Báo cáo khoa học: Apoptosis-inhibiting activities of BIR family proteins in Xenopus egg extracts ppt

Apoptosis-inhibiting activities of BIR family proteins
in Xenopus egg extracts
Yuichi Tsuchiya, Shin Murai and Shigeru Yamashita
Department of Biochemistry, Toho University School of Medicine, Tokyo, Japan
The survival of oocytes and eggs is crucial to the
reproduction of multicellular organisms (reviewed in
[1,2]). Whereas oocytes grow and survive in nurturing
conditions, ovulated eggs lose environmental support
and have to survive on their own until successful fertil-
ization. Eggs that fail to be fertilized for a long time
need to be removed because they may suffer from
aging and degeneration of maternal materials, leading
to abortive or abnormal development. Recent studies
on various species suggest that eggs possess a maternal
system of apoptotic execution [3–8]. This machinery is
inhibited for a limited time by unknown mechanisms,
and only successfully developed embryos can switch
off this embryonic time bomb. However, abnormally
developed embryos, aged unfertilized eggs, and
parthenogenetically activated eggs fail to stop the timer
and commit suicide by an intrinsic apoptotic mechan-
ism [9,10]. In Xenopus laevis (referred to simply as
Xenopus hereafter) embryos, a drastic developmental
change occurs after the 12th cell division, called the
midblastula transition (MBT) stage. Checkpoint sys-
tems are overridden before MBT in this system
because of the rapid embryonic cell cycle, and embryos
challenged by environmental insults (DNA replication
block, DNA damage, or transcriptional ⁄ translational
inhibition) still continue to divide until MBT. When
the somatic cell cycle starts after MBT, checkpoint sys-

tems begin to work properly, but cells with an unre-
coverable accumulation of defects activate the suicide
program and die by apoptosis at this stage. Aged and
Keywords
apoptosis inhibitor; survivin; Xenopus egg
extracts; XIAP; XLX
Correspondence
S. Yamashita, Department of Biochemistry,
Toho University School of Medicine, 5-21-16
Omori-nishi, Ota-ku, 143-8540 Tokyo, Japan
Fax: +81 3 5493 5412
Tel: +81 3 3762 4151 ext. 2356
E-mail:
Note
Nucleotide sequence data are available in
the DDBJ ⁄ EMBL ⁄ GenBank databases under
the accession numbers AB197247,
AB197249, AB197251, and AB197252.
(Received 11 January 2005, revised 1 March
2005, accepted 7 March 2005)
doi:10.1111/j.1742-4658.2005.04648.x
In many animal species including Xenopus, ovulated eggs possess an intrin-
sic apoptotic execution system. This program is inhibited for a limited time
by some maternal apoptosis inhibitors, although their molecular properties
remain uncharacterized. Baculovirus IAP repeat (BIR) family proteins con-
tain evolutionarily conserved BIR domains and play important roles in
apoptosis suppression, and are therefore good candidates as maternal
apoptosis inhibitors. We identified four maternal BIR family proteins in
Xenopus eggs and, using the biochemical advantages of egg extracts, exam-
ined their physiological functions. These molecules included two survivin-

related proteins, xEIAP ⁄ XLX, and a possible ortholog of XIAP named
xXIAP. The addition of recombinant xXIAP greatly delayed apoptotic exe-
cution, whereas the immunodepletion of endogenous xXIAP significantly
accelerated the onset of apoptosis. In contrast, xEIAP ⁄ XLX was a poor
apoptosis inhibitor, and neither of the survivin orthologs showed anti-
apoptotic activity in our assay. Both xEIAP ⁄ XLX and xXIAP were degra-
ded by activated caspases, and also by a novel proteolytic system that
required the presence of C-terminal RING finger domain but was insensit-
ive to proteasome inhibition. Our data suggest that the regulation of
endogenous xXIAP concentration is important for the survival of Xenopus
eggs.
Abbreviations
BIR, baculovirus IAP repeat; CSF, cytostatic factor; IAP, inhibitor of apoptosis protein; MAPK, mitogen-activated protein kinase; MBT,
midblastula transition; MBP, maltose-binding protein; MG-132, carbobenzoxy-
L-leucyl-L-leucyl-L-leucinal; Z-VAD-FMK, carbobenzoxy-L-valyl-
L-alanyl-b-methyl-L-aspart-1-yl fluoromethane.
FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS 2237
parthenogenetically activated eggs are also thought to
be killed by the same maternal mechanism, albeit with
different timing. Stack & Newport [3] hypothesized
that a maternal apoptosis inhibitor functions until
MBT, although the nature of this inhibitor is not
known.
A physiologically relevant, sudden initiation of
apoptotic execution can be reproduced with an in vitro
system derived from Xenopus egg extracts [11]. When
interphase egg extracts are incubated in the presence
of sperm nuclei and the protein synthesis inhibitor
cycloheximide (to prevent cyclin B synthesis and arrest
the cell cycle) for several hours at room temperature,

spontaneous nuclear fragmentation begins to appear,
followed by the disappearance of nuclear membranes
and the scattering of fragmented chromatin [12,13].
This change in nuclear morphology is known to be
due to the activation of a cytochrome c-induced apop-
totic pathway, as this phenomenon is accompanied by
the release of cytochrome c from mitochondria, oligo-
nucleosomal degradation of genomic DNA, activation
of apoptotic caspases, and proteolytic cleavage of
caspase substrates. Although this process has been
reported to be effectively inhibited by the addition of
exogenous Bcl-2 and chemical caspase inhibitors [11–
19], the presence of endogenous apoptosis inhibitors
has not been investigated. If they do exist, then it is
necessary to evaluate their functional contribution to
this widely used experimental system.
The BIR (baculovirus IAP repeat, in which IAP
stands for inhibitor of apoptosis protein) domain is an
evolutionarily conserved zinc-binding motif. Many, but
not all, proteins containing the BIR domain (BIR fam-
ily proteins) play important roles in apoptosis suppres-
sion (reviewed in [20–25]). These proteins possess one
or more repeats of the BIR domain, some of which are
important for caspase inhibition. Some members also
contain the C-terminal RING finger domain, which is
reported to be required for the ubiquitylation of them-
selves and bound caspases [26–28]. They are therefore
good candidates as maternal apoptosis inhibitors if
expressed in eggs. In this study, we identified four
members of the BIR family of proteins from Xenopus

eggs and characterized their biochemical behavior and
functions.
Results
By a combination of EST database searches, PCR,
and cDNA library screening, we isolated four cDNAs
coding for BIR family proteins from Xenopus oocytes
and eggs. Of these, two clones are related to mamma-
lian survivin. One cDNA encodes a 160-amino-acid
protein with a single BIR domain, which we named
xSvv1 for Xenopus survivin1. This clone is nearly iden-
tical with the Xenopus survivin ortholog recently iden-
tified by several groups [29–31]. The other cDNA,
which we designated xSvv2 for Xenopus survivin2,
encodes a similar but distinct molecule of 157 amino-
acid residues containing a single BIR domain. As a
very similar protein, named SIX, was recently identi-
fied as an RXRa-interacting protein [32], we refer to
this molecule as xSvv2⁄ SIX. Amino-acid sequence
alignment of xSvv1 and xSvv2 ⁄ SIX with human survi-
vin is shown in Fig. 1A. The residues critical for zinc
co-ordination, as well as the Thr residue phosphorylated
by Cdc2 ⁄ cyclin B, are conserved in xSvv1 and xSvv2 ⁄
SIX. Putative orthologs of xSvv1 and xSvv2 ⁄ SIX were
also identified in diploid Xenopus tropicalis (GenBank
CR761050 and CR761512, respectively). Thus, xSvv1
and xSvv2 ⁄ SIX are distinct proteins rather than the
nonallelic isoforms of a single protein in pseudotetra-
ploid X. laevis.
Another cDNA encodes a 401-residue protein con-
taining two N-terminal BIR domains and a C-terminal

RING finger domain, which we initially termed xEIAP
for egg or embryonic IAP because of its strong expres-
sion in oocytes and eggs. This domain organization is
most similar to those of IAPs from pig, insect or viral
origins, but no corresponding orthologs have been
identified from human or mouse. While we were pre-
paring this manuscript, Holley et al . [33] reported a
new Xenopus BIR family protein, XLX, which is
almost identical with xEIAP, and we refer to this
molecule as xEIAP ⁄ XLX hereafter.
The fourth cDNA encodes a protein of 412 amino
acids with three N-terminal BIR domains and a C-ter-
minal RING finger domain, the composition of which
is most closely related to that of mammalian XIAP. We
therefore named this molecule xXIAP for Xenopus or-
tholog of XIAP, although we did not examine whether
this gene is also linked to the X chromosome. Amino-
acid sequence alignment of xEIAP ⁄ XLX and xXIAP
with human XIAP is shown in Fig. 1B. xEIAP ⁄ XLX
is moderately similar to human XIAP but lacks one
BIR domain. xXIAP shows significant homology with
human XIAP along the entire length despite the shorter
spacer region between BIR3 and RING finger domains.
All of the zinc co-ordinating residues in BIR and
RING finger domains are completely conserved in both
clones. For both xEIAP ⁄ XLX and xXIAP, several
highly related sequences were found in the Xenopus
tropicalis EST database. We therefore conclude that
xXIAP is the structural ortholog of mammalian XIAP
and that xEIAP ⁄ XLX is a new member of the BIR

family of proteins in Xenopus.
Xenopus egg apoptosis inhibitors Y. Tsuchiya et al.
2238 FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS
Notably, amino-acid sequence analyses using the
PESTfind program ( />tools/bio/PESTfind/) revealed potential PEST sequen-
ces in xSvv1 (+14.17 in 71–86 residues) and xEIAP ⁄
XLX (+8.21 in 246–259 residues and +5.58 in 333–
346 residues). PEST sequence is defined as a region
enriched in Pro ⁄ Glu ⁄ Ser ⁄ Thr, and some proteins
containing PEST sequences are rapidly degraded [34].
The presence of PEST sequences in xSvv1 and xEIAP ⁄
XLX suggests their instability in vivo.
To carry out biochemical characterization, we bacte-
rially expressed and purified a pair of His
6
-tagged and
maltose-binding protein (MBP)-tagged recombinant
fusions for each Xenopus BIR family protein (Fig. 2A).
His
6
-tagged recombinants were all insoluble and used
only for antibody production and purification, whereas
Fig. 1. Primary sequences of Xenopus BIR family proteins. (A) Amino-acid sequence alignment of xSvv1, xSvv2, and human survivin. (B)
Amino-acid sequence alignment of xEIAP ⁄ XLX, xXIAP, and human XIAP. Identical and similar residues are boxed and shaded, respectively.
Zinc-co-ordinating Cys ⁄ His are indicated by asterisks at the bottom. The putative Cdc2 ⁄ cyclin B phosphorylation site in survivin is shown by
(p). Xl, Xenopus laevis;Hs,Homo sapiens. The GenBank accession numbers of xSvv1, xSvv2 ⁄ SIX, human survivin, xEIAP ⁄ XLX, xXIAP, and
human XIAP are AB197247, AB197249, NM_001168, AB197251, AB197252, and NM_001167, respectively.
Y. Tsuchiya et al. Xenopus egg apoptosis inhibitors
FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS 2239
MBP-tagged recombinants were all soluble and used

for functional analyses as below. Our affinity-purified
antibodies were all able to recognize as little as 1 ng of
the corresponding recombinant antigens (Fig. 2B), but
we could not detect the endogenous proteins in egg
extracts directly by western blot, suggesting their low
content. To investigate the existence of these proteins,
the immunoprecipitated materials from 100 lL egg
extracts with specific antibodies were similarly ana-
lyzed. Although previous reports identified endogenous
xSvv1 in egg extracts [29,30], we failed to detect it
with our antibody (Fig. 2B). However, xSvv2 ⁄ SIX,
xEIAP ⁄ XLX, and xXIAP were clearly detected with
this procedure. Up to 5–10 ng xXIAP and smaller
amounts of xSvv2 ⁄ SIX and xEIAP ⁄ XLX could be pre-
cipitated from 100 lL cytostatic factor (CSF)-arrested
egg extracts, although the recoveries were variable.
These results indicate that xSvv2 ⁄ SIX, xEIAP ⁄ XLX,
and xXIAP are present in egg extracts at low concen-
tration (less than 1 lgÆmL
)1
).
Recent studies have firmly established that the mor-
phological change in sperm nuclei is a hallmark of
apoptotic execution in Xenopus egg extracts [11–19].
The incubation of egg extracts at room temperature
for 3–5 h converts spherical nuclei (I, Fig. 3A left)
into degenerated ones (A, Fig. 3A left), although the
time required depends on egg quality. This apoptotic
change is accompanied by chromatin DNA degrada-
tion (data not shown). Moreover, caspase-dependent

partial (but not complete) proteolysis of p150
Glued
,
an endogenous apoptotic substrate in Xenopus egg
extracts, is reproducibly detectable with a commercially
available antibody (Fig. 3A right and [35]). All these
events occur simultaneously and can be specifically
inhibited by a pan-caspase inhibitor carbobenzoxy-
l-valyl-l-alanyl-b-methyl-l-aspart-1-yl fluoromethane
(Z-VAD-FMK), as previously reported [11–19]. We
therefore used nuclear morphology and p150
Glued
pro-
teolysis as reliable indicators of apoptotic execution in
this study. The molecule capable of preventing these
processes can be regarded as an inhibitor of apoptosis.
As all recombinants were fused with MBP, we selected
nonfused MBP as a control protein. Preliminary stud-
ies indicated that the addition of up to 10 lgÆmL
)1
MBP had no effect on apoptotic timing and nuclear
morphology in egg extracts. Therefore, all recombi-
nants were added at the same protein (rather than
equimolar) concentration at 10 lgÆmL
)1
. The presence
of 10 lgÆmL
)1
control MBP, MBP-xSvv1, or MBP-
xSvv2 showed little inhibition of this process, and

MBP-xEIAP showed only limited inhibition (Fig. 3B).
In contrast, the addition of MBP-xXIAP effectively
inhibited apoptotic nuclear fragmentation. Moreover,
caspase-dependent partial proteolysis of p150
Glued
was
also prevented only by MBP-xXIAP (Fig. 3C). Finally,
we examined the dose–response of MBP-xXIAP, the
only effective molecule in our assay. As shown in
Fig. 3D, anti-apoptosis activity of MBP-xXIAP was
strong at 10 lgÆmL
)1
but decreased at 3 lgÆmL
)1
,
and became undetectable at 1 lgÆmL
)1
. These results
indicate that only xXIAP shows strong apoptosis-
inhibiting activity in our assay with recombinant
proteins.
A
B
Fig. 2. Western blot analysis of Xenopus BIR family proteins in egg
extracts. (A) Purity of recombinant BIR family proteins. Purified
His
6
-tagged (left four lanes) and MBP-fused (right four lanes) rec-
ombinants were resolved by SDS ⁄ PAGE and stained with Coomas-
sie Brilliant blue. Lanes S1, xSvv1; lanes S2, xSvv2; lanes E,

xEIAP ⁄ XLX, and lanes X, xXIAP. Positions of size markers (Mr) are
indicated on the left. (B) Quantification of BIR family proteins in
egg extracts. Immunoprecipitated materials from 100 lL CSF-arres-
ted egg extracts with control (C) and the respective affinity-purified
specific (I) antibodies were resolved by SDS ⁄ PAGE, followed by
western blot with the same antibodies. For the quantification of
each protein, 10, 3, and 1 ng of the corresponding recombinant
His
6
-tagged antigen was subjected to western blot analysis at the
same time. Positions of size markers are indicated on the left.
Immunoprecipitated antigens and IgG heavy chain are denoted by
arrowheads and asterisks, respectively.
Xenopus egg apoptosis inhibitors Y. Tsuchiya et al.
2240 FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS
Although exogenous xXIAP strongly suppressed
apoptotic change in egg extracts, it is still not certain
whether the same molecule actually functions as an
endogenous apoptosis inhibitor in vivo. To examine the
physiological roles biochemically, we blocked the func-
tions of endogenous BIR family proteins by immuno-
depletion with specific antibodies. The efficiency of
depletion could not be accurately estimated because of
the low contents of endogenous molecules, but excess
amounts of antibodies were used to deplete the corres-
ponding proteins from egg extracts (see Experimental
procedures). The first immunodepletion appeared near
complete for each BIR family protein because no
endogenous molecule was detected when immunode-
pletion was repeated and the second immunoprecipi-

tate was subjected to western blot (data not shown).
The nuclei in xXIAP-depleted extract became fragmen-
ted significantly earlier than those in other immunode-
pleted samples (Fig. 4A). Western blotting of p150
Glued
also revealed the accelerated apoptotic cleavage only
in xXIAP-depleted extract (Fig. 4B). These results
strongly suggest that the physiological role of xXIAP
is as a maternal apoptosis inhibitor in Xenopus egg
extracts.
The above results reveal that xSvv1, xSvv2 ⁄ SIX, and
xEIAP ⁄ XLX could not inhibit apoptosis in egg
extracts (Fig. 3B). However, these proteins may be
unstable and completely degraded before the onset of
apoptotic change. To examine the stability of these
proteins, we first tried to detect the endogenous mole-
cules by western blot to follow their quantitative chan-
ges. However, reliable assay was not possible because
of the low concentrations of the endogenous mole-
cules. As an alternative approach, each of four MBP
fusions was added at 1 lgÆmL
)1
at the start of incuba-
tion, and the remaining recombinants were detected
by western blot with anti-MBP serum. As shown in
Fig. 3D, all MBP fusions at this low concentration did
A
B
C
D

Fig. 3. Apoptosis-inhibiting activities of recombinant BIR family pro-
teins in egg extracts. (A) Indications of apoptosis in Xenopus egg
extracts. Left, typical images of normal interphase (I) and fragmen-
ted apoptotic (A ) nuclei stained with 4,6-diamidino-2-phenylindole.
Bar, 10 lm. Right, Partial cleavage of p150
Glued
in apoptotic
extracts. p150
Glued
in interphase (I) and apoptotic (A) extracts were
detected by western blot. (B) Time course of apoptotic nuclear
fragmentation in the presence of recombinant BIR family proteins.
Each recombinant MBP fusion protein was added to interphase
egg extracts at 10 lgÆmL
)1
, and morphological changes in 50 nuclei
were counted every hour for each sample. The percentages of
apoptotic nuclei are represented as mean ± SEM from three inde-
pendent experiments. (C) Apoptotic cleavage of p150
Glued
in the
presence of recombinant BIR family proteins. The extracts at 4 h
incubation in one experiment in (B) were analyzed by western blot
as shown in Experimental procedures. In B and C, the addition of
each protein is denoted as below: s (lane C), control MBP; n (lane
S1), MBP-xSvv1; m (lane S2), MBP-xSvv2; h (lane E), MBP-xEIAP;
(lane X), MBP-xXIAP. (D) Dose–response of MBP-xXIAP. Various
amounts of MBP-xXIAP were added to egg extracts, and the mor-
phological changes in nuclei were counted as in (B). s,0lgÆmL
)1

;
d,1lgÆmL
)1
; h,3lgÆmL
)1
; ,10lgÆmL
)1
. Data are represented
as mean ± SEM from three independent experiments.
Y. Tsuchiya et al. Xenopus egg apoptosis inhibitors
FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS 2241
not affect the timing of apoptotic onset. The amount
of MBP-xSvv1 decreased only slightly, and that of
MBP-xSvv2 was virtually unchanged during a 4-h
incubation (Fig. 5A, upper). These results indicate that
both recombinants were stable and remained in suffi-
cient amounts in extracts, but that they could not inhi-
bit apoptosis (Fig. 3B,C).
In contrast, the signal of MBP-xEIAP rapidly dimin-
ished before the onset of apoptotic change and had
completely disappeared after 4 h of incubation. MBP-
xXIAP was also gradually degraded but more slowly
than MBP-xEIAP. These results raised the possibility
that anti-apoptotic activities of xEIAP ⁄ XLX and
xXIAP may be fully or partially masked by their insta-
bilities. To confirm the above results, another set of
studies was carried out using recombinants translated
in vitro in rabbit reticulocyte lysates. Although the sta-
bility of His
6

-xSvv1 was difficult to analyze because of
low translation efficiency, the three other recombinants
showed similar behaviors to corresponding MBP
fusions; His
6
-xSvv2 was stable, His
6
-xEIAP was rap-
idly degraded, and His
6
-xXIAP was relatively stable at
2 h but degraded at 4 h (Fig. 5A, lower). Similar
results were obtained with untagged recombinants
(data not shown), excluding the possibility of struc-
tural interference by the N-terminal tags.
These data suggest that the lack of apoptotic inhibi-
tion by MBP-xEIAP, as well as MBP-xXIAP at low
concentration, may be due to its rapid proteolysis.
Thus, we focused our attention on the stabilities of
xEIAP ⁄ XLX and xXIAP. Recent studies have identi-
fied at least four independent mechanisms that control
the stability of BIR family proteins; RING finger-
dependent ubiquitylation and subsequent degradation
by proteasome [26–28], caspase-dependent cleavage
[36–38], calpain-dependent degradation [39], and pro-
teolysis by a serine protease Omi ⁄ HtrA2 released from
mitochondria [40–42]. Moreover, proteolytic fragments
may be further degraded by the N-end rule pathway in
Drosophila [43]. To evaluate the contribution of these
proteolytic systems, the effects of specific protease

inhibitors were tested against the degradation of the
two BIR family proteins as well as apoptotic p150
Glued
cleavage.
Although the pan-caspase inhibitor Z-VAD-FMK at
100 lm effectively blocked the apoptotic cleavage of
p150
Glued
and nuclear fragmentation, the initial
decrease in the two BIR family proteins within 2 h
was not inhibited (Fig. 5B,C). However, the amounts
of MBP-xEIAP and MBP-xXIAP did not change sig-
nificantly during the next 2 h, indicating that cleavage
of these proteins during this period was blocked by the
inhibitor. These results suggest that the cleavage of
these proteins proceeded in two steps: a caspase-inde-
pendent first step and caspase-dependent second step.
In contrast, a proteasome ⁄ calpain inhibitor, MG-132
(carbobenzoxy-l-leucyl-l-leucyl-l-leucinal), at 100 lm
could not prevent, or even accelerated, the apoptotic
cleavage of p150
Glued
, the decreases in MBP-xEIAP ⁄
XLX and MBP-xXIAP, and nuclear fragmentation
(Fig. 5B,C). With the same inhibitor concentration,
proteolysis of endogenous cyclin B2 (a typical sub-
strate of the ubiquitin–proteasome pathway) during
the calcium-induced metaphase exit was effectively
blocked (data not shown). UCF-101, a recently devel-
oped specific inhibitor of HtrA family proteases [44],

had no effect at 100 lm (data not shown) and acceler-
ated the cleavage of these three proteins and nuclear
fragmentation at 1 mm. It should be noted that
B
A
Fig. 4. Acceleration of apoptotic onset in xXIAP-depleted egg
extracts. (A) Time course of apoptotic nuclear fragmentation in
interphase extracts depleted of each BIR family protein. The mor-
phological changes in the nuclei were counted as in Fig. 3B. Data
are represented as mean ± SEM from three independent experi-
ments. (B) Apoptotic cleavage of p150
Glued
in interphase extracts
depleted of each BIR family protein. The extracts at 3 h incubation
in one experiment in (A) were analyzed as in Fig. 3C. Depletion of
each protein is denoted as follows: s (lane C), control-depleted;
n (lane S1), xSvv1-depleted; m (lane S2), xSvv2-depleted; h (lane
E), xEIAP ⁄ XLX-depleted;
(lane X), xXIAP-depleted.
Xenopus egg apoptosis inhibitors Y. Tsuchiya et al.
2242 FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS
inhibitors of the lysosomal proteases (leupeptin, chy-
mostatin, and pepstatin at 10 lgÆmL
)1
) were also
included in the extracts, excluding the possibility of
nonspecific lysosomal proteolysis. These data suggest
that both proteins are degraded at least in two steps,
initially by an unknown proteolytic system and subse-
quently by activated caspases.

To define the domains that regulate the function
and stability of xEIAP ⁄ XLX and xXIAP, we further
analyzed the stabilities of three C-terminal deletion
mutants, ED1, ED2, and XD1, as in Fig. 5. ED1 lacks
the second PEST sequence (PEST2) and RING finger
domain of xEIAP ⁄ XLX, and the first PEST sequence
(PEST1) is further deleted in ED2 (Fig. 6A). Similarly,
the XD1 mutant lacks the RING finger domain and
C-terminal half of the BIR3 domain (Fig. 6A). Each
of these three mutants was fused with N-terminal
MBP, expressed in Escherichia coli, and purified for
biochemical characterization (Fig. 6B). Western blot
revealed that, in contrast with the full-length version,
both MBP-ED1 and MBP-ED2 were stable during the
first 2 h in interphase egg extracts without any detect-
able decreases, indicating that the C-terminal region
confers susceptibility on the initial phase of degrada-
tion (Fig. 6C). During the next 2 h, however, MBP-
fused ED1 and ED2 both showed remarkable decreases.
Most of the MBP-ED2 was cleaved into smaller pep-
tides during this period, whereas only about a half of
the MBP-ED1 was degraded. These data indicate that
MBP-ED1 is more stable than MBP-ED2 because of
the existence of residues 219–269. Moreover, cleaved
fragments from MBP-ED1 and MBP-ED2 generated
after 4 h incubation appeared more stable than those
derived from full-length MBP-xEIAP (compare
Figs 5A and 6C). MBP-XD1 was also resistant to ini-
tial degradation within 2 h but was cleaved during the
next 2 h (Fig. 6C). Similar to the cases of MBP-

ED1 ⁄ ED2, cleavage products from MBP-XD1 were also
more stable than those from full-length MBP-xXIAP
(compare Figs 5A and 6C). Thus, in both xEIAP ⁄ XLX
and xXIAP, the C-terminal region containing the
RING finger domain is required for the initial phase
of proteolysis within 2 h and also for the final degra-
dation of caspase-generated fragments.
A
B
C
Fig. 5. Different stabilities of BIR family proteins in Xenopus inter-
phase egg extracts. (A) Stability of BIR family proteins in interphase
egg extracts. Purified MBP-tagged recombinants at 1 lgÆmL
)1
(upper) or rabbit reticulocyte lysates containing
35
S-labeled His
6
-
tagged recombinants at 0.1 volume (lower) were supplied to inter-
phase egg extracts. The remaining proteins after 0, 2, and 4 h
incubation were detected by western blot using anti-MBP serum
(aMBP) and image analyzer (
35
S), respectively. (B) Effects of prote-
ase inhibitors on the degradation of p150
Glued
, MBP-xEIAP, and
MBP-xXIAP. Interphase egg extracts containing 1 lgÆmL
)1

MBP-
xEIAP or MBP-xXIAP were incubated for 0, 2, and 4 h in the pres-
ence of dimethylsulfoxide, 100 l
M Z-VAD-FMK, 100 lM MG-132,
and 1 m
M UCF-101, respectively. p150
Glued
and the remaining MBP
fusion proteins were detected as in Figs 3C and 5A, respectively.
(C) Effects of protease inhibitors on apoptotic nuclear fragmenta-
tion. The morphological changes in nuclei were counted as in
Fig. 3B. s, control dimethylsulfoxide; d, 100 l
M MG-132; n,
100 l
M Z-VAD-FMK; m,1mM UCF-101. Data are represented as
mean ± SEM from three independent experiments.
Y. Tsuchiya et al. Xenopus egg apoptosis inhibitors
FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS 2243
Nuclear fragmentation and p150
Glued
cleavage assays
indicate that, although MBP-ED1 did not inhibit apop-
tosis despite its stability, MBP-ED2 significantly pre-
vented apoptosis (Fig. 6D,E). These data suggest that
the apoptosis-inhibiting potential of the two N-ter-
minal BIR domains in xEIAP ⁄ XLX is self-inhibited by
residues 219–269. In contrast, MBP-XD1 retained anti-
apoptotic function as strong as full-length MBP-
xXIAP despite the lack of functional BIR3 and RING
finger domains (Fig. 6D,E).

A recent report indicated that Xenopus CSF-arres-
ted metaphase egg extracts are more resistant to
apoptosis than interphase extracts by a mitogen-acti-
vated protein kinase (MAPK)-dependent, post-cyto-
chrome c release mechanism [45]. To check whether
this resistance involves any of the BIR family pro-
teins, we first immunodepleted endogenous molecules
and studied the timing of apoptotic onset in CSF-
arrested egg extracts containing cycloheximide.
Because nuclear morphology could not be observed
because of mitotic chromatin condensation in meta-
phase extracts, only the apoptotic cleavage of
p150
Glued
was examined. In control-depleted CSF-
arrested extract, apoptotic p150
Glued
cleavage became
evident after 6 h incubation, significantly later than
in interphase extract (Fig. 7A). Immunodepletion of
xSvv1, xSvv2, or xEIAP ⁄ XLX showed no effect on
the timing of apoptotic onset (data not shown). In
contrast, p150
Glued
cleavage started after only 2 h
incubation in xXIAP-depleted CSF-arrested extract,
as quickly as in xXIAP-depleted interphase extract
(compare Figs 4B and 7A). Therefore, in the absence
of xXIAP, both CSF-arrested and interphase egg
extracts executed apoptosis with similar timing.

To examine whether this difference between CSF-
arrested and interphase egg extracts can be attributed
to the activities of MAPK or other kinases, we used
several protein kinase inhibitors and compared the
apoptotic cleavage of p150
Glued
as well as the stabil-
ity of MBP-xXIAP. Apoptotic p150
Glued
cleavage did
AB
C
E
D
Fig. 6. Apoptosis-inhibiting activities of MBP-xEIAP and MBP-xXIAP C-terminal deletion mutants. (A) Structures of deletion mutants. BIR
domain (gray ovals), RING finger domain (black boxes) and PEST sequence (gray boxes) are schematically presented. (B) Purity of recombin-
ant proteins. Purified MBP-tagged mutants were detected as in Fig. 2B. Positions of size markers are indicated on the left. (C) Stability of
deletion mutants in interphase egg extracts. Purified MBP-tagged mutants at 1 lgÆmL
)1
were supplied to interphase egg extracts. The
remaining proteins after 0, 2, and 4 h incubation were detected as in Fig. 5A. In XD1, a smaller truncated product was also observed, which
was poorly detected by protein staining in B. (D) Time course of apoptotic nuclear fragmentation in the presence of recombinant mutants.
The morphological changes in nuclei were counted as in Fig. 3B. Data are represented as mean ± SEM from three independent experi-
ments. (E) Apoptotic cleavage of p150
Glued
in the presence of recombinant mutants. The extracts at 3 h incubation in one experiment in (D)
were analyzed as in Fig. 3C. (C, D, E) The addition of each protein is denoted as follows: s (lane C), MBP; d (lane ED1), MBP-ED1; n (lane
ED2), MBP-ED2; m (lane XD1), MBP- XD1.
Xenopus egg apoptosis inhibitors Y. Tsuchiya et al.
2244 FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS

not occur until 6 h in dimethylsulfoxide-treated
control CSF-arrested egg extracts (Fig. 7B, left).
Whereas a Cdk inhibitor, roscovitine, at 100 lm had
little effect, an MEK inhibitor, U0126, at 100 lm
significantly accelerated caspase-dependent cleavage
of p150
Glued
to interphase level (4 h). The degrada-
tion of MBP-xXIAP was also delayed in CSF-arres-
ted egg extracts (compare right, upper panels of
Figs 5B and 7B; dimethylsulfoxide). This delay was
mainly due to the suppression of the second phase
of the cleavage because the initial degradation within
2 h in CSF-arrested egg extracts was similar to that
in interphase egg extracts. The presence of dimethyl-
sulfoxide or roscovitine did not affect these results
(Fig. 7B, right). However, the second caspase-depend-
ent apoptotic cleavage of MBP-xXIAP during 2–4 h
incubation, which had been almost completely sup-
pressed in CSF-arrested egg extracts, was recovered
in the presence of U0126 (Fig. 7B, right). In con-
trast, the initial degradation of MBP-xXIAP was not
affected by U0126. However, MAPK did not phos-
phorylate MBP-xXIAP in vitro (data not shown).
Thus, in CSF-arrested egg extracts, MAPK appears
to suppress apoptosis by delaying caspase activation
rather than stabilizing xXIAP by direct phosphoryla-
tion.
Discussion
xXIAP is the central apoptosis inhibitor

in Xenopus egg extracts
In this study, we identified four BIR family proteins
from Xenopus eggs and evaluated their biochemical
and physiological roles as apoptosis inhibitors. Our
recombinant protein addition (Fig. 3) and immunode-
pletion (Figs 4 and 7A) analyses indicated that xXIAP,
a putative ortholog of mammalian XIAP, functions as
a genuine maternal apoptosis inhibitor in egg extracts,
and probably also in eggs in vivo. Numerous studies
have indicated that XIAP is the most potent IAP in
mammals (reviewed in [20–25]). Thus, the central role
of XIAP as a physiological apoptosis inhibitor may be
conserved among animal species. BIR3 and RING fin-
ger domains of xXIAP were dispensable for apoptosis
inhibition in our assay (Fig. 6). This result is consistent
with the structural data for mammalian XIAP showing
that effector caspases (caspases-3 ⁄ 7) are inhibited by
the linker peptide between BIR1 and BIR2 domains,
which is also conserved in xXIAP.
xEIAP/XLX is a potential apoptosis inhibitor
in Xenopus egg extracts
xEIAP ⁄ XLX was less effective in apoptotic inhibition
(Fig. 3), and its immunodepletion did not affect the
timing of apoptotic onset in egg extracts (Fig. 4).
Although Holley et al. [33] did not examine the func-
tion of xEIAP ⁄ XLX, our assay using deletion mutants
indicates that residues 1–218 of xEIAP ⁄ XLX contain-
ing two BIR domains could inhibit apoptosis (Fig. 6).
However, the presence of the residue 219–269-contain-
ing PEST1 blocked its apoptosis-inhibiting function,

and the further addition of the residue 270–401-con-
taining PEST2 and RING finger domain resulted in
rapid proteolysis. Therefore, the apoptosis-inhibiting
potential of xXIAP ⁄ XLX may be normally hindered
by both self-inhibition and rapid degradation in Xen-
opus eggs. However, we do not exclude possible func-
tions of xEIAP ⁄ XLX in other cell death pathways. We
are currently searching physiological conditions in
which xEIAP ⁄ XLX becomes functional.
Multiple proteolytic pathways regulate the
concentrations of BIR family proteins
Both xEIAP ⁄ XLX and xXIAP were degraded in a
multistep process in egg extracts. During the first step
operating within 2 h of incubation, xEIAP ⁄ XLX was
degraded rapidly, whereas xXIAP was degraded rather
A
B
Fig. 7. Critical role of xXIAP in the increased resistance against
apoptosis in CSF-arrested egg extracts. (A) Delay of apoptotic onset
in CSF-arrested extracts was compromised by xXIAP immunodeple-
tion. CSF-arrested extracts were depleted with control (left) or anti-
xXIAP (right) antibody, supplied with cycloheximide and incubated.
The time course of apoptotic p150
Glued
cleavage every 2 h was
analyzed by western blot as in Fig. 3C. (B) MAPK-dependent indi-
rect stabilization of xXIAP in CSF-arrested egg extracts. MBP-xXIAP
was supplied at 1 lgÆmL
)1
to CSF-arrested metaphase egg extracts

containing cycloheximide and incubated for 0, 2, and 4 h in the
presence of dimethylsulfoxide (DMSO), 100 l
M roscovitine, and
100 l
M U0126. p150
Glued
and the remaining MBP fusion proteins
were similarly detected as in Figs 3C and 5A, respectively.
Y. Tsuchiya et al. Xenopus egg apoptosis inhibitors
FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS 2245
slowly. For both molecules, this initial degradation
step required the RING finger domain-containing
C-terminal region, and could not be blocked by inhibi-
tors of caspases, proteasome ⁄ calpain, and HtrA family
proteases. After longer incubation, the second caspase-
dependent step further cleaved the remaining molecules
(Fig. 5) and apoptosis was triggered. In addition, the
final digestion of cleavage fragments also partially
depended on the RING finger domain because
caspase-generated fragments were detected more
clearly in deletion mutants than in full-length versions
(compare Figs 5 and 6). This final step was also resist-
ant to MG-132 and UCF-101 (our unpublished result).
The involvement of these multiple proteolytic systems
can explain why recombinant MBP-xXIAP at
10 lgÆmL
)1
, but not at 1 lgÆmL
)1
, effectively delayed

apoptosis onset (compare Figs 3 and 5). Although the
second step was clearly caspase-dependent, we were
not able to determine which proteolytic mechanisms
were responsible for the first and the last degradation
steps. Whereas previous reports suggested the involve-
ment of the ubiquitin–proteasome system in the RING
finger-dependent and N-end rule pathways [26–28,43],
our data indicate that neither the initial nor the final
degradation step was blocked by proteasome inhibi-
tion. Holley et al. [33] also reported the reduction of
xEIAP ⁄ XLX by caspase-dependent and translational
inhibition-dependent dual mechanisms. Cycloheximide,
a protein synthesis inhibitor, is required for Xenopus
egg extracts to be used for in vitro apoptosis study.
The main purpose of this drug is to prevent cyclin B
reaccumulation and to arrest the cell cycle in inter-
phase, but general protein translation is also inhibited.
Holley et al. found that, in Xenopus egg extracts, the
cell death induced by reaper involves general trans-
lation inhibition, probably in a similar manner to
cycloheximide-induced apoptosis. Otherwise, the stabil-
ities of these proteins may be regulated by conforma-
tional changes or stoichiometric interaction with other
molecules. Further studies are being performed to
address these issues.
As the number of mitochondria greatly increases
during Xenopus oogenesis, unwanted cytochrome c
release from mitochondria may occur spontaneously
even in healthy eggs. The level of endogenous xXIAP
may be sufficient to prevent caspase amplification

induced by a low level of cytochrome c release.
Caspase inhibition can further prevent the caspase-
dependent degradation of xXIAP, thereby forming a
positive feedback loop. Once translation inhibition or
aging ⁄ damage-induced enhanced cytochrome c release
leads to consumption of all endogenous xXIAP,
caspase amplification may occur rapidly. This
mechanism can convert gradual cytochrome c release
into all-or-none apoptotic execution. Therefore, unper-
turbed synthesis of xXIAP may be at least in part
necessary for apoptosis suppression in egg extracts
in vitro and for egg survival in vivo.
xXIAP stability and MAPK-dependent apoptosis
inhibition
A current model of CSF function is to inhibit the ana-
phase-promoting complex to protect several cell cycle
regulators (cyclin B and securin, for example) from
ubiquitylation and proteasome-dependent proteolysis,
leading to cell cycle arrest at metaphase. In CSF-arres-
ted egg extracts, apoptotic onset was delayed in a
MAPK-dependent manner (Fig. 7B and [45]). How-
ever, in the absence of endogenous xXIAP, both CSF-
arrested and interphase egg extracts executed apoptosis
with similar timing (2 h; compare Figs 4B and 7A),
indicating that MAPK-induced delay of apoptosis
depends on the presence of xXIAP. Moreover, as the
degradation of xXIAP is accelerated by MAPK kinase
(MEK) inhibition, this also indicates that MAPK
increases the stability of xXIAP. Therefore, it is rea-
sonable to consider that MAPK delays apoptosis by

stabilizing xXIAP. This MAPK-induced delay of
xXIAP degradation is mainly due to the inhibition of
the caspase-dependent second phase because the initial
caspase-independent degradation of xXIAP is not sig-
nificantly altered in CSF-arrested egg extracts. At first,
we speculated that MAPK stabilizes xXIAP by direct
phosphorylation of the protein. However, recombinant
MBP-xXIAP was not significantly phosphorylated by
MAPK or CSF-arrested egg extracts (our unpublished
result). MAPK may stabilize xXIAP indirectly by sup-
pressing caspase activation or by inhibiting caspase
activities themselves. Further studies will be necessary
to identify the molecular determinant and signal-trans-
duction pathway for MAPK-dependent egg survival.
Survivin orthologs do not inhibit apoptosis
in Xenopus egg extracts
We also isolated two survivin-related molecules, xSvv1
and xSvv2 ⁄ SIX, but failed to show their anti-apoptosis
activities. Whereas survivin was originally identified as
an apoptosis inhibitor, recent studies have suggested
that survivin-related proteins of yeasts, insects (except
Drosophila deterin) and vertebrates play important
roles in cell cycle progression rather than caspase inhi-
bition (reviewed in [20–25]). Survivin forms a ‘chromo-
somal passenger complex’ with INCENP and Aurora
B kinase to regulate sister chromatid segregation and
Xenopus egg apoptosis inhibitors Y. Tsuchiya et al.
2246 FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS
cytokinesis. Although we could not detect xSvv1 by
our western blot, Bolton et al. [29] and Losada et al.

[30] identified xSvv1 in egg extracts as a stoichiometric
binding partner of INCENP and Aurora B kinase.
The reason for this discrepancy is unknown and under
investigation. Instead, our western blot detected
xSvv2 ⁄ SIX in egg extracts [32]. As Xenopus egg
extracts are also useful for studying the biochemical
basis of cell-cycle-related events, we are currently
examining whether the biochemical properties of
xSvv2 ⁄ SIX are similar to those reported for xSvv1
[29,30]. It will be important to clarify the functional
links between cell cycle and apoptosis, the two crucial
elements of life. For example, Wee1, an important cell-
cycle-regulating protein tyrosine kinase, was found to
be necessary for Crk-mediated apoptotic signal trans-
duction in Xenopus egg extracts [46].
Experimental procedures
Chemical reagents
Unless otherwise stated, all the reagents were purchased
from Wako Pure Chemicals (Osaka, Japan).
cDNA cloning of Xenopus BIR family proteins
During the course of another unrelated study, we unexpect-
edly obtained a Xenopus egg cDNA fragment coding for a
RING finger motif highly homologous to those of verteb-
rate IAPs. This fragment was used for screening of a
Xenopus oocyte kZapII cDNA library (kindly provided by
Professor Haruhiko Takisawa and Dr Satoru Mimura,
Osaka University, Osaka, Japan). We identified an ORF
encoding two N-terminal BIR domains and a C-terminal
RING finger motif, which was designated xEIAP for
Xenopus egg or embryonic IAP. While the initial phase of

this study was in progress, many Xenopus EST clones
homologous to mammalian BIR family proteins were
deposited in the GenBank database. Homology searches
revealed the presence of other BIR family proteins
expressed in Xenopus oocytes or eggs. By exploiting this
information, we were also able to isolate the clones enco-
ding survivin1 (xSvv1), survivin2 (xSvv2), and an apparent
ortholog of mammalian XIAP (xXIAP) from the same
oocyte cDNA library. The longest clone from each group
was completely sequenced using an ABI373 DNA sequencer
and dye terminator cycle sequencing kit (Applied Biosys-
tems, Tokyo, Japan) for both strands.
Recombinant protein expression and purification
The coding sequence of xSvv1 was amplified by PCR using
ExTaq polymerase and the following primers: xSvv1-F,
5¢-CATATGTATTCTGCCAAGAACAGGTTTG-3¢ and
xSvv1-R, 5¢-GGATCCCATGCTATTGGTCTTTGCT-3¢.
The amplified fragment was cloned into pGEM-T Easy
(Promega, Tokyo, Japan), verified for nucleotide sequence,
and subcloned into pET-15b or pET-22(+) (Novagen,
Madison, WI, USA) using NdeI and BamHI restriction sites
to produce the recombinants with or without N-terminal
hexahistidine tag (His
6
), respectively. To produce MBP
fusion protein, the coding sequence for His
6
-xSvv1 was
excised as a blunt-ended NcoI–BamHI fragment, and sub-
cloned into the blunt-ended EcoRI–BamHI site of pMALc-

RI (New England Biolabs, Beverly, MA, USA). Similar
procedures were performed to produce His
6
- ⁄ untagged-
⁄ MBP- fusion sets of xSvv2, xEIAP, and xXIAP using the
following primers: xSvv2-F, 5¢-CATATGATGAGTATTT
CTCCGATTG-3¢ and xSvv2-R, 5¢-GGATCCTCACTGTG
TTTCGTCAGCATC-3¢; xEIAP-F, 5¢-CATATGAGTCGC
TGGTCCGTATCC-3¢ and xEIAP-R, 5¢-GGATCCTCAG
GACATGAAGGCTCTAAC-3¢; xXIAP-F, 5¢-CATATGA
CATGCCAGTGTCCAAAG-3¢ and xXIAP-R, 5¢-GGATC
CAACTGAGCTTTGGCATTTC-3¢. To produce MBP-ED1
(1–269 residues of xEIAP and a vector-derived extra 11 res-
idues, lacking PEST2 and RING finger), the pMALc-RI
vector encoding full-length MBP-xEIAP was digested with
PstI and HindIII, blunt-ended with Klenow polymerase,
and self-ligated. Similar procedures were used to create the
vector expressing MBP-ED2 (1–218 residues of xEIAP and
an extra 11 residues, lacking PEST1, PEST2 and RING fin-
ger) using StuI and HindIII, and MBP-XD1 (1–301 residues
of xXIAP and an extra 9 residues derived from an alternat-
ive reading frame, lacking RING finger and a part of the
BIR3 domain) using SphI.
For large-scale preparation of recombinant proteins, the
vectors were transformed into E. coli BL21 (DE3) strain,
and bacterial overnight culture was inoculated into Luria–
Bertani medium and grown at 37 °C with agitation until
mid-exponential phase. To express His
6
-BIR family pro-

teins, 1 mm isopropyl b-d-thiogalactoside and 1 mm ZnSO
4
were added, and protein expression was induced for 3 h at
37 °C with vigorous shaking. As all the His
6
-BIR family
proteins were insoluble, inclusion body fractions were pre-
pared according to the manufacturer’s instructions (Nova-
gen). Each recombinant was solubilized with binding buffer
(0.1 m Tris ⁄ HCl, pH 8.0, 0.5 m NaCl) containing 6 m guani-
dine hydrochloride, and manually injected using a syringe
into a nickel-loaded HiTrap chelating column (1 mL; Amer-
sham Biosciences, Tokyo, Japan) equilibrated with binding
buffer containing 6 m urea. The column was washed with
2· Hepes buffer (20 mm Hepes ⁄ KOH, pH 7.8, 200 mm
KCl, 2 mm MgCl
2
) containing 6 m urea and 20 mm imida-
zole, and recombinant protein was eluted with the same
buffer containing 6 m urea and 200 mm imidazole. Several
attempts to refold these denatured proteins have failed.
Soluble MBP-BIR family proteins were expressed with
0.3 mm isopropyl b-d-thiogalactoside and 1 m m ZnSO
4
at
Y. Tsuchiya et al. Xenopus egg apoptosis inhibitors
FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS 2247
18 °C overnight. Each soluble fraction was loaded on to
amylose resin (1 mL; New England Biolabs) packed in an
Econo-Pac column (Bio-Rad, Tokyo, Japan) and equili-

brated with 2· Hepes buffer. MBP fusion protein was elut-
ed with 2· Hepes buffer containing 10 mm maltose. After
protein concentration had been determined with BCA rea-
gent (Pierce, Rockford, IL, USA) using BSA as a standard,
an equal volume of glycerol containing 20 mm 2-mercapto-
ethanol was added, and proteins were stored in )20 °Cor
)80 °C as 50% glycerol solution.
Both pET-15b-encoded and pET-22(+)-encoded clones
were also used to produce radiolabeled recombinants with
and without His
6
tag, respectively, using the TNT T7 Quick
coupled transcription ⁄ translation system (Promega) and
Pro-mix L-[
35
S] in vitro cell labeling mix (Amersham Bio-
sciences) according to the manufacturer’s instructions.
Production of specific antibodies and immuno-
depletion
Each denatured His
6
-BIR family protein in urea-containing
buffer was supplied with SDS and 2-mercaptoethanol both
at 0.1% to maintain solubility, dialyzed extensively against
phosphate buffered saline, and used for rabbit immuniza-
tion to raise polyclonal antiserum. The same method was
used to immobilize the recombinants on to HiTrap NHS-
activated columns (1 mL; Amersham Biosciences) except
that proteins were dialyzed against 0.2 m NaHCO
3

(pH 8.3) ⁄ 0.5 m NaCl. The resulting columns were used for
the affinity purification of specific antibodies according to
the manufacturer’s instructions.
To perform western blot, we electrically transferred
proteins resolved by SDS ⁄ PAGE on to Immobilon P
poly(vinylidene difluoride) membrane (Millipore). Mem-
branes were first blocked in TBST (10 mm Tris ⁄ HCl,
pH 8.0, 150 mm NaCl, 0.1% Tween 20) containing 5% dry
skimmed milk for 1 h at room temperature and then incu-
bated for 1 h at room temperature in TBST containing
each antiserum (1 : 10 000 dilution) and 3% BSA. After
washing in TBST, membranes were incubated in TBST
containing the corresponding secondary antibody–alkaline
phosphatase conjugate (1 : 1000 dilution; New England
Biolabs) and 5% dry skimmed milk for 1 h at room tem-
perature. Immunocomplexes were detected using nitro blue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as
substrates.
For immunoprecipitation ⁄ immunodepletion analyses,
each affinity-purified antibody was cross-linked on Affi-
Prep protein A (Bio-Rad) with dimethylpimelimidate at
2 mg antibody per mL beads, followed by equilibration
with CSF-XB (10 mm Hepes ⁄ KOH, pH 7.8, 100 mm KCl,
0.1 mm CaCl
2
,2mm MgCl
2
,5mm EGTA, 50 mm sucrose).
For immunoprecipitation-coupled western blot, 1 lL beads
was mixed with 100 lL CSF-arrested extracts for 1 h on ice

with occasional flicking. After centrifugation at 15 000 g
for 30 s, beads were extensively washed and subjected to
western blot using the corresponding antibodies. For immuno-
depletion, 2.5 lL beads was mixed with 25 lL CSF-arrested
extracts and treated similarly. After centrifugation, 20 lL
supernatant was carefully removed and used for further
analyses.
Egg, sperm and extract manipulation
Priming and ovulation of female Xenopus frogs and prepar-
ation of egg CSF-arrested extracts and demembranated
sperm nuclei were according to the published protocols
[13,16,47]. We first prepared CSF-arrested extracts and then
released them into interphase by the addition of 0.4 mm
CaCl
2
and 0.1 mgÆmL
)1
cycloheximide, followed by incuba-
tion at 23 °C for 30 min. Under our experimental conditions,
these extracts gave clearer spherical nuclear morphology and
greater stability during the immunodepletion process than
did interphase extracts directly prepared without EGTA.
Observation of apoptotic change in egg extracts
MBP-BIR family proteins or control MBP were added at
10 lgÆmL
)1
to interphase extracts supplemented with sperm
nuclei (500 lL
)1
). During the incubation at 23 °C, 3 lLof

the extracts was taken out every hour, spotted on to a glass
slide, and mixed with 3 lL fixing solution consisting of
1· MMR salts (0.1 m NaCl, 2 mm KCl, 1 mm MgCl
2
,
2mm CaCl
2
, 0.1 mm EDTA and 5 mm Hepes ⁄ KOH,
pH 7.8), 60% glycerol, 10% formaldehyde and 10 lgÆmL
)1
4,6-diamidino-2-phenylindole. At each time point, the mor-
phology of 50 nuclei was observed for every sample using a
confocal laser scanning microscope (OZ with INTERVI-
SION; Noran Instruments, Middleton, WI, USA) and an
optical microscope (Zeiss, Tokyo, Japan). Fluorescent ima-
ges were processed with photoshop 5.0LE (Adobe, Tokyo,
Japan). For the detection of p150
Glued
cleavage, samples
corresponding to 0.4 lL egg extracts were subjected to
western blot using monoclonal anti-p150
Glued
Ig (1 : 1000
dilution; BD Transduction Laboratory, San Diego, CA,
USA) as described previously [35].
Stability analyses of BIR proteins in CSF-arrested
and interphase egg extracts
MBP-fused BIR family proteins were supplied at 1 lgÆmL
)1
to CSF-arrested or interphase extracts, both containing

0.1 mgÆmL
)1
cycloheximide. After 0, 2, and 4 h incubation,
extracts were diluted with SDS ⁄ PAGE buffer and subjected
to western blot using anti-MBP serum (1 : 5000 dilution;
New England Biolabs). Where indicated, Z-VAD-FMK
(Peptide Institute, Osaka, Japan), MG-132 (Peptide Insti-
tute), UCF-101 (Calbiochem), roscovitine (Calbiochem), or
U0126 (Sigma-Aldrich) dissolved in dimethylsulfoxide was
Xenopus egg apoptosis inhibitors Y. Tsuchiya et al.
2248 FEBS Journal 272 (2005) 2237–2250 ª 2005 FEBS
added to extracts (1% dimethylsulfoxide) and similarly ana-
lyzed after 2 and 4 h of incubation. All egg extracts also
contained 10 lgÆmL
)1
leupeptin, pepstatin, and chymostatin
(Peptide Institute) to inhibit nonspecific lysosomal proteo-
lysis [13,16,47]. For the stability assay of radiolabeled
proteins, in vitro translated rabbit reticulocyte lysates and
egg extracts were mixed at 1 : 9 and similarly incubated.
After SDS ⁄ PAGE, gels were dried and analyzed with a
BAS2000II image analyzer (Fuji, Tokyo, Japan).
Acknowledgements
We thank Professor Haruhiko Takisawa and Dr Satoru
Mimura (Osaka University) for providing the Xenopus
oocyte cDNA library, and Drs Sanae Miyake, Yutaka
Deguchi, and Osamu Nakabayashi for helpful discus-
sions. The provision of Xenopus demembranated sperm
nuclei by Dr Norio Masuya (Toho University Sakura
Hospital) is greatly appreciated. This work was sup-

ported by Project Research Grant no. 14-29 from
Toho University School of Medicine.
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