The Journal of Cell Biology
The Journal of Cell Biology, Volume 160, Number 7, March 31, 2003 1115–1127
The Rockefeller University Press, 0021-9525/2003/03/1115/13 $8.00
Caspase cleavage product of BAP31 induces
mitochondrial fission through endoplasmic reticulum
calcium signals, enhancing cytochrome
to the cytosol
David G. Breckenridge,
Richard C. Marcellus,
and Gordon C. Shore
Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
Gemin X Biotechnologies Inc., Montreal, Quebec, Canada H2X 3P9
timulation of cell surface death receptors activates
caspase-8, which targets a limited number of sub-
strates including BAP31, an integral membrane protein
of the endoplasmic reticulum (ER). Recently, we reported
that a caspase-resistant BAP31 mutant inhibited several
features of Fas-induced apoptosis, including the release of
c (cyt.c) from mitochondria (Nguyen, M., D.G.
Breckenridge, A. Ducret, and G.C. Shore. 2000.
20:6731–6740), implicating ER-mitochondria crosstalk
in this pathway. Here, we report that the p20 caspase
cleavage fragment of BAP31 can direct pro-apoptotic signals
between the ER and mitochondria. Adenoviral expression of
p20 caused an early release of Ca
from the ER, concomitant
uptake of Ca
ment of Drp1, a dynamin-related protein that mediates
scission of the outer mitochondrial membrane, resulting in
dramatic fragmentation and fission of the mitochondrial
network. Inhibition of Drp1 or ER-mitochondrial Ca
naling prevented p20-induced fission of mitochondria.
p20 strongly sensitized mitochondria to caspase-8–induced
cyt.c release, whereas prolonged expression of p20 on its
own ultimately induced caspase activation and apoptosis
through the mitochondrial apoptosome stress pathway.
Therefore, caspase-8 cleavage of BAP31 at the ER stimulates
-dependent mitochondrial fission, enhancing the release
of cyt.c in response to this initiator caspase.
into mitochondria, and mitochondrial recruit-
Mitochondria are key regulators of apoptosis that integrate
diverse apoptotic stimuli into a core death pathway (Green
and Reed, 1998). Mitochondrial control of apoptosis is gov-
erned by the BCL-2 family of proteins, which include anti-
apoptotic BCL-2 and BCL-x
BAK; the balance between these opposing members is regulated
by a third subgroup called the “BH3-only” proteins (Cory
and Adams, 2002). Current models hold that certain BH3-only
proteins invoke a mitochondrial phase of apoptosis by directing
the insertion of BAX into mitochondria and inducing oligo-
and pro-apoptotic BAX and
merization of BAX and BAK in the outer mitochondrial
membrane (OMM),* causing an efflux of intermembrane
space proteins, including cytochrome
al., 2000). Once in the cytosol, cyt.c complexes with Apaf-1
and procaspase-9 forming the apoptosome, a direct activator
of downstream effector caspases 3 and 7 (Budihardjo et al.,
1999). Activation of the TNF family of cell surface death
receptors is coupled to the mitochondrial phase of apoptosis by
the BH3-only protein BID. Binding of Fas to its ligand or
agonistic antibody induces the recruitment and autoactivation
of initiator procaspase-8 (Krammer, 2000). In turn, caspase-8
cleaves BID, generating tBID, which translocates from the
cytosol to mitochondria and induces organelle dysfunction
and cyt.c release (Li et al., 1998; Luo et al., 1998). BID plays
an obligate role in transducing signals from death receptors
to mitochondria in at least some cell types because hepatocytes
mice do not release cyt.c in response to
Fas, despite normal activation of caspase-8 (Yin et al., 1999). In
some contexts, caspase-8 can bypass mitochondria and directly
cleave downstream caspases. However, in many cell types,
c (cyt.c; Korsmeyer et
The online version of this article includes supplemental material.
Address correspondence to Gordon C. Shore, 3655 Promenade Sir
William Osler, McIntyre Medical Sciences Building (906), Dept. of
Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6.
Tel.: (514) 398-7282. Fax: (514) 398-7384. E-mail: firstname.lastname@example.org
*Abbreviations used in this paper: crBAP31, caspase-resistant BAP31;
c ; HA, hemagglutinin; OMM, outer mitochondrial
membrane; RTA, reverse tet transactivating protein; TG, thapsigargin.
Key words: apoptosis; caspase-8; BID; BAX; Drp1
The Journal of Cell Biology
1116 The Journal of Cell Biology
Volume 160, Number 7, 2003
the BID-dependent mitochondrial loop is required to amplify
weak death receptor signals and relieve the inhibitory effect
of IAP proteins on caspase activity (Scaffidi et al., 1998; Yin
et al., 1999; Deng et al., 2002; Fulda et al., 2002).
Although it is clear that BCL-2 family members govern
mitochondrial dysfunction, it remains unclear at what point
the functions of these proteins intercede with gross alter-
ations in mitochondrial morphology that occur during ap-
optosis. Normal mitochondrial morphology can vary dra-
matically between cell types, but in most cases mitochondria
form long “wormlike” tubules that may (Rizzuto et al.,
1998) or may not (Collins et al., 2002) make up intercon-
nected networks. The distribution of mitochondria depends
on interactions with microtubules whereas mitochondrial
size and shape is the result of constant fusion and fission
processes (Bereiter-Hahn and Voth, 1994). Little is known
about the mechanism of mitochondrial fission and fusion
except that it is regulated by a group of evolutionary con-
served GTPases; fusion is dependent on Fzo/Mfn, whereas
fission relies on a dynamin related protein, Drp1 (Oster-
young, 2001; Shaw and Nunnari, 2002). During apoptosis
mitochondria remodel inner membrane cristae (Scorrano et
al., 2002), fragment into small punctiform organelles that
sometimes cluster in the perinuclear region (Desagher and
Martinou, 2000; Frank et al., 2001; Pinton et al., 2001),
and eventually undergo matrix swelling leading to OMM
rupture (Petit et al., 1998; Mootha et al., 2001). Recently,
Frank et al. (2001) demonstrated that fragmentation of the
mitochondrial network during apoptosis is caused by large-
scale activation of Drp1-dependent mitochondrial fission,
and that this event is requisite for the mitochondrial phase
of apoptosis. How apoptotic signals converge on the fission
machinery, however, is unclear.
In the current paper, we present evidence that caspase
cleavage of BAP31 at the ER can trigger the onset of mito-
chondrial fission. BAP31 is a polytopic integral protein of
the ER membrane that forms a large hetero-oligomeric
complex with the related BAP29 protein and components
of the actomyosin network (Adachi et al., 1996; Ng et al.,
1997; Nguyen et al., 2000). After activation of cell surface
death receptors, human BAP31 is cleaved at two identical
caspase recognition sites in its cytosolic tail, generating a
membrane-embedded fragment, called p20, which induces
apoptosis when expressed ectopically (Ng et al., 1997;
Nguyen et al., 2000). Cleavage of BAP31 seems to be an
important event in the Fas pathway because cells expressing
a caspase-resistant BAP31 (crBAP31) mutant retain a near
normal morphology after stimulation and resist apoptotic
membrane blebbing/fragmentation, disruption of the actin
network, and irreversible loss of cell growth potential after
removal of the Fas stimulus (Nguyen et al., 2000). In addi-
tion, crBAP31 prevents mitochondrial remodeling and the
release of cyt.c in the face of activated caspases, suggesting
that events at the ER can modulate mitochondrial dysfunc-
tion in intact cells (Nguyen et al., 2000). To better under-
stand this communication between ER and mitochondria,
and how BAP31 contributes to Fas signaling in general,
we investigated the role of p20 in apoptotic progression.
We find that p20 stimulates ER Ca
the activation of Drp1-dependent fission of mitochondria,
release, resulting in
which ultimately sensitizes this organelle to caspase-8–
induced cyt.c release.
Caspase cleavage of BAP31 during Fas-mediated
apoptosis generates p20
BAP31 and its cellular homologue and heterodimerizing part-
ner, BAP29 (Adachi et al., 1996), are structurally conserved
proteins sharing identical topology in the ER membrane and
47% sequence identity in man. Both proteins initiate with
terminus facing the lumen, followed by three trans-
membrane regions and a cytosolic tail containing a long
coiled coil domain ending in a canonical KKXX ER retrieval
sequence (Fig. 1 A). Human BAP31 contains two identical
caspase cleavage sites (AAVD.G) at D164 and D238 that are
preferentially cleaved by caspase-8 (Ng et al., 1997; Wang et
al., 2003). Fig. 1 B shows that in human KB cells stimulated
with agonistic anti-Fas antibody BAP31 was cleaved generat-
ing the p27 and p20 membrane-embedded fragments. Only
the former cleavage site is conserved in mouse Bap31, sug-
gesting that cleavage at D164 is critical. The two caspase
cleavage sequences are not conserved in BAP29, which re-
mained structurally intact during apoptosis (Fig. 1 B).
Previously, we observed that the p20 caspase cleavage frag-
ment of BAP31 is cytotoxic when expressed ectopically (Ng
et al., 1997), indicating that caspase cleavage of BAP31
might generate a pro-apoptotic gain of function. To study
the mechanism of action of p20, we created an adenoviral
vector (Adp20) expressing this fragment (aa 1–164 of hu-
man BAP31) with a COOH-terminal hemagglutinin (HA)
tag. The endogenous p20 protein generated during Fas-
mediated apoptosis was associated with microsomes and re-
mained resistant to alkali extraction (pH 11.5), indicative of
a membrane-integrated protein (unpublished data). Immu-
nofluorescence microscopic analysis of Adp20-infected hu-
man H1299 cells revealed that exogenous p20 strongly colo-
calized with endogenous calreticulin, a resident ER lumen
protein, but p20 did not colocalize with TOM20, a marker
of the OMM. Therefore, caspase cleavage of BAP31 gener-
ates a pro-apoptotic p20 fragment that remains at the ER.
Prolonged expression of p20 induces apoptosis
Expression of p20 was observed by 10 h post-infection of
KB cells with Adp20, and remained stable for over 50 h
(Fig. 2 A). 30–40 h after infection, Adp20 induced the acti-
vation of caspases, measured by the hydrolysis of the caspase
substrate DEVD-amc and by processing of procaspase-3, in
many cell types including KB, H1299, HeLa, and Rat1 cells
(Fig. 2 B; unpublished data). The mechanism of this caspase
activation seemed to occur via the classical mitochondrial
apoptosome stress pathway. For example, p20 expression re-
sulted in the insertion of BAX into the OMM, homo-oligo-
merization of BAK, and release of cyt.c from mitochondria
in the presence of the pan-caspase inhibitor, zVAD-fmk
(Fig. 2 C; unpublished data). In contrast, p20-induced
caspase activation was abrogated in
(Fig. S1, available at http://www.jcb.org/cgi/content/full/
jcb.200212059/DC1). Control adenovectors expressing ei-
The Journal of Cell Biology
Caspase-cleaved BAP31 induces mitochondrial fission |
Breckenridge et al. 1117
cleavage product, and BAP29 in the ER membrane. Both BAP31 and BAP29 contain three transmembrane domains, a cytosolic tail containing a
coiled coil domain (boxed region), and terminate with a canonical KKXX ER retrieval sequence. The caspase-8 recognition sites in BAP31 are
shown. (B) KB cells were untreated or stimulated with 500 ng/ml anti-Fas activating antibody (CH11) and 10 ?g/ml cycloheximide (CHX) for 7 h,
and cell lysates were analyzed by SDS-PAGE and immunoblotting with anti-BAP31 (left) or anti-BAP29 (right) pAbs. The positions of BAP31, its p27
and p20 cleavage products, and BAP29 are indicated. (C) Adenoviral-expressed p20-HA (Adp20) localizes to the ER. H1299 cells were infected
with Adp20 for 20 h, then fixed and double stained with anti-HA and anti-calreticulin antibodies or anti-HA and anti-TOM20 antibodies.
BAP31, but not BAP29, is cleaved during Fas-mediated apoptosis. (A) Schematic representation of human BAP31, the p20 caspase
The Journal of Cell Biology
1118 The Journal of Cell Biology
Volume 160, Number 7, 2003
ther LacZ or the reverse tet transactivating protein (RTA)
did not cause any of the aforementioned apoptotic changes
(unpublished data). Inhibition of caspases using zVAD-fmk,
or overexpression of BCL-2 or BCL-x
morphological features of apoptosis including loss of plasma
membrane integrity as assessed by trypan blue uptake (Fig. 2
D). In the absence of these inhibitors, cells showed typical
signs of apoptosis, including nuclear condensation and frag-
mentation, membrane blebbing and cell surface exposure of
phosphatidylserine (unpublished data).
p20 strongly heterodimerizes with full-length BAP31
(Nguyen et al., 2000) and therefore, might cause apoptosis
by exerting a dominant-negative influence on endogenous
BAP31 or BAP29. However, as shown in Fig. 2 E, cell death
was observed in Bap31 or Bap29,31 double-deleted mouse
cells (Breckenridge et al., 2002) infected with Adp20, dem-
onstrating that p20 has an intrinsic pro-apoptotic activity at
the ER that is separate from the functions of BAP31 and
BAP29. Moreover, export of ectopic VSV G protein from
the ER occurred at 22 h after Adp20 infection, suggesting
that p20 does not exert a gross influence on ER-Golgi traf-
ficking at this early time (Fig. S2). Collectively, these results
indicate that p20 can activate mitochondrial apoptosis.
However, it is noteworthy that this pathway did not culmi-
nate until at least 30–40 h after infection (Fig. 2), whereas
ectopic tBID induces cyt.c release within several hours of its
expression (Li et al., 1998). Therefore, a more relevant func-
tion for p20 in physiological cell death might relate to an
early sensitization of mitochondria to a costimulus.
, blocked downstream
p20 sensitizes mitochondria to caspase-8–induced
Given that BAP31 is a caspase-8 substrate, p20 might coop-
erate with other products generated by caspase-8, such as
tBID, to enhance mitochondrial dysfunction. According to
this model, immediately after its expression, p20 should ac-
tivate a signal that is slow to induce cyt.c release on its own,
but able to synergize with other apoptotic signals during
Fas-mediated apoptosis. Therefore, we investigated whether
p20 could enhance caspase-8–driven cyt.c release. Death re-
ceptor–dependent caspase-8 activation was mimicked by in-
fecting H1299 cells with adenovector-expressing triplicate
copies of F
(a mutant of FKBP) fused to the catalytic sub-
units of caspase-8 (AdMF
3FLICE; Muzio et al., 1998). Af-
ter its expression in cells, oligomerization and autoactivation
of the caspase-8 fusion protein was induced with the F
dimerizing compound, FK1012Z. This approach has the
benefit of delivering synchronized caspase-8 signals to cells
without stimulating caspase-8–independent pathways acti-
Adp20 and cell lysates were collected and analyzed by immuno-
blotting at the times indicated post-infection. (B) KB and H1299
cells were infected with Adp20, and effector caspase (DEVDase)
activity was measured at the indicated times post-infection by the
ability of cell lysates to hydrolyze the fluorogenic caspase substrate
DEVD-amc. Shown is a representative experiment. (C) KB cells
were mock infected or infected with Adp20 for 35–40 h in the
Prolonged expression of p20 induces mitochondrial
(A) Expression of p20 in KB cells. Cells were infected with
absence or presence of 50
of post-mitochondrial supernatants were analyzed for the presence
of cyt.c by SDS-PAGE and immunoblotting. The membrane was
reprobed with anti-actin antibody to confirm equal loading.
(D) Parental KB cells, or KB cells stably overexpressing BCL-2 or
, were mock infected or infected with Adp20 in the absence
or presence of 50
M zVAD-fmk, and at 45 h post infection, cell
death was assessed by trypan blue staining. Shown is mean
three independent experiments. (E) Wt,
null mouse ES cells were treated and analyzed as in D.
M zVAD-fmk, and equivalent amounts
Bap31 -null, and Bap29,31-
The Journal of Cell Biology
Caspase-cleaved BAP31 induces mitochondrial fission |
Breckenridge et al. 1119
vated by death receptors (Schulze-Osthoff et al., 1998;
Wang et al., 2001). In Fig. 3, H1299 cells were co-infected
with AdRTA (control adenovector) and AdMF
with Adp20 and AdMF
3FLICE. 16 h after infection, a
time when Adp20 alone did not induce cyt.c release or
caspase activation (Figs. 2 and 3), the cells were exposed to a
short treatment (45 or 90 min) with FK1012Z or vehicle
(DMSO) alone, and the mitochondrial and post-mitochon-
drial fractions were isolated. Compared with caspase-8 acti-
vation in the presence of the control protein RTA, caspase-8
activation in the presence of ectopic p20 strongly induced
release of cyt.c to the cytosol (Fig. 3 A), but it did not affect
the amount of caspase-8–generated tBID that was recovered
in the mitochondrial fraction (Fig. 3 B). In all cases, equiva-
lent amounts of MF
3FLICE were produced (unpublished
data). Therefore, these results suggest that p20-mediated sig-
nals from the ER might cooperate with other caspase-8–gen-
erated signals to increase cyt.c release from mitochondria.
p20 mediates its effect through an early release
of Ca from the ER
Next, we sought to identify the early ER signaling events af-
ter p20 expression. Release of Ca
early event during many forms of apoptosis, including the
Fas pathway, and Ca
has been implicated as a second mes-
senger between ER and mitochondria during apoptosis
(Hajnoczky et al., 2000; Breckenridge and Shore, 2002).
We tested whether p20 expression altered ER Ca
stasis by loading Adp20-infected cells with the Ca
tive fluorescent indicator Fura2-AM and measuring the
from the ER occurs as an
increase in cytosolic Ca
(TG)-induced depletion of ER stores. TG invokes a rapid
emptying of ER Ca
stores to the cytosol by irreversibly
inhibiting SERCA pumps that normally maintain the con-
centration of ER Ca
above that of the cytosol ([Ca
pression of p20 in H1299 cells in the presence of zVAD-fmk
caused an early, time-dependent decrease in ER Ca
The kinetics of ER Ca
release was concomitant with
an increase in the concentration of mitochondrial Ca
), measured by Rhod2 fluorescence. These changes
in ER and mitochondrial Ca
early as 12–14 h post-infection (i.e., 2–4 h after p20 protein
appears; Fig. 2 A), making them the earliest events we ob-
served in the p20 pathway.
To determine whether the release of ER Ca
early responses of mitochondria to p20, we examined the
consequence of inhibiting Ca
and mitochondria. We began by adopting two experimental
conditions that reduce the amount of Ca
leased from the ER by p20 (Pinton et al., 2001). In the first
case, H1299 cells were incubated with a low concentration
of TG (50 nM). Addition of TG resulted in an immediate
emptying of ER Ca
stores, which remained depleted for
over 24 h (Fig. S3 A). In a second approach, we took ad-
vantage of the ability of BCL-2 to lower the [Ca
increasing the passive leak of Ca
youzi-Youssefi et al., 2000; Pinton et al., 2000). However,
to minimize the antiapoptotic activity of BCL-2 at the mito-
chondria, we created H1229 cells stably overexpressing BCL-2
that results from thapsigargin
) several orders of magnitude
). Fig. 4 A reveals that ex-
levels could be measured as
signaling between the ER
that could be re-
from the organelle (Fo-
to caspase-8–induced cyt.c release.
H1299 cells were mock infected, or
co-infected with AdRTA (control) and
AdMFpk3FLICE or with Adp20 and
AdMFpk3FLICE. 16 h post-infection,
FK1012Z or vehicle alone (DMSO;
Wang et al., 2003) were added for 45 or
90 min, and the amount of cyt.c in the
post-mitochondrial supernatant and
tBID in the mitochondrial fraction were
assessed by SDS-PAGE and Western
blot. The intensity of the cyt.c (A) and tBID
(B) signals, relative to loading controls,
was determined using ImageQuantTM
software (Amersham Biosciences) and is
expressed in arbitrary units. Shown is a
representative of three independent
p20 sensitizes mitochondria
The Journal of Cell Biology
1120 The Journal of Cell Biology
Volume 160, Number 7, 2003
selectively targeted to the ER with the membrane insertion
sequence of cytochrome b5 (H1299 b5-BCL-2; Zhu et al.,
1996). H1299 b5-BCL-2 cells had an
resting ER Ca
levels, resulting in a substantial decrease in
the total amount of Ca
released in response to Adp20 (Fig.
S3 B). We also tested the effect of two other agents:
BAPTA-AM, a cytosolic Ca
chelator that can inhibit Ca
transmission between the ER and mitochondria (Byrne et
al., 1999; Sharma et al., 2000); and Ru360, an inhibitor of
uptake (Matlib et al., 1998).
H1299 cells treated with Adp20
and stained with cyt.c displayed dramatically fragmented mi-
tochondria compared with mock-infected cells (Fig. 4 B). Re-
markably, in cells pre-treated with TG or expressing b5-BCL-2,
the mitochondrial network remained intact and highly
interconnected with no signs of mitochondrial fragmentation
(Fig. 4 B; unpublished data). Quantification of the two mito-
chondrial phenotypes revealed that TG and b5-BCL-2 re-
40% reduction in
zVAD-fmk for 24 h
duced the number of cells showing signs of mitochondrial
fragmentation from 52% to 10 and 13%, respectively (Fig. 4
C). Pretreatment of cells with BAPTA or Ru360 also reduced
the number of cells manifesting fragmented mitochondria in
response to p20 (Fig. 4 C). The expression of p20 was not af-
fected by any of the treatments (unpublished data). TG, b5-
BCL-2, BAPTA, and Ru360 also inhibited p20-induced re-
lease of cyt.c from mitochondria, which occurred subsequent
to fragmentation (unpublished data; see following section).
Therefore, inhibition of Ca
transport between the ER and
mitochondria inhibits the effect of p20 on mitochondrial
morphology and redistribution of cyt.c.
of the mitochondrial network
The observation that p20 caused mitochondrial fragmenta-
tion was extended in Fig. 5. p20 induced an early fragmenta-
tion of the mitochondrial network into small punctiform or-
release of Ca2? from the ER. H1299 cells were infected with Adp20 in the presence of 50 ?M zVAD-fmk and at the indicated times post-infection,
cells were loaded with Fura2-AM in Ca2?-free buffer and ER calcium stores were measured as the sudden difference in Fura2 fluorescence
recorded after the addition of TG (see Materials and methods). Shown is the mean and SD of five independent experiments. Bottom, elevated
[Ca2?]m after Adp20 infection. HeLa cells were treated as in A, except cells were loaded with Rhod2-AM and the [Ca2?]m was estimated as
described in the Materials and methods. (B) p20 induces dramatic fragmentation of mitochondria, which is inhibited by predepletion of ER Ca2?
stores with TG. H1299 cells were infected with 50 ?M Adp20 ? zVAD-fmk in the absence or presence of 50 nm TG for 24 h, and mitochondria
were visualized by anti-cyt.c staining. Representative images are shown. (C) Reducing ER Ca2? stores, chelating cytosolic Ca2?, or preventing
mitochondrial Ca2? uptake inhibits p20-induced fragmentation of mitochondria. As in B, but H1229 cells, or H1299 cells pretreated with
50 nm TG, 2 ?M BAPTA-AM, or 20 ?M Ru360, or H1299 b5-BCL-2 cells were infected with Adp20 ? zVAD-fmk for 24 h and the number of
cells showing signs of mitochondrial fragmentation was quantified. Shown is the mean ? SD of five independent experiments.
p20-induced mitochondrial fragmentation is mediated by an ER-mitochondria Ca2? signal. (A) Top, p20 induces a time dependent
The Journal of Cell Biology
Caspase-cleaved BAP31 induces mitochondrial fission |
Breckenridge et al. 1121
ganelles in all cell types tested, including H1299, Rat1, and
HeLa cells (Figs. 4 and 5; unpublished data). The gross mor-
phological changes in the mitochondrial network could be
observed 15–16 h after Adp20 infection (i.e., 2–3 h after the
onset of Ca2? release), a time when p20 sensitized mito-
chondria to caspase-8–induced cyt.c release (Fig. 3). Induc-
tion of mitochondrial fragmentation by p20 occurred in the
absence of zVAD-fmk–sensitive caspase activation and cell
shrinkage or disruption of microtubules. For example, Fig. 5
A shows that Rat1 fibroblasts expressing p20 ? zVAD-fmk
for 20 h and costained with anti-tubulin and anti-TOM20
antibodies displayed a normal microtubule distribution de-
spite having fragmented mitochondria. The punctiform mi-
tochondria could be observed in living cells stained with
MitoTracker® Red (unpublished data), indicating that frag-
mented mitochondria maintain membrane potential and
were not an artifact of fixation. Co-staining of Rat1 fibro-
blasts expressing p20 ? zVAD-fmk with antibody to cyt.c
and antibody selective for the active conformation of BAX
(Desagher et al., 1999) revealed that the transition of mito-
chondria into punctiform organelles preceded cyt.c release
and activation of BAX. As exemplified in Fig. 5 B, most cells
expressing p20 ? zVAD-fmk for 25 h displayed fragmented
mitochondria but showed no signs of cyt.c release or BAX
conformation-specific immunoreactivity. BAX immunore-
activity could only be observed in apoptotic cells that had re-
leased cyt.c from mitochondria, and all cells that had under-
gone cyt.c release stained positive for BAX. These results
suggest that p20 induces early fragmentation of mitochon-
dria, which precedes BAX activation and cyt.c release. Given
that disintegration of the mitochondrial network has been
demonstrated to contribute to apoptotic progression (De-
sagher and Martinou, 2000; Frank et al., 2001) but that
BAX/BAK activation and cyt.c release are normally stimu-
lated by BH3-only molecules (Korsmeyer et al., 2000), it is
likely that p20 mediates its sensitizing effect by inducing
early fragmentation of mitochondria.
p20 induces Drp1 translocation to mitochondria
Recently, fragmentation of mitochondria during stauro-
sporin-induced apoptosis was demonstrated to be dependent
on mitochondrial fission mediated by Drp1 (Frank et al.,
2001). Therefore, we investigated whether Drp1-dependent
fission played a role in the p20 pathway. We began by exam-
ining the subcellular localization of Drp1 because GFP-
tagged Drp1 was shown to redistribute from a predomi-
nately cytosolic location to predicted sites of division along
mitochondrial tubules after treatment with staurosporin
(Frank et al., 2001). Fig. 6 A documents that endogenous
Drp1 was recruited to mitochondria before the onset of
mitochondrial fragmentation in HeLa cells treated with
Adp20. HeLa cells were used for this experiment because
their mitochondria form long, clearly defined tubules ideal
for colocalization studies; however, the results were also
confirmed in Rat1 and H1299 cells. In mock-infected
HeLa cells, Drp1 was distributed throughout the cytosol
and showed only minor colocalization with mitochondria
stained with TOM20 (Fig. 6 A), likely because Drp1 nor-
mally cycles on and off mitochondria continuously (Frank et
al., 2001; Smirnova et al., 2001). In contrast, Drp1 showed
a strong colocalization with mitochondria in HeLa cells in-
fected with Adp20 ? zVAD-fmk for 17 h (Fig. 6 A, bottom
panels). Enlargement of the merged image revealed that
Drp1 formed clusters along the surface of mitochondrial tu-
bules before the onset of fragmentation. Interestingly, in
Caenorhabditis elegans, similar clusters of GFP-Drp1 on mi-
in the absence of cell shrinkage. Rat1 fibroblasts were infected with Adp20 in the presence of 50 ?M zVAD-fmk (to prevent caspase activation
and cell detachment) for 20 h, fixed, and double stained with anti-tubulin and anti-TOM20 antibodies. (B) Mitochondrial fragmentation occurs
before activation of BAX and cyt.c release. As in A, except cells were infected for 25 h and double stained with anti-cyt.c antibody (arrows,
lower left) and the active conformation-specific anti-BAX-NT antibody (aa 1–21, Upstate Biotechnology; arrows, lower right).
p20 induces fragmentation of the mitochondrial network as an early event. (A) Mitochondrial restructuring and fragmentation occur
The Journal of Cell Biology
1122 The Journal of Cell Biology | Volume 160, Number 7, 2003
tochondrial tubules were shown to coincide with future sites
of membrane scission (Labrousse et al., 1999). Pretreatment
of H1299 cells with TG or expression of b5-BCL-2 reduced
the amount of endogenous Drp1 recovered in the mito-
chondrial fraction after p20 expression (Fig. 6 B, compare
lane 2 with lanes 3 and 4) and inhibited mitochondrial fis-
sion (Fig. 4 C).
Dominant-negative Drp1K38E prevents p20-induced
To confirm that p20 mediates its sensitizing effect on mito-
chondria through Drp1, we examined the effect of a domi-
nant-negative Drp1 mutant on p20-induced mitochondrial
changes. Mutation of a conserved lysine (K38) in the GTP
binding domain of Drp1 is predicted to reduce GTPase ac-
tivity (Smirnova et al., 1998; Bleazard et al., 1999), and ex-
pression of such a mutant inhibits OMM scission (Labrousse
et al., 1999). Ectopic expression of CFP-Drp1K38E in Rat1
cells offset the normal balance between mitochondrial fission
and fusion and increased the connectivity of mitochondria
compared with untransfected cells or cells transfected with
wild-type CFP-Drp1 (Fig. 7 A, top panels; transfected
[CFP-positive] cells are indicated by arrows). Overexpression
of wild-type Drp1 does not induce fission in mammalian
cells (Smirnova et al., 1998; Frank et al., 2001), and accord-
ingly, Rat1 cells transiently transfected with CFP-Drp1 ex-
hibited a normal mitochondrial phenotype and underwent
fragmentation in response to Adp20 (Fig. 7 A). Cells trans-
fected with CFP-Drp1K38E, on the other hand, resisted
Adp20-induced mitochondrial fission and the highly inter-
connected network remained intact. As shown in Fig. 7 (B
and C), CFP-Drp1K38E also inhibited p20-induced cyt.c re-
lease and caspase activation. Based on morphological criteria,
as well as the recruitment of endogenous Drp-1 to mito-
chondria and dominant interference by the Drp1K38E mu-
tant, we conclude that p20 activates Drp1-dependent mito-
chondrial fission, sensitizing this organelle for cyt.c release.
Engagement of the TNF receptor family of death receptors,
including TNF-R1, Fas, Trail-R1, and Trail-R2, with their
cognate ligands leads to the recruitment and autoactivation
of initiator procaspase-8 (Krammer, 2000). Recent studies
implicate that caspase-8 substrates located at distinct cellu-
lar loci play key roles in mediating death receptor–induced
apoptosis. For example, caspase-8 cleavage of the BH3-
only molecule BID promotes mitochondrial release of cyt.c
and Smac/Diablo (Yin et al., 1999; Li et al., 2002); cleav-
age of RIP prevents the activation of NF-?B survival re-
Drp1 to mitochondria. HeLa cells were mock infected (top) or infected with Adp20 (bottom)
in the presence of zVAD-fmk, and 17 h post-infection, cells were fixed, double stained with
anti-Drp1 (green) and anti-TOM20 (red) antibodies, and imaged by confocal immunofluorescence
microscopy. Enlargement of the merged overlay revealed that clusters of Drp1 relocate along
mitochondrial filaments before the onset of fission. (B) H1299 cells, H1299 cells treated with
TG, or H1299 b5-BCL-2 cells were infected with Adp20?zVAD for 18 h and the mitochondrial
fraction was isolated and analyzed for the presence of Drp1 by SDS-PAGE and immunoblotting.
The blot was reprobed with anti-TOM20 antibody to demonstrate equal protein loading.
Drp1 mediates p20-induced mitochondrial fission. (A) Recruitment of endogenous
The Journal of Cell Biology
Caspase-cleaved BAP31 induces mitochondrial fission | Breckenridge et al. 1123
sponses (Lin et al., 1999); and cleavage of the cytolinker
plectin is important for disassembly of microfilaments
(Stegh et al., 2000). In this work, we investigated the con-
sequence of caspase-8 cleavage of BAP31 at the ER by ex-
pressing the pro-apoptotic p20 cleavage fragment in cells
using an adenovirus vector. This approach allowed us to
isolate and delineate a predicted branch of the death recep-
tor signaling cascade. Specifically, we found that p20 could
mediate Ca2?-dependent apoptotic crosstalk between the
ER and mitochondria, stimulating mitochondrial fission
and sensitization of this organelle to caspase-8–induced
The importance of BAP31 cleavage during Fas-mediated
apoptosis was first highlighted by the observation that ex-
pression of crBAP31 strongly inhibited apoptotic membrane
blebbing and release of cyt.c from mitochondria (Nguyen
et al., 2000), suggesting that ER-mitochondrial signaling
played a role in this pathway. When we reexamined photo-
graphs of mitochondria in crBAP31 cells undergoing Fas-
induced apoptosis, it was apparent that mitochondrial frag-
mentation was also strongly inhibited (Nguyen et al., 2000).
Thus, full-length BAP31 and p20 have opposing functions
during Fas-mediated apoptosis, the former inhibiting mito-
chondrial fission and egress of cyt.c from mitochondria, and
the latter stimulating these events. Importantly, however,
p20 operates independently of BAP31 and BAP29 because
p20 caused apoptosis in Bap31- and Bap29,31-null cells
(Fig. 2 E). Therefore, caspase-8 cleavage of BAP31 converts
it from an inhibitor to an activator of cell death; a paradigm
that has been ascribed to other caspase targets such as BCL-2
(Cheng et al., 1997), BCL-xL (Clem et al., 1998), and RIP
(Lin et al., 1999).
Cleavage of BAP31 may contribute to other cell death
pathways that signal through caspase-8. For example, we re-
cently reported that BAP31 and BAP29 play a role in the re-
cruitment and activation of procaspase-8L at the ER during
E1A-induced apoptosis (Breckenridge et al., 2002). The ki-
netics of procaspase-8L processing strongly correlated with
BAP31 cleavage in response to E1A, suggesting that acti-
vated procaspase-8L may hydrolyze BAP31. The ensuing
p20-induced Ca2? release and mitochondrial fission might
then enhance cyt.c release by other pro-apoptotic regulators
that are activated by E1A, including BIK (Breckenridge and
Shore, 2000; Mathai et al., 2002).
fibroblasts were transiently transfected with CFP-Drp1 or CFP- Drp1K38E, then either mock infected or infected with Adp20 in the presence of
zVAD-fmk. 24 h post-infection cells were fixed, stained with anti-TOM20, and analyzed by fluorescence microscopy. Cells expressing
CFP-Drp1 or CFP- Drp1K38E were identified under the cyan filter and are indicated with an arrow. (B) CFP- Drp1K38E inhibits cyt.c release.
H1299 cells were treated as in B for 36 h, and immunofluorescence microscopy was used to assess the distribution of cyt.c in cells positive
for CFP fluorescence. Shown is the mean ? SD of four independent experiments. (C) H1299 cells were transiently cotransfected with the
indicated constructs and 36 h post-transfection cell lysates were collected and processed for DEVDase activity, shown is the mean ? SD of
three independent experiments.
Expression of a Drp1K38E dominant-negative mutant inhibits p20 induced disruption of the mitochondrial network. (A) Rat1
The Journal of Cell Biology
1124 The Journal of Cell Biology | Volume 160, Number 7, 2003
Based on studies using pharmacological modulators of
Ca2? signaling and inhibitors of apoptosis and mitochon-
drial fission, our results suggest that p20 induces an apop-
totic pathway between the ER and mitochondria (Fig. 8 A).
This is initiated by ER Ca2? release coupled to mitochon-
drial Ca2? uptake. An important caveat, of course, is that
such conclusions rely on the specificity of the inhibitors that
are widely used to interfere with Ca2? signaling. Moreover,
it cannot be ruled out that additional mechanisms are also
involved. Importantly, however, it has been demonstrated
that Drp1 recruitment to mitochondria initiates fission (La-
brousse et al., 1999; Smirnova et al., 2001). Because either
the lowering of ER Ca2? stores, or chelating cytosolic Ca2?,
or preventing mitochondrial Ca2? uptake all prevented
p20-induced fission of mitochondria, it is likely that ER-
mitochondrial Ca2? transmission acts upstream of Drp1
translocation in this context. Drp1 recruitment is likely me-
diated by an OMM receptor protein(s), and this complex
likely cooperates with an inner mitochondrial membrane
reorganizing enzyme(s) to mediate organelle fission (Shaw
and Nunnari, 2002). Mitochondrial membranes are often
in close proximity and privileged Ca2? exchange between
the two organelles has previously been implicated during
apoptosis. For example, IP3 receptor– and ryanodine re-
ceptor–mediated Ca2? spikes that modulate mitochondrial
metabolism in healthy cells also sensitize mitochondria to
pro-apoptotic stimuli during cell death (Szalai et al., 1999;
Hajnoczky et al., 2000). Moreover, manipulations that in-
crease [Ca2?]ER also increase agonist-induced Ca2? spikes
and enhance mitochondrial cyt.c release and apoptosis,
whereas a lowering of ER Ca2? stores has the opposite effect
(Nakamura et al., 2000; Pinton et al., 2001). Modulation
of the frequency, amplitude and spatio-temporal pattern of
ER Ca2? release during apoptosis may determine how mito-
chondria respond to Ca2? signals (Berridge et al., 2000;
Pacher and Hajnoczky, 2001). Our results suggest that
caspase cleavage of BAP31 may be one mechanism to gener-
ate such pro-apoptotic ER-mitochondrial Ca2?-dependent
crosstalk in the Fas pathway.
In isolation, p20 caused ER Ca2? release soon after its ex-
pression and Drp1 redistribution and mitochondrial fission
were apparent within several hours of this event, but BAX
activation, cyt.c release, and caspase activation were signifi-
cantly delayed. Therefore, in the absence of a parallel BH3-
dependent hit, mitochondria undergo fission in response to
p20 and probably remain in a fragmented state (without re-
leasing cyt.c) until a second signal responds and activates
BAX/BAK. However, in a normal death receptor signaling
context, simultaneous processing of BAP31 and BID by
caspase-8 would be predicted to mount a dual attack on mi-
tochondria, with p20 causing mitochondrial fission and
tBID inducing cristae remodeling and activation of BAX
and BAK (Scorrano et al., 2002; Fig. 8 B). Apoptotic cristae
remodeling and mitochondrial fission may be intimately
by mitochondria. Mitochondria, in turn, recruit Drp1, which initiates organelle fission. Lowering ER Ca2? stores by pretreatment with TG
or expression of b5-BCL-2, chelating the Ca2? released to the cytosol with BAPTA, blocking mitochondrial uptake of Ca2? with Ru360, or
inhibition of Drp1 by expression of Drp1K38E all prevent p20-induced mitochondrial fission. (B) A model depicting how in intact cells, cleavage
of BAP31 at the ER sensitizes mitochondria to caspase-8–driven cyt.c release. Stimulation of Fas leads to caspase-8–dependent processing of
BAP31 and BID, generating p20 and tBID. tBID translocates to mitochondria, where it induces the oligomerization of BAX/BAK into pores in
the OMM. Simultaneously, p20 triggers ER Ca2? release, causing Drp-1 translocation to mitochondria and subsequent organelle fission,
enhancing the release of cyt.c to the cytosol.
A proposed mechanism of p20-induced mitochondrial fission. (A) p20 triggers a specific Ca2? signal from the ER that is decoded
The Journal of Cell Biology
Caspase-cleaved BAP31 induces mitochondrial fission | Breckenridge et al. 1125
linked because cristae reorganization occurs during normal
fission and fusion events in healthy cells (Bereiter-Hahn and
Voth, 1994; Shaw and Nunnari, 2002) and mitochondrial
fission is a requisite for cyt.c release (Frank et al., 2001). A
“two hit” model in which an ER–mitochondrial Ca2? signal
and a direct mitochondrial insult synergize to promote the
mitochondrial phase of apoptosis likely functions in other
apoptosis pathways (Szalai et al., 1999; Pinton et al., 2001).
Of note, tBID was reported to induce caspase-independent
mitochondrial fragmentation on its own (Li et al., 1998),
and therefore, p20 signaling may not be an obligate require-
ment for cyt.c release on the death receptor pathway but
rather a sensitizer of this event. Indeed, the combined ac-
tions of p20 and tBID could cooperate in vivo because p20
strongly enhanced the ability of caspase-8 to promote cyt.c
release without affecting the extent of BID cleavage (Fig. 3).
This duality in signaling may be particularly relevant in
physiological situations where apoptotic stimuli are sub-
optimal or are countered by opposing survival signals, and
the fate of the cell hinges on the balance of pro-apoptotic
and anti-apoptotic signals received by mitochondria.
Materials and methods
Antibodies, plasmids, and reagents
The following antibodies were used in this work: chicken anti–human
BAP31 (Ng et al., 1997), rabbit pAbs raised against the recombinant hu-
man BAP29, human TOM-20 (Goping et al., 1995), ?-actin (a gift from P.
Braun, McGill University, Montreal, PQ), and human BAX aa 1–21 (Up-
state Biotechnology); Rabbit pAb raised against the p15 caspase cleavage
product of BID and purified by affinity selection; and mouse mAbs to pi-
geon cyt.c (BD Biosciences), chicken ?-tubulin (clone DM1A; Sigma-
Aldrich), rodent Drp1 (BD Biosciences), and HA (clone 16B12; BAbCO).
Goat anti-calreticulin was provided by M. Michalak (University of Alberta,
Edmonton, AB). Anti-human Fas activating antibody (Clone CH-11) was
from Upstate Biotechnology. Standard PCR techniques were used to gener-
ate cDNA encoding p20 (aa 1–164 of human BAP31) with a COOH-termi-
nal HA tag that was cloned in to pcDNA3. Plasmids encoding Drp1 and
Drp1K38E fused to CFP at the NH2 terminus were gifts from H. McBride (Ot-
tawa Heart institute, Ottawa, ON). Carbobenzoxy-valvy-alanyl-aspartyl-
methyl ester-fluormethyl ketone (zVAD-fmk) was purchased from Enzyme
System Products. Fura2-AM, Rhod2-AM, and BAPTA-AM were from Mo-
lecular Probes, Inc., and Ru360 was from Calbiochem. All other chemicals
were purchased from Sigma-Aldrich, unless otherwise noted.
Cell culture, virus infection, and transfection
KB epithelial cells and H1299 lung carcinoma cells were maintained in
MEM-? supplemented with 10% FBS and 100 U/ml streptomycin and pen-
icillin. Rat1 fibroblast, CHO, and HeLa cells were grown in DME supple-
mented as above. KB cells stably expressing HA-Bcl-2 and HA-Bcl-XL have
been described previously (Nguyen et al., 1998; Ruffolo et al., 2000).
H1299 b5-BCL-2 cells were created by transfecting H1299 cells with
pcDNA3 vector encoding human HA-Bcl-2 with amino acids 215–239
swapped with the transmembrane sequence of human cytochrome B5
(amino acids 107–134) and selecting for resistance to geneticin. Bap31-
and Bap29-null mouse embryonic stem cells were maintained as de-
scribed previously (Breckenridge et al., 2002).
For the construction of Adp20, AdMFpk3FLICE, and AdRTA, cDNAs en-
coding p20-HA, MFpk3FLICE (Muzio et al., 1998) and RTA were subcloned
into a variant of pCA14 containing the T-REx™ promoter (Invitrogen),
which functioned as a shuttle to produce the adenoviral vectors as de-
scribed previously (Bett et al., 1994; Mathai et al., 2002). All adenoviral in-
fections were conducted at a multiplicity of infection of 100 pfu/cell as de-
scribed previously (Ng et al., 1997), except for in Fig. 3, where the viruses
were mixed to generate a total of 100 pfu/cell. LipofectAMINE™ Plus (In-
vitrogen) was used for all transfections using the manufacturer’s protocols.
In experiments where Adp20 infection followed transfection of CFP-Drp1
or CFP-Drp1K38E, the transfection medium was removed 3 h after transfec-
tion and medium containing serum and Adp20 virus was added back.
DEVDase activity was measured from 25 ?g of cell lysate protein accord-
ing to the manufacturer’s protocol (Upstate Biotechnology). For statistical
analysis of mitochondrial fission and cyt.c release, cells were stained for
TOM20 or cyt.c, and the distribution of cyt.c and the morphology of mito-
chondria were analyzed by conventional immunofluorescence micros-
copy. In all cases, at least five independent experiments were conducted,
where three counts of 150 randomly selected cells was done per experi-
ment. In Fig. 7 B, only cells showing CFP expression were assessed for
cyt.c release. Biochemical isolation of the heavy membrane fraction en-
riched in mitochondria or post-mitochondrial supernatant for measure-
ment of cyt.c release and Drp1 recruitment was done as described previ-
ously (Ruffolo et al., 2000). In Fig. 3, the intensity of each cyt.c and tBID
Western blot signal was quantified using ImageQuantTM (Amersham Bio-
sciences) software and compared with the intensity of a loading control
signal in the same lane (actin or TOM20, respectively) after the subtraction
of background. The relative values were expressed as arbitrary units.
Cells were typically seeded at 50% confluency on glass coverslips and
mock infected or infected with Adp20, always in the presence of zVAD-
fmk to prevent apoptosis and cell detachment. At the indicated times after
infection, cells were washed in PBS, and were then fixed in PFA solution
(4% PFA, 23 mM NaH2PO4, and 77 mM Na2HPO4, pH 7.3). Cells were
briefly permeabilized in PBS/0.2% Triton X-100, then blocked in blocking
solution (PBS containing 10% FCS and 0.1% Triton X-100). Primary and
secondary antibody incubations were done in blocking solution for 1 h at
RT using the indicated antibodies and goat anti–mouse IgG or goat anti–
rabbit IgG secondary antibody coupled to Alexa® 488 (green) or Alexa®
594 (red; Molecular Probes, Inc.). Cells were visualized by confocal mi-
croscopy or by conventional fluorescence microscopy on an inverted mi-
croscope (TE-FM Epi-fl; Nikon).
Measurement of ER Ca2? content
The ER Ca2? store was measured as the sudden increase in [Ca2?]c on addi-
tion of TG. [Ca2?]c was measured by the cell permeable fluorescent indica-
tor Fura2-AM. In brief, 2 ? 106 cells were washed in Ca2?-free buffer (20
mM Hepes, pH 7.4, 143 mM NaCl, 6 mM KCL, 1 mM MgSO4, 0.1% glu-
cose, and 250 ?M sulfinpyrazone), then loaded with 3 mM Fura2-AM for
30 min at 37?C in Ca2?-free buffer containing 0.02% pluronic acid and
0.1% BSA. After a final wash, cells were resuspended in Ca2?-free buffer
and [Ca2?]c was measured as 340/380 nm excitation wavelength ratio at
510 nm wavelength emission (340/380 ratio) in a luminescence spectro-
photometer (model LS 50B; PerkinElmer). The ER calcium content was
measured as the difference between the baseline 340/380 ratio before TG
addition and the peak 340/380 ratio after TG addition. This value was arbi-
trarily set at 100% for untreated cells.
Measurement of [Ca2?]m
5 ? 105 cells were collected, washed once in PBS, then resuspended in 1
ml Earl’s balanced salt solution and loaded with 2 ?M Rhod2-AM in the
presence of 0.02% pluronic acid for 20 min at RT. Cells were washed
twice in the same buffer and Rhod2 fluorescence (F) was measured as
above at 550/580 excitation/emission wavelengths. Minimum and maxi-
mum fluorescence values (Fmax and Fmin, respectively) were then obtained
on the sequential addition of EGTA and saturating amounts of CaCl2 in the
presence of detergent. [Ca2?]m was determined by the equation
[Ca2?] ? Kd (F ? Fmin)/(Fmax ? F) where Kd is the dissociation constant of
Rhod2. Rhod2 was judged to be localized to mitochondria based on analysis
by immunofluorescence microscopy and by the fact that fluorescence was re-
duced to basal levels on the addition of the mitochondrial uncoupler, CCCP.
Online supplemental material
Fig. S1 documents that Adp20-induced release of cyt.c from mitochondria,
procaspase-3 processing, and caspase activation are blocked in APAF-1–
null MEFs (Yoshida et al., 1998; a gift from T. Mak, Ontario Cancer Insti-
tute, Toronto, Canada). Fig. S2 shows that Adp20 infection does not affect
ER-Golgi trafficking of temperature-sensitive VSV-G-EGFP. Fig. S3 docu-
ments that TG and b5-BCL-2 effectively lowered resting ER Ca2? stores.
Immunofluorescence microscopy confirmed that b5-BCL-2 was located
exclusively at the ER. Online supplemental material available at http://
We are very grateful to J. Lynch and M. Michalak for Fura2 protocols and
The Journal of Cell Biology
1126 The Journal of Cell Biology | Volume 160, Number 7, 2003
D.G. Breckenridge is a recipient of a Canadian Institutes of Health Re-
search Doctoral award. This work was supported by grants to G.C. Shore
from the National Cancer Institute of Canada, and the Canadian Institutes
of Health Research.
Submitted: 10 December 2002
Revised: 6 February 2003
Accepted: 11 February 2003
Adachi, T., W.W. Schamel, K.M. Kim, T. Watanabe, B. Becker, P.J. Nielsen, and
M. Reth. 1996. The specificity of association of the IgD molecule with the
accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence.
EMBO J. 15:1534–1541.
Bereiter-Hahn, J., and M. Voth. 1994. Dynamics of mitochondria in living cells:
shape changes, dislocations, fusion, and fission of mitochondria. Microsc.
Res. Tech. 27:198–219.
Berridge, M.J., P. Lipp, and M.D. Bootman. 2000. The versatility and universality
of calcium signalling. Nat. Rev. Mol. Cell Biol. 1:11–21.
Bett, A.J., W. Haddara, L. Prevec, and F.L. Graham. 1994. An efficient and flexi-
ble system for construction of adenovirus vectors with insertions or deletions
in early regions 1 and 3. Proc. Natl. Acad. Sci. USA. 91:8802–8806.
Bleazard, W., J.M. McCaffery, E.J. King, S. Bale, A. Mozdy, Q. Tieu, J. Nunnari,
and J.M. Shaw. 1999. The dynamin-related GTPase Dnm1 regulates mito-
chondrial fission in yeast. Nat. Cell Biol. 1:298–304.
Breckenridge, D.G., and G.C. Shore. 2000. Regulation of apoptosis by E1A and
Myc oncoproteins. Crit. Rev. Eukaryot. Gene Exp. 10:273–280.
Breckenridge, D.G., and G.C. Shore. 2002. The endoplasmic reticulum and apop-
tosis. In Genetics of Apoptosis. S. Grimm, editor. BIOS Scientific Publishers
Inc., Oxford. 95–113.
Breckenridge, D.G., M. Nguyen, S. Kuppig, M. Reth, and G.C. Shore. 2002. The
procaspase-8 isoform, procaspase-8L, recruited to the BAP31 complex at the
endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 99:4331–4336.
Budihardjo, I., H. Oliver, M. Lutter, X. Luo, and X. Wang. 1999. Biochemical
pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol.
Byrne, A.M., J.J. Lemasters, and A.L. Nieminen. 1999. Contribution of increased
mitochondrial free Ca2? to the mitochondrial permeability transition induced
by tert-butylhydroperoxide in rat hepatocytes. Hepatology. 29:1523–1531.
Cheng, E.H., D.G. Kirsch, R.J. Clem, R. Ravi, M.B. Kastan, A. Bedi, K. Ueno,
and J.M. Hardwick. 1997. Conversion of Bcl-2 to a Bax-like death effector
by caspases. Science. 278:1966–1968.
Clem, R.J., E.H. Cheng, C.L. Karp, D.G. Kirsch, K. Ueno, A. Takahashi, M.B.
Kastan, D.E. Griffin, W.C. Earnshaw, M.A. Veliuona, and J.M. Hardwick.
1998. Modulation of cell death by Bcl-XL through caspase interaction. Proc.
Natl. Acad. Sci. USA. 95:554–559.
Collins, T.J., M.J. Berridge, P. Lipp, and M.D. Bootman. 2002. Mitochondria are
morphologically and functionally heterogeneous within cells. EMBO J. 21:
Cory, S., and J.M. Adams. 2002. The Bcl2 family: regulators of the cellular life-or-
death switch. Nat. Rev. Cancer. 2:647–656.
Deng, Y., Y. Lin, and X. Wu. 2002. TRAIL-induced apoptosis requires Bax-depen-
dent mitochondrial release of Smac/DIABLO. Genes Dev. 16:33–45.
Desagher, S., and J.C. Martinou. 2000. Mitochondria as the central control point
of apoptosis. Trends Cell Biol. 10:369–377.
Desagher, S., A. Osen-Sand, A. Nichols, R. Eskes, S. Montessuit, S. Lauper, K.
Maundrell, B. Antonsson, and J.C. Martinou. 1999. Bid-induced conforma-
tional change of Bax is responsible for mitochondrial cytochrome c release
during apoptosis. J. Cell Biol. 144:891–901.
Foyouzi-Youssefi, R., S. Arnaudeau, C. Borner, W.L. Kelley, J. Tschopp, D.P.
Lew, N. Demaurex, and K.H. Krause. 2000. Bcl-2 decreases the free Ca2?
concentration within the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA.
Frank, S., B. Gaume, E.S. Bergmann-Leitner, W.W. Leitner, E.G. Robert, F. Catez,
C.L. Smith, and R.J. Youle. 2001. The role of dynamin-related protein 1, a
mediator of mitochondrial fission, in apoptosis. Dev. Cell. 1:515–525.
Fulda, S., W. Wick, M. Weller, and K.M. Debatin. 2002. Smac agonists sensitize
for Apo2L/TRAIL- or anticancer drug-induced apoptosis and induce regres-
sion of malignant glioma in vivo. Nat. Med. 8:808–815.
Goping, I.S., D.G. Millar, and G.C. Shore. 1995. Identification of the human mi-
tochondrial protein import receptor, huMas20p. Complementation of delta
mas20 in yeast. FEBS Lett. 373:45–50.
Green, D.R., and J.C. Reed. 1998. Mitochondria and apoptosis. Science. 281:
Hajnoczky, G., G. Csordas, M. Madesh, and P. Pacher. 2000. Control of apoptosis
by IP(3) and ryanodine receptor driven calcium signals. Cell Calcium. 28:
Korsmeyer, S.J., M.C. Wei, M. Saito, S. Weiler, K.J. Oh, and P.H. Schlesinger.
2000. Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX
into pores that result in the release of cytochrome c. Cell Death Differ.
Krammer, P.H. 2000. CD95’s deadly mission in the immune system. Nature. 407:
Labrousse, A.M., M.D. Zappaterra, D.A. Rube, and A.M. van der Bliek. 1999. C.
elegans dynamin-related protein DRP-1 controls severing of the mitochon-
drial outer membrane. Mol. Cell. 4:815–826.
Li, H., H. Zhu, C.J. Xu, and J. Yuan. 1998. Cleavage of BID by caspase 8 mediates
the mitochondrial damage in the Fas pathway of apoptosis. Cell. 94:491–501.
Li, S., Y. Zhao, X. He, T.H. Kim, D.K. Kuharsky, H. Rabinowich, J. Chen, C.
Du, and X.M. Yin. 2002. Relief of extrinsic pathway inhibition by the Bid-
dependent mitochondrial release of Smac in Fas-mediated hepatocyte apop-
tosis. J. Biol. Chem. 277:26912–26920.
Lin, Y., A. Devin, Y. Rodriguez, and Z.G. Liu. 1999. Cleavage of the death do-
main kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes Dev.
Luo, X., I. Budihardjo, H. Zou, C. Slaughter, and X. Wang. 1998. Bid, a Bcl2 in-
teracting protein, mediates cytochrome c release from mitochondria in re-
sponse to activation of cell surface death receptors. Cell. 94:481–490.
Mathai, J.P., M. Germain, R.C. Marcellus, and G.C. Shore. 2002. Induction and
endoplasmic reticulum location of BIK/NBK in response to apoptotic sig-
naling by E1A and p53. Oncogene. 21:2534–2544.
Matlib, M.A., Z. Zhou, S. Knight, S. Ahmed, K.M. Choi, J. Krause-Bauer, R.
Phillips, R. Altschuld, Y. Katsube, N. Sperelakis, and D.M. Bers. 1998. Ox-
ygen-bridged dinuclear ruthenium amine complex specifically inhibits Ca2?
uptake into mitochondria in vitro and in situ in single cardiac myocytes. J.
Biol. Chem. 273:10223–10231.
Mootha, V.K., M.C. Wei, K.F. Buttle, L. Scorrano, V. Panoutsakopoulou, C.A.
Mannella, and S.J. Korsmeyer. 2001. A reversible component of mitochon-
drial respiratory dysfunction in apoptosis can be rescued by exogenous cyto-
chrome c. EMBO J. 20:661–671.
Muzio, M., B.R. Stockwell, H.R. Stennicke, G.S. Salvesen, and V.M. Dixit. 1998.
An induced proximity model for caspase-8 activation. J. Biol. Chem. 273:
Nakamura, K., E. Bossy-Wetzel, K. Burns, M.P. Fadel, M. Lozyk, I.S. Goping, M.
Opas, R.C. Bleackley, D.R. Green, and M. Michalak. 2000. Changes in en-
doplasmic reticulum luminal environment affect cell sensitivity to apoptosis.
J. Cell Biol. 150:731–740.
Ng, F.W., M. Nguyen, T. Kwan, P.E. Branton, D.W. Nicholson, J.A. Cromlish,
and G.C. Shore. 1997. p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-asso-
ciated protein in the endoplasmic reticulum. J. Cell Biol. 139:327–338.
Nguyen, M., P.E. Branton, S. Roy, D.W. Nicholson, E.S. Alnemri, W.C. Yeh,
T.W. Mak, and G.C. Shore. 1998. E1A-induced processing of procaspase-8
can occur independently of FADD and is inhibited by Bcl-2. J. Biol. Chem.
Nguyen, M., D.G. Breckenridge, A. Ducret, and G.C. Shore. 2000. Caspase-resis-
tant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and
release of cytochrome c from mitochondria. Mol. Cell. Biol. 20:6731–6740.
Osteryoung, K.W. 2001. Organelle fission in eukaryotes. Curr. Opin. Microbiol.
Pacher, P., and G. Hajnoczky. 2001. Propagation of the apoptotic signal by mito-
chondrial waves. EMBO J. 20:4107–4021.
Petit, P.X., M. Goubern, P. Diolez, S.A. Susin, N. Zamzami, and G. Kroemer.
1998. Disruption of the outer mitochondrial membrane as a result of large
amplitude swelling: the impact of irreversible permeability transition. FEBS
Pinton, P., D. Ferrari, P. Magalhaes, K. Schulze-Osthoff, F. Di Virgilio, T. Pozzan,
and R. Rizzuto. 2000. Reduced loading of intracellular Ca2? stores and
downregulation of capacitative Ca2? influx in Bcl-2-overexpressing cells. J.
Cell Biol. 148:857–862.
Pinton, P., D. Ferrari, E. Rapizzi, F.D. Di Virgilio, T. Pozzan, and R. Rizzuto.
2001. The Ca2? concentration of the endoplasmic reticulum is a key deter-
minant of ceramide-induced apoptosis: significance for the molecular mech-
anism of Bcl-2. EMBO J. 20:2690–2701.
The Journal of Cell Biology Download full-text
Caspase-cleaved BAP31 induces mitochondrial fission | Breckenridge et al. 1127
Rizzuto, R., P. Pinton, W. Carrington, F.S. Fay, K.E. Fogarty, L.M. Lifshitz, R.A.
Tuft, and T. Pozzan. 1998. Close contacts with the endoplasmic reticulum
as determinants of mitochondrial Ca2? responses. Science. 280:1763–1766.
Ruffolo, S.C., D.G. Breckenridge, M. Nguyen, I.S. Goping, A. Gross, S.J. Kors-
meyer, H. Li, J. Yuan, and G.C. Shore. 2000. BID-dependent and BID-
independent pathways for BAX insertion into mitochondria. Cell Death Dif-
Scaffidi, C., S. Fulda, A. Srinivasan, C. Friesen, F. Li, K.J. Tomaselli, K.M. Deba-
tin, P.H. Krammer, and M.E. Peter. 1998. Two CD95 (APO-1/Fas) signal-
ing pathways. EMBO J. 17:1675–1687.
Schulze-Osthoff, K., D. Ferrari, M. Los, S. Wesselborg, and M.E. Peter. 1998.
Apoptosis signaling by death receptors. Eur. J. Biochem. 254:439–459.
Scorrano, L., M. Ashiya, K. Buttle, S. Weiler, S.A. Oakes, C.A. Mannella, and S.J.
Korsmeyer. 2002. A distinct pathway remodels mitochondrial cristae and
mobilizes cytochrome c during apoptosis. Dev. Cell. 2:55–67.
Sharma, V.K., V. Ramesh, C. Franzini-Armstrong, and S.S. Sheu. 2000. Transport
of Ca2? from sarcoplasmic reticulum to mitochondria in rat ventricular
myocytes. J. Bioenerg. Biomembr. 32:97–104.
Shaw, J.M., and J. Nunnari. 2002. Mitochondrial dynamics and division in bud-
ding yeast. Trends Cell Biol. 12:178–184.
Smirnova, E., L. Griparic, D.L. Shurland, and A.M. van der Bliek. 2001. Dy-
namin-related protein Drp1 is required for mitochondrial division in mam-
malian cells. Mol. Biol. Cell. 12:2245–2256.
Smirnova, E., D.L. Shurland, S.N. Ryazantsev, and A.M. van der Bliek. 1998. A
human dynamin-related protein controls the distribution of mitochondria.
J. Cell Biol. 143:351–358.
Stegh, A.H., H. Herrmann, S. Lampel, D. Weisenberger, K. Andra, M. Seper, G.
Wiche, P.H. Krammer, and M.E. Peter. 2000. Identification of the cy-
tolinker plectin as a major early in vivo substrate for caspase 8 during CD95-
and tumor necrosis factor receptor-mediated apoptosis. Mol. Cell. Biol. 20:
Szalai, G., R. Krishnamurthy, and G. Hajnoczky. 1999. Apoptosis driven by IP(3)-
linked mitochondrial calcium signals. EMBO J. 18:6349–6361.
Wang, B., M. Nguyen, D.G. Breckenridge, M. Stojanovic, P.A. Clemons, S. Kup-
pig, and G.C. Shore. 2003. Uncleaved BAP31 in association with A4 pro-
tein at the endoplasmic reticulum is an inhibitor of Fas-initiated release of
cytochrome c from mitochondria. J. Biol. Chem. In press.
Wang, J., H.J. Chun, W. Wong, D.M. Spencer, and M.J. Lenardo. 2001. Caspase-
10 is an initiator caspase in death receptor signaling. Proc. Natl. Acad. Sci.
Yin, X.M., K. Wang, A. Gross, Y. Zhao, S. Zinkel, B. Klocke, K.A. Roth, and S.J.
Korsmeyer. 1999. Bid-deficient mice are resistant to Fas-induced hepatocel-
lular apoptosis. Nature. 400:886–891.
Yoshida, H., Y.Y. Kong, R. Yoshida, A.J. Elia, A. Hakem, R. Hakem, J.M. Pen-
ninger, and T.W. Mak. 1998. Apaf1 is required for mitochondrial pathways
of apoptosis and brain development. Cell. 94:739–750.
Zhu, W., A. Cowie, G.W. Wasfy, L.Z. Penn, B. Leber, and D.W. Andrews. 1996.
Bcl-2 mutants with restricted subcellular location reveal spatially distinct
pathways for apoptosis in different cell types. EMBO J. 15:4130–4141.