1 (Fig.
5A). Similar results were obtained with Cdh1 as the APC activator
(Fig. 12C). These results indicate that the C terminus of Emi2,
which bears the most identity with Emi1, is sufficient to inhibit
We determined whether Emi2 is sufficient to prevent calcium-
induced CSF release. Addition of 2 ?M MBP-Emi2 to CSF extract
sperm nuclei decondensation (Fig. 5B). The slow partial decline in
cyclin B levels is most likely the result of calcium-induced Emi2
destruction. The 2 ?M concentration of MBP-Emi2 used here
appears to saturate the destruction machinery and suffices to block
CSF release in response to calcium. Finally, we examined whether
Emi2 destruction is a prerequisite for CSF release in vivo. Calcium
ionophore A23187 triggers the decline in cyclin B2 levels and H1
kinase activity in matured eggs injected at G2with water or WT
5C). Together with the finding that Emi2 inhibits the APC in vitro,
these results suggest that Emi2 is an APC inhibitor that must be
destroyed for CSF release.
Emi2 Is a Plk1 Target During CSF Release. Another potential parallel
between Emi1 and Emi2 is regulation by Plk1. Given that Plx1 is
required for Emi1 proteolysis in mitotic egg extract (17), we
supposed that Emi2 destruction during egg activation is a Plx1-
dependent process. Two rounds of immunodepletion using anti-
Plx1 antibodies effectively removed Plx1 from CSF extract (Fig.
6A). Consistent with prior work (13), calcium addition to Plx1-
depleted, but not mock IgG-depleted, CSF extract failed to trigger
cyclin B2 destruction and H1 kinase inactivation (Fig. 6B). Radio-
labeled IVT myc-Emi2 is rapidly destroyed upon calcium addition
to mock-depleted CSF extract, but remains stable in Plx1-depleted
anti-MBP immunoprecipitates shows that constitutively active
GST-Plk1 (T210D mutant) protein enhances the binding of radio-
labeled ?TrCP to full-length MBP-Emi2 fusion protein but not to
the C terminus of Emi2 lacking ?TrCP degrons (Fig. 6D). These
to calcium signaling by stimulating Emi2 and ?TrCP binding.
During our studies we learned that Ohsumi et al. (18) raised an
antibody against Emi1 and failed to detect a 44-kDa band by
immunoblot in the egg or developing embryo until gastrulation.
Consistent with our results, Ohsumi et al. observed the destruction
of exogenous Emi1 in CSF extract and maturing oocytes. However,
they concluded from the inability to detect endogenous Emi1 and
the observation that exogenous Emi1 is unstable in the egg that
Emi1 does not and cannot possibly exist in Xenopus until gastru-
lation. However, no evidence is provided that the ?44-kDa band,
which Ohsumi et al. only see appearing at 10–12 h postfertilization
in the embryo, is indeed Emi1. The species they see is induced at
gastrulation, shortly after zygotic transcription is activated (24), but
no specific validation or blocking experiment of the endogenous
band shows this species is Emi1.
To shed light on this discrepancy in greater detail, we charac-
terized four antibodies raised against Emi1 and present evidence
that the 44-kDa species detected by these antibodies, presumably
Emi1, is present in the egg. Importantly, another research group
Emi2 is an APC inhibitor. Recombinant hEmi1, hEmi2, an hEmi2 C-terminal
fragment (residues 541–708), or control (MBP) proteins (2 ?M) were tested for
complex. (B) Emi2 is sufficient to prevent CSF release. Addition of excess hEmi2
protein blocks the calcium-induced exit from MII in CSF extract. CSF extract was
initiated to exit MII by calcium addition and assayed for cyclin B2 destruction
extract blocked MII exit. (C) CSF release requires Emi2 destruction. Injection of
nondestructible xEmi2, but not WT, into maturing oocytes prevents MII exit. In
vitro-transcribed xEmi2 (WT or nondestructable 2x DS-AA mutant) was injected
into oocytes. Oocytes were matured with progesterone and harvested at GVBD
and various times afterward. At 3 h post-GVBD, oocytes were released from MII
arrest by addition of calcium ionophore A23187. Samples were immunoblotted
for cyclin B2 and assayed for H1 kinase activity.
Emi2 inhibits the APCCdc20complex and blocks exit from CSF arrest. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0501108102Tung et al.
readily detects Emi1 at constant levels during oocyte maturation Download full-text
with an independently raised antibody (T. Lorca, personal com-
munication). Ohsumi et al. propose that Emi1 cannot exist in the
egg because exogenous Emi1 is destroyed. However, we show here
that, although unstable, newly synthesized Emi1 can accumulate to
detectable steady-state levels. During its synthesis, Emi1 could be
sequestered by some cellular structure or stabilizing factor that
would allow higher levels of accumulation. The instability of
exogenous ?-catenin in egg extract provides an example of an
unstable protein that is sequestered in a stable complex (organized
is that the antibody used in the study failed to detect endogenous
Emi1 and these negative data by themselves are insufficient evi-
dence to support the idea that the protein does not exist.
On the other hand, we remain cautiously skeptical that the
44-kDa species detected by our antibodies is unambiguously Emi1
for the simple reason that Xenopus laevis is not an organism
allowing a direct gene knockout strategy to definitively settle this
matter. Nonetheless, our functional evidence strongly suggests that
in CSF arrest.
Bearing in mind that new synthesis of B-type cyclins was origi-
until the discovery of three additional cyclin B members a decade
later (26), we considered the existence of unidentified Emi1
homologs highly plausible. In this study, we identify Emi2 as an
Emi1 homolog that crossreacts with antibodies raised against
full-length Emi1 in immunoprecipitation experiments but not in
immunoblots. Thus, we conclude that the 44-kDa band detected by
anti-Emi1 antibodies in eggs is unlikely to be a form of Emi2, but
the possibility that the calcium-independent CSF release caused by
Emi1 immunodepletion in our previous work (9) and antibody
is a candidate CSF maintenance protein. In support for a role of
Emi2 in CSF arrest, we show here that (i) Emi2 is an APC inhibitor
sufficient to prevent CSF release, (ii) Emi2 destruction through its
SCF?TrCPrecognition sequence is a requirement for CSF release in
response to calcium signaling, and (iii) Emi2 is targeted for de-
struction by Plk1 upon CSF release. These observations strongly
demonstrated that disabling either Emi1 or Emi2 function without
perturbing the other causes loss of CSF maintenance.
Do the negative data from Ohsumi et al. (18) and the identifi-
cation of Emi2 justify the dismissal of the role of Emi1 in main-
ablation by small interfering RNA causes spontaneous egg activa-
tion, providing plausible genetic evidence that Emi1 is indeed
essential for CSF arrest (10). As was the case for B-type cyclins,
without the luxury of a complete X. laevis genome sequence
database, there may be additional members of the early mitotic
inhibitor family of proteins awaiting identification. As our current
knowledge stands, the production of Emi1?/?, Emi2?/?, and
double homozygous null mice will most constructively resolve the
relative importance of these two homologs in CSF arrest.
We thank James Nelson (Stanford University, Stanford, CA) for ?-catenin
antibody, William Dunphy (California Institute of Technology, Pasadena)
cyclin B2 antibody, and Thierry Lorca for communicating unpublished
results. This work was supported by Public Health Service Grants 5T32
CA09302-27 (to J.J.T.) and RO1 GM60439 and GM54811 (to P.K.J.).
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Tung et al. PNAS ?
March 22, 2005 ?
vol. 102 ?
no. 12 ?