Endoplasmic reticulum protein BI-1
regulates Ca2+-mediated bioenergetics
to promote autophagy
Renata Sano,1Ying-Chen Claire Hou,1Michael Hedvat,1Ricardo G. Correa,1Chih-Wen Shu,1
Maryla Krajewska,1Paul W. Diaz,1Craig M. Tamble,1Giovanni Quarato,2Roberta A. Gottlieb,2
Masaya Yamaguchi,3Victor Nizet,3,4Russell Dahl,1David D. Thomas,5Stephen W. Tait,6
Douglas R. Green,6Paul B. Fisher,7Shu-Ichi Matsuzawa,1and John C. Reed1,8
1Sanford-Burnham Medical Research Institute, La Jolla, California 92037, USA;2BioScience Center, San Diego State University,
San Diego, California 92182, USA;
Pharmaceutical Sciences, University of California at San Diego, La Jolla, California, 92093 USA;5Department of Biochemistry,
Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA;6Department of Immunology,
St. Jude Children’s Research Hospital, Memphis, Tennessee 92105, USA;7Department of Human and Molecular Genetics, VCU
Institute of Molecular Medicine, Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, USA
3Department of Pediatrics, School of Medicine,
4Skaggs School of Pharmacy and
Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy
production during nutrient-scarce conditions such as those encountered in tumor microenvironments.
Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca2+mediated by inositol triphosphate
receptors (IP3Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane
protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP3R-dependent manner. By reducing steady-state levels
of ER Ca2+via IP3Rs, BI-1 influences mitochondrial bioenergetics, reducing oxygen consumption, impacting
cellular ATP levels, and stimulating autophagy. Furthermore, BI-1-deficient mice show reduced basal
autophagy, and experimentally reducing BI-1 expression impairs tumor xenograft growth in vivo. BI-1’s ability
to promote autophagy could be dissociated from its known function as a modulator of IRE1 signaling in the
context of ER stress. The results reveal BI-1 as a novel autophagy regulator that bridges Ca2+signaling between
ER and mitochondria, reducing cellular oxygen consumption and contributing to cellular resilience in the face
of metabolic stress.
[Keywords: BI-1; autophagy; ER stress; Ca2+; bioenergetics]
Supplemental material is available for this article.
Received November 28, 2011; revised version accepted March 29, 2012.
Autophagy is a catabolic process characterized by the
formation of double-membraned vesicles that deliver
intracellular components to lysosomes for degradation
(Klionsky 2007, 2010; Mizushima 2010). Under basal con-
ditions, autophagy provides a mechanism for the disposal
keeping’’ function. However, during metabolic stresses,
such as nutrient deprivation and hypoxia, autophagy is
also employed to catabolize macromolecules and thereby
generate substrates for production of ATP (Kroemer et al.
2010; Rabinowitz and White 2010). Dysregulation of
autophagy has been implicated in aging and numerous
diseases, including neurodegeneration, inflammation,
host–pathogen interactions, cardiovascular diseases, and
cancer (Mizushima et al. 2008). In the context of cancer,
autophagy is hypothesized to play various roles in tu-
mor development, progression, and responses to therapy
(Kimmelman 2011). Among these potential roles of
autophagy is promoting cancer cell survival under condi-
tions where rapidly growing tumors outstrip their vas-
cular supply, becoming malnourished and hypoxic (Boya
et al. 2005; Degenhardt et al. 2006; Tsuchihara et al. 2009).
Inducers of autophagy include endoplasmic reticulum
(ER) stress, a condition precipitated by hypoxia, oxidative
stress, fluctuations in glucose, and other conditions, which
causes accumulation of unfolded proteins in the ER lu-
men, thus triggering the unfolded protein response (UPR).
stasis in the ER by limiting new protein translation, in-
creasing ER protein-folding capacity through the induction
of various ER chaperones, and promoting degradation of
unfolded ER proteins (Malhotra and Kaufman 2007). Many
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.184325.111.
GENES & DEVELOPMENT 26:1041–1054 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org1041
stress is common in tumor microenvironments (Tsai and
Weissman 2010; Mahadevan and Zanetti 2011).
Recent studies suggest that ER Ca2+participates in the
regulation of autophagy (Hoyer-Hansen et al. 2007). Pro-
posed mechanisms for linking ER Ca2+to autophagy
include (1) modulation of Ca2+-dependent protein kinases
(e.g., Ca2+/calmodulin kinase kinase-b) and (2) inositol
triphosphate receptors (IP3Rs), which are responsible for
transporting Ca2+from ER into mitochondria to promote
bioenergetics (Criollo et al. 2007). IP3Rs are also regulated
by members of the Bcl-2 family, with anti-apoptotic pro-
teins Bcl-2 and Bcl-XLpromoting IP3-independent leak-
age of Ca2+from the ER via physical interactions with
IP3Rs (Kuo et al. 1998; Oakes et al. 2005; Rong et al. 2009).
Interestingly, mitochondria and ER form interconnected
membrane networks that influence various cellular pro-
cesses, including metabolism and cell death (Csordas
et al. 2006). In this regard, mitochondria preferentially
accumulate Ca2+in microdomains where ER and mito-
chondria are found in close apposition, termed mitochon-
dria-associated microdomains (MAMs) (Rizzuto et al.
1998; Decuypere et al. 2011). The physiological signif-
icance of the Ca2+-rich microdomains at the sites of
ER–mitochondria contact has recently been elucidated.
For instance, elegant work from Foskett and colleagues
(Cardenas et al. 2010) demonstrated that IP3R knock-
down or IP3R inhibition reduces Ca2+transport between
ER and mitochondria (mitochondrial bioenergetics), low-
ering cellular ATP and consequently activating autophagy
via an AMPK-dependent and mTOR-independent mech-
anism. Thus, apart from their role in second messenger
(IP3)-mediated intracellular signaling, IP3Rs function as
facilitators of mitochondrial bioenergetics.
BI-1 (Bax inhibitor-1) is an anti-apoptotic protein that
was first discovered by functional screening of cDNA
libraries for inhibitors of yeast cell death induced by ec-
topic expression of mammalian Bax (Xu and Reed 1998).
BI-1 is overexpressed in several types of human cancers,
and survival in culture of some tumor cell lines is BI-1-
dependent (Reimers et al. 2008). BI-1 associates with the
anti-apoptotic proteins Bcl-2 and Bcl-XL in ER mem-
branes and operates downstream from Bcl-2 family pro-
teins to control ER Ca2+homeostasis (Chae et al. 2004;
Xu et al. 2008). BI-1 also associates with IRE-1a com-
plexes, suppressing IRE1a’s intrinsic endoribonuclease
activity (responsible for production of transcription fac-
tor XBP-1) and blunting IRE1a-mediated activation of
stress kinases (Lisbona et al. 2009; Bailly-Maitre et al.
2010). Studies of genetically engineered mice (BI-1 trans-
genics and knockouts) have documented protective roles
for BI-1 in several diseases where ER stress makes im-
portant contributions (Bailly-Maitre et al. 2006, 2010;
Hunsberger et al. 2011; Krajewska et al. 2011). However,
the roles of BI-1 in cancer remain poorly understood, and
the mechanisms by which BI-1 impacts cellular processes
that determine tumor cell survival in harsh microenvi-
ronments are undefined. Here, we document the impor-
tance of BI-1 for tumorigenesis and report a novel role
for BI-1 as a regulator of IP3R-dependent Ca2+transfer from
ER to mitochondria, thereby impacting mitochondrial bio-
energetics and promoting autophagy. Together with its pre-
viously identified function as a modulator of UPR signaling,
the ability of BI-1 to reduce dependence on oxidative phos-
phorylation suggests that BI-1 contributes to tumorigenesis
by promoting cellular resilience during metabolic stress.
Tissues of BI-1-deficient mice show changes
in autophagy markers
We compared levels of the autophagy marker protein
p62 in tissues of wild-type and age-matched BI-1 knock-
out mice (littermates of the same sex) with and without
in vivo treatment with autophagy inducer rapamycin
(1 mg/kg) for various times (6–72 h). Due to its degrada-
tion by the autophagy lysosome system (Ichimura et al.
2008), conditions that suppress autophagy cause accu-
mulation of p62, resulting in the formation of p62-positive
inclusions in cells (Komatsu and Ichimura 2010). Eleva-
tions of p62 were seen at baseline in the hearts, livers, and
lungs of BI-1 knockout mice (as measured by immuno-
blotting after normalization for total protein content), sug-
gesting that basal autophagy is impaired in BI-1-deficient
mice (Fig. 1). Treatment with rapamycin resulted in de-
creases in p62 in both wild-type and BI-1 knockout mice,
which were more significant for BI-1-deficient mice due
to the starting high levels of p62. In contrast to p62 protein,
similar levels of p62 mRNA were observed in both wild-
type and BI-1 knockout mice, regardless of rapamycin treat-
ment (Supplemental Fig. S1), suggesting that p62 protein
accumulation is not due to elevated transcriptional activ-
ity. Furthermore, numerous p62 inclusions were found in
BI-1 knockout mouse tissues, including the kidneys and
brains of BI-1 knockout mice (Supplemental Fig. 2).
LC3 (Atg8 ortholog) is a major constituent of
autophagosomes (Kabeya et al. 2000). The proteolytically
processed cytoplasmic form of LC3 (LC3-I, 16 kDa) be-
comes lipid conjugated to generate LC3-II (14 kDa), which
is inserted into autophagosome membranes. After fu-
sion of autophagosomes with lysosomes, those LC3-II
molecules that are bound to the inner membrane of
autophagosomes are degraded by lysosomal proteases. Tis-
sues of BI-1 knockout mice showed reduced LC3-II levels
at baseline and after rapamycin treatment (Fig. 1B,C).
Finally, BI-1’s impact on autophagy in vivo was also
documented by quantification of autophagic vesicles (AVs)
using electron microscopy to analyze the heart tissue of
wild-type and BI-1 knockout mice. After 24 h of treat-
ment with rapamycin (1 mg/kg), AVs, mainly in the form
of autophagolysosomes with partially degraded material
in their lumen, were detected throughout the cytoplasm
of wild-type mice. In contrast, significantly fewer AVs
were observed in BI-1-deficient hearts (Fig. 1D; see Sup-
plemental Fig. S3 for representative images). We conclude,
therefore, that BI-1 deficiency alters autophagy in vivo.
BI-1 modulates autophagy in tumor xenografts
To assess the role of BI-1 on autophagy in the context of
tumorigenesis, we employed a xenograft model in which
Sano et al.
1042 GENES & DEVELOPMENT
the lung cancer cell line H322M (which contains high
endogenous levels of BI-1 mRNA, consistent with prior
reports for non-small-cell lung cancers) (Tanaka et al.
2006) was stably transduced with control or BI-1 shRNA
knockdown vectors and injected subcutaneously into im-
munocompromised (nu/nu) mice. Whereas cells infected
with the shRNA control rapidly generated large palpable
tumors, H322M cells containing shRNA BI-1 produced
much smaller tumors (Fig. 1E,F). Tumors were processed
for immunohistochemical evaluation of autophagy mark-
ers LC3 and p62, showing reduced percentages of tumor
cells with punctate LC3 staining and elevated percent-
ages with p62 staining, suggesting reduced autophagy in
BI-1 knockdown tumors (Fig. 1G; Supplemental Fig. S4).
BI-1 deficiency impairs autophagy in cultured cells
In addition to evaluating the effects of BI-1 deficiency
in vivo, we used shRNA vectors to knock down BI-1
expression in cultured tumor cell lines and then mon-
itored effects on autophagy markers in vitro (Fig. 2). Using
the same genetically modified H322M lung cancer cells
employed for in vivo tumor xenografts in which en-
dogenous BI-1 mRNA was reduced by >80% by shRNA
(Fig. 2B), levels of p62 were assessed before and after
various stressors. At basal conditions, BI-1 knockdown
H322M cells showed markedly elevated levels of p62,
suggesting that autophagy was impaired. Challenging the
stably transduced control (green fluorescent protein [GFP]
shRNA) and BI-1 shRNA cells with various inducers of
autophagy resulted in decreases in p62, suggesting that
although BI-1-deficient cells have reduced autophagy, they
still remain responsive to autophagy induction by the
classical pathways that sense nutrient availability (Fig. 2A).
To translate the results found in mammalian cells to
other organisms, we knocked down BI-1 in Drosophila S2
cells and monitored p62 levels after 2 h of starvation
(glucose deprivation). Once again, experimentally reduc-
ing BI-1 expression caused an accumulation of p62 protein
type (wt) and BI-1 knockout (ko) mice treated with rapamycin for various times as indicated (in hours). Lysates were normalized for total
protein content and analyzed by SDS-PAGE/immunoblotting using anti-p62 or anti-tubulin antibodies. Wild-type (wt) and BI-1 knockout
(ko) livers (B) and lungs (C) were collected after 24 h of injection with rapamycin. Tissues lysates were analyzed by immunoblotting as
above using anti-LC3, anti-p62, and anti-tubulin antibodies. (D) Wild-type (wt) and BI-1 knockout (ko) mice were injected with rapamycin.
After 24 h, hearts were dissected, processed, and examined by transmission electron microscopy. From 20 images each for wild-type (wt)
and knockout specimens, the total number of autophagosomes was determined using ImageJ software. Mice were injected with 1 mg of
rapamycin per kilogram of body weight. Horizontal bars indicate mean. Data are statistically significant by t-test (P = 0.009). (E) Female
BALB/c nu/nu mice were injected subcutaneously with H322M cells (5 3 106) containing scrambled control or BI-1 shRNAvectors. Tumor
volumes were measured over time (mean 6 SD; n = 10). (F) Seven weeks post-transplantation, animals were sacrificed, and tumors were
excised and weighed (mean 6 SD; n = 10 animals per group). (G) Tumor sections were analyzed for LC3 and p62 staining by quantitative
immunohistochemistry. The overall low percentage of cells showing punctate LC3 immunostaining may reflect the antibody conditions
employed, which were designed to avoid detection of diffuse cytosolic staining of nonautophagic cells.
BI-1-deficient mice have reduced autophagy. (A) Protein levels of p62 were assessed by immunoblot analysis of hearts from wild-
BI-1 modulates autophagy
GENES & DEVELOPMENT1043
(Supplemental Fig. S5A). Comparable elevations in p62
were found in BI-1 knockdown S2 cells and in cells where
the essential autophagy gene Atg1 was silenced, thus
serving as a positive control.
To determine LC3 flux, we cultured control and BI-1
knockdown H322M cells in the absence or presence of the
lysosomal inhibitors (LIs) NH4Cl and leupeptin for various
times to measure autophagic flux (Martinez-Vicente et al.
2011). Specifically, the ratio of LC3-II levels at 2 h versus
0 h of exposure to LIs was calculated as a representation
of LC3 flux (Martinez-Vicente et al. 2011). H322M cells
in which BI-1 was knocked down showed reduced levels
of LC3 flux, indicating decreased autophagy (Fig. 2C,D).
Moreover, comparison of changes in LC3-II at two differ-
ent times upon blockage of degradation (which can be
used as a direct measurement of autophagosome forma-
tion) revealed a lower net increase in LC3-II content in
the BI-1-deficient cells. Thus, the decrease in autophagic
flux seen in BI-1-deficient cells is probably not due to
compromised clearance of autophagosomes, but rather
is a reflection of reduced autophagosome formation.
BI-1 modulates antibacterial autophagy
Autophagy plays an important role in host defense by
eradicating intracellular bacteria through lysosomal de-
struction (Noda and Yoshimori 2010). We used primary
macrophages infected with group A Streptococcus (GAS)
as a model for functional assessment of the effects of BI-1
deficiency on autophagy. Accordingly, macrophage cul-
tures were prepared from wild-type versus BI-1 knockout
mice and infected with GAS, and the numbers of in-
tracellular viable bacteria were enumerated at various
times thereafter. Compared with wild-type cells, the BI-1-
deficient macrophages contained significantly higher
numbers of GAS (Supplemental Fig. 6), thus providing
further evidence that BI-1 deficiency impairs autophagy.
BI-1 overexpression promotes autophagy
in cancer cells
In addition to the studies of BI-1 deficiency, we also
ascertained the effects of BI-1 overexpression using cul-
tured cancer cell lines. For these experiments, 293T cells
(Fig. 3A) were transiently transfected with either empty
vector or pDNA3-BI-1-HA and then treated with vari-
ous autophagy inducers. Overexpressing BI-1 increased
the steady-state levels of LC3-II, in agreement with
higher autophagic activity in these cells (also supported
by higher rates of conversion of LC3-I to LC3-II in these
cells). This increase in basal autophagic activity was also
observed upon autophagy induction by culturing cells
without nutrients (Hank’s buffered salt solution [HBSS])
and, to a lesser extent, by rapamycin.
To further evaluate the role of BI-1 in autophagy
regulation, we used HeLa cells, which we previously en-
gineered to inducibly express BI-1 under control of a
tetracycline/doxycycline-regulated promoter (Wang et al.
2004).Upon addition to cultures of doxycycline at various
concentrations, BI-1 protein expression was induced (Fig.
3B). We compared the levels of autophagy biomarkers
p62 and LC3-I/II in these cells before and after inducing
BI-1 at basal conditions (Fig. 3B, left) and upon inducing
autophagy with rapamycin (Fig. 3B, right). Inducing BI-1
resulted in a decline in p62 in both the basal conditions
and following autophagy induction with rapamycin. Over-
expressing BI-I increased the steady-state levels of LC3-II,
in agreement with higher autophagic activity in these
cells (also supported by higher rates of conversion of
LC3-I to LC3-II in these cells) (Fig. 3B). Similar to results
seen in human cancer cells, when either Drosophila or
human BI-1 protein was expressed in fly S2 cells, a reduc-
tion in p62 levels was observed (Supplemental Fig. S5B).
In addition to the end-point studies, we also ascer-
tained the effects of BI-1 overexpression on autophagy
flux. (A) Stably transduced H322M cells were
cultured in standard rich medium or with se-
rum- and glucose-deficient medium ([H] HBSS;
[E] EBSS) alone or with various agents, including
rapamycin (R; 25 mg/mL), thapsigargin (TG;
5 mM), and tamoxifen (TAM; 10 mM), for 16 h.
Lysates were prepared, normalized for total pro-
tein content, and analyzed by SDS-PAGE/im-
munoblotting using anti-p62 and anti-tubulin
antibodies. (B) H322M cells were stably trans-
duced with recombinant shRNA lentiviruses
targeting BI-1 or GFP. Relative levels of endoge-
nous BI-1 mRNA were assessed by quantitative
RT–PCR (expressing data as a percentage of con-
trol relative to cells transduced with GPF shRNA
control vector). (C) H322M cells that were stably
transduced with recombinant shRNA lentivi-
ruses targeting BI-1 or GFP (control) were treated
with LIs (20 mM NH4Cl and 100 mM leupeptin)
for either 2 h or 4 h, as indicated. Levels of LC3-I
and LC3-II were analyzed by immunoblotting.
BI-1 knockdown reduces autophagic
Blots were reprobed with anti-b-actin antibody as a loading control. (D) Bands were quantified by densitometry, and measurements
were used to calculate LC3 flux (mean 6 SD; n = 3 experiments; P = 0.017).
Sano et al.
1044GENES & DEVELOPMENT
flux. Similar to experiments performed in BI-1 knock-
down cells, control and BI-1-overexpressing cells were
cultured in the absence or presence of the LIs NH4Cl and
leupeptin (Fig. 3C). Quantitative analysis of LC3-II levels
demonstrated that BI-1-overexpressing cells have in-
creased LC3 flux (Fig. 3D).
As another measure of autophagy, we generated a sta-
ble tumor cell line overexpressing GFP-LC3 combined
with BI-1 under control of a tetracycline-inducible pro-
moter. Localization of LC3 was assessed by high-content
imaging using GFP-tagged protein. During autophagy,
GFP-LC3 distribution changes from cytosolic diffuse to
punctate, reflecting its attachment to AVs (Kabeya et al.
2000). Using a high-throughput microscopy system, we
quantified both the number of ‘‘spots’’ of GFP-LC3 and
the ‘‘spot’’ intensity per cell. We observed that BI-1 in-
duction significantly increased both the average number
of spots per cell and the average spot intensity per cell,
with similar results obtained using both live-cell imaging
and an analysis of fixed cells (Supplemental Fig. S7A–D).
We conclude, therefore, that overexpression of BI-1 in-
BI-1 does not require autophagy for cytoprotection
Previous studies have shown that BI-1 is cytoprotective
against apoptosis, particularly triggers of ER stress (Chae
et al. 2004). Given that BI-1 modulates autophagy, we
next addressed whether this property might account
for BI-1’s cytoprotective activity. For this purpose, we
stably transduced BI-1 into autophagy-deficient Atg7?/?
mouse embryonic fibroblasts (MEFs) using recombinant
lentivirus (Fig. 4B). After 48 h, cells were treated with
apoptosis inducers, and then the percentage of annexin
V-positive (dead and dying) cells was determined by flow
cytometry. The percentage of annexin V-positive cells in
cultures of wild-type and Atg7?/?knockout cells over-
expressing BI-1 were reduced comparably, suggesting that
BI-1 does not require autophagy for its cytoprotective
activity (Fig. 4A). Similar results were obtained measur-
ing cell death based on propidium iodide (PI) staining
(Supplemental Fig. S8).
Clonogenic survival assays were also performed, wherein
cells were subjected to the stress of culture for 4 h in
nutrient-depleted medium (Eagle’s balanced salt solution
[EBSS]) and then cultured for 10 d in complete medium
before counting surviving colonies (Fig. 4C). In agreement
with the annexin V staining, BI-1-overexpressing Atg7?/?
MEFs showed significantly increased numbers of colonies
compared with control Atg7?/?MEFs (P = 0.013), indicat-
ing that BI-1 retains the ability to promote cell survival
even in autophagy-defective cells.
Extensive cross-talk occurs between the mechanisms
regulating apoptosis, ER stress, and autophagy (Kang et al.
using lysates prepared from 293T cells transiently transfected with empty vector or BI-1-HA vector. Where indicated, cells were pre-
treated with bafilomycin (bafilo; 200 nM) or rapamycin (rapa; 25 mg/mL) for 16 h, or HBSS for 4 h. (B) The levels of p62 and LC3 were
measured in HeLa cells in which BI-1 expression was conditionally driven using a doxycycline-inducible system. Cells were cultured
with various concentrations (0, 50, 100, 250, 500, and 1000 ng/mL) for 12 h. (Left) Untreated cell. (Right) Rapamycin-treated cells
(25 mg/mL for 12 h). Cell extracts were normalized for total protein content before analysis by SDS-PAGE/immunoblotting using anti-
HA (to detect HA-BI-1), anti-p62, and anti-LC3 antibodies. Tubulin was used to verify equal protein loading. (C) HeLa cells in which BI-1
expression was conditionally driven using a doxycycline-inducible system (1 mg/mL) were treated with LIs (20 mM NH4Cl and 100 mM
leupeptin) for either 2 h or 4 h. Levels of LC3-I and LC3-II were analyzed by immunoblotting of cell lysates. b-Actin served as a loading
control. (D) LC3-II bands were quantified by densitometry, and measurements were used to calculate LC3 flux. LC3 flux was quantified by
dividing levels of LC3-II after 2 h of LI treatment per level of LC3-II without LI (mean 6 SD; n = 3 independent experiments; P = 0.026).
BI-1 overexpression increases autophagic flux. (A) The relative levels of LC3-I and LC3-II were assessed by immunoblotting
BI-1 modulates autophagy
GENES & DEVELOPMENT1045
2011; Rubinstein et al. 2011; Verma and Datta 2012). We
tested whether minimal medium elicits an ER stress
response in Atg7?/?MEFs. Using BiP (Grp78) as a marker
of UPR signaling, we observed that Atg7?/?cells are more
sensitive to ER stress induction by nutrient deprivation
compared with wild-type cells (Supplemental Fig. S9).
Interestingly, BI-1 overexpression suppressed UPR marker
expression (BiP) in wild-type but not Atg7?/?cells, imply-
ing that autophagy influences the ability of BI-1 to impact
UPR signaling—even though autophagy is not required
for BI-1-mediated protection from ER stress-induced cell
stably transduced with empty or BI-1-encoding lentiviruses. Cells were cultured under basal conditions (NT) or for 24 h with various
cell stress agents ([TAM] 20 mM tamoxifen; [Rapa] 35 mg/mL rapamycin; [GSD] glucose and serum [FBS] deprivation; [TM] 10 mg/mL
tunicamycin) in complete medium or nutrient-depleted medium (HBSS) for 8 h. Cells were then stained with annexin V–FITC and
analyzed by FACS. Results are expressed as percentage of annexin V+cells. (B) To verify defective autophagy in Atg7?/?MEFs, LC3
levels were analyzed by immunoblotting. Tubulin was used as a loading control. (C) A clonogenic survival assay was performed using
Atg7?/?(ko) cells stably transduced with empty or BI-1-encoding lentivirus. Cells were cultured (200 cells per six-well plate) in
nutrient-depleted medium (EBSS) for 4 h, then washed and cultured for an additional 10 d in complete medium. Cells were stained with
crystal violet, and the number of colonies (>1 mm) per well was counted (mean 6 SD; n = 3; P = 0.001 for EBSS-treated control vs. BI-1).
(D) Saturated overnight cultures of BY4741 wild-type (wt) yeast or the indicated gene deletion strains containing a galactose-inducible
Bax expression vector and either BI-1 or control plasmids were normalized to OD600= 1.0 and then serially diluted in increments of
1:10. Yeast cultures were spotted onto glucose (Bax-off) or galactose (Bax-on) plates and grown for 3 d at 30°C. (E) Ire1a?/?(?) cells were
stably transduced with either GFP or BI-1 lentiviruses. Cells were cultured for 24 h with various cell stress agents ([TG] 5 mM
thapsigargin; [TM] 10 mM tunicamcyin; [Rapa] 35 mg/mL rapamycin; [GSD] glucose and FBS deprivation) or in nutrient-depleted
medium (HBSS) for 8 h. Cell lysates were normalized for total protein content and subjected to immunoblot analysis to detect p62, Ire1,
and tubulin (loading control). (F) Ire1a?/?(knockout [ko]) or Ire1a+/+(wild-type [wt]) MEFs were stably transduced with either GFP or
BI-1 lentiviruses and cultured with cell stress agents as in E. To assess cell death, cells were stained with annexin V–FITC, enumerating
the percentage of annexin V+(dead) cells by flow cytometry (mean 6 SD; n = 3 independent experiments). (G) Clonogenic survival
assays were performed using Ire1a?/?MEFs stably transduced with GFP control or BI-1 lentiviruses. Cells were seeded (200 cells per
six-well plate) in nutrient-depleted medium (EBSS) for 4 h and then cultured for an additional 10 d in nutrient-rich medium. Cells were
stained with crystal violet, and the number of colonies (>1 mm) per well was counted (mean 6 SD; n = 3 independent experiments).
Differences between control and BI-1-overexpressing cells were not significantly different (P = 0.09 by t-test).
BI-1 requires IRE1a but not autophagy for cytoprotective activity. (A) Wild-type (wt) and Atg7?/?(knockout [ko]) MEFs were
Sano et al.
1046GENES & DEVELOPMENT
BI-1 requires IRE1a for cytoprotection
but not for autophagy modulation
Because BI-1 is reported to bind to and suppress IRE1
signaling (Lisbona et al. 2009; Bailly-Maitre et al. 2010),
we explored the impact of IRE1 on BI-1-mediated cyto-
protection and autophagy. We conducted initial experi-
ments in yeast, where the cytoprotective activity of
human BI-1 was first demonstrated by showing that it
blocks yeast cell death induced by ectopic expression of
the mammalian proapoptotic protein Bax (Xu and Reed
1998). In yeast, only the IRE1 component of the UPR
machinery is present (unlike mammalian cells). We ob-
served that yeast IRE1 binds human BI-1 by coimmuno-
precipitation (co-IP) experiments (data not shown), in-
dicating that human BI-1 interacts with both yeast and
human IRE1. Using various gene deletion mutants, we
determined that IRE1 and its downstream target, HAC
(yeast ortholog of XBP1), are required for BI-1-mediated
protection from Bax in yeast, but not the ERAD compo-
nent UBX2 or the autophagy gene ATG12 (Fig. 4D). Thus,
the IRE1/HAC pathway is required for BI-1 to protect
against Bax in yeast, while autophagy is not.
Because IRE1a reportedly modulates autophagy (Ogata
et al. 2006), we next explored the role of IRE1a for BI-1’s
proautophagic and cytoprotective activities by using
Ire1a?/?MEFs that had been stably transduced with
BI-1 versus control lentiviruses. Levels of LC3 and p62
were evaluated by immunoblot analysis in basal condi-
tions and following treatment with various ER stress
and autophagy inducers (Fig. 4E). Reduced p62 levels in
BI-1-overexpressing Ire1a?/?cells suggested that IRE1a
is not necessary for BI-1 to induce autophagy. BI-1 also
retained its proautophagic activity in Perk?/?cells (Sup-
plemental Fig. S10), further distinguishing components
of the ER stress response from BI-1’s autophagic activity.
To test the cytoprotective function of BI-1 in Ire1a?/?
cells, we treated cells with various autophagy inducers
(rapamycin, tamoxifen, glucose, and FBS deprivation, or
HBSS)or theERstress inducer thapsigarginandthenmea-
sured cell viability by annexin V staining (Fig. 4F). Un-
der these conditions, most of the cell death probably oc-
curred via apoptosis, based on costaining FITC–annexin V
plus PI costaining analysis (<3% PI-positive cells) (data
not shown). While BI-1 overexpression increased the
percentages of surviving Ire1+/+cells, it did not rescue
Ire1?/?cells. Similar results were obtained using a clono-
genic survival assay where nutrient deprivation was
applied as a cellular stress (Fig. 4G). Thus, while other
mechanisms may make contributions, we conclude that
BI-1’s cytoprotective activity is largely dependent on
BI-1 modulates ER Ca2+and mitochondria
BI-1 is known to impact ER Ca2+in a manner that
phenocopies anti-apoptotic proteins Bcl-2 and Bcl-XL,
causing reductions in resting steady-state levels of free
Ca2+in the ER lumen (Xu et al. 2008; Weston and
Puthalakath 2010; Hunsberger et al. 2011; Rodriguez
et al. 2011). We therefore considered that BI-1’s ability
to regulate ER Ca2+might account for its proautophagic
activity. To lay a foundation for these studies, we com-
pared the pools of ER Ca2+in HeLa cells containing
tetracycline/doxycycline-inducible BI-1 (with vs. without
BI-1 induction) using the irreversible ER Ca2+ATPase
inhibitor thapsigargin. Cells were loaded with the cyto-
solic Ca2+-sensing dye Indo1 or the mitochondrial Ca2+-
sensing dye Rhod2 and then placed into Ca2+-free me-
dium to assess intracellular Ca2+fluxes upon treatment
with thapsigargin. Inducing BI-1 expression by addition
of the tetracycline analog doxycycline resulted in re-
duced levels of ER (thapsigargin-releasable) Ca2+(Fig.
5A) and, correspondingly, reduced mitochondrial Ca2+
uptake (Fig. 5B). These data are consistent with prior re-
ports showing reduced sequestration of Ca2+in the ER of
BI-1-overexpressing cells, such that release of ER Ca2+by
thapsigargin treatment results in less Ca2+efflux into the
cytosol and therefore less uptake into mitochondria (Xu
et al. 2008).
To assess the role of altered ER Ca2+in the cellular
phenotypes of BI-1, we re-established ER Ca2+in BI-1-
overexpressing cells by either overexpressing sarco/endo-
plasmic reticulum Ca2+ATPase (SERCA) (ER Ca2+pump)
agonist (Supplemental Fig. S11). By overexpressing
SERCA or stimulating endogenous SERCA, our goal
was to overcome the Ca2+leakage from ER created by
BI-1, thus re-establishing ER Ca2+levels toward normal.
Indeed, SERCA overexpression by gene transfer and
endogenous SERCA activation by pharmacological ag-
onist treatment re-established pools of thapsigargin-
releasable (ER) Ca2+to normal levels in BI-1-overex-
pressing cells, as measured by Indo1 fluorescence in cells
cultured in Ca2+-free medium (Fig. 5C,D). These results
were independently confirmed using an ER targeted Ca2+-
sensing fluorescent protein (‘‘cameleon’’) (Fig. 5E) previ-
ously used as a probe for assessing the impact of BI-1 and
Bcl-2 family proteins on ER Ca2+(Xu and Reed 1998;
Palmer et al. 2004). Both SERCA overexpression and
pharmacological stimulation of endogenous SERCA
overcame the effect of BI-1, elevating ER CA2+(Fig. 5F).
Mitochondria require Ca2+to maintain the tricarbox-
ylic acid (TCA) cycle, which provides reducing equiva-
lents to support oxidative phosphorylation and ATP pro-
duction. Given that BI-1-overexpressing cells showed
reduced levels of mitochondrial Ca2+, we assessed
whether mitochondrial metabolism was affected. To this
end, we first measured levels of total mitochondrial
dehydrogenases that are normally activated by Ca2+. Of
note, cells overexpressing BI-1 showed a significant re-
duction in the activity of these enzymes, suggesting
a reduction of the TCA cycle activity (Fig. 6A). Isocitrate
dehydrogenase (IDH), a mitochondrial matrix protein
that represents a component of the TCA cycle, is re-
sponsible for catalyzing the reversible conversion of
isocitrate to a-ketoglutarate and CO2in a two-step re-
action. Because the activity of this enzyme is regulated by
changes in mitochondrial matrix Ca2+ion concentration,
we measured its activity in BI-1-overexpressing cells.
BI-1 modulates autophagy
GENES & DEVELOPMENT1047
Activity of IDH was significantly lower in BI-1-over-
expressing cells compared with control cells (Supplemen-
tal Fig. S12), a result that was in agreement with lower
levels of mitochondrial Ca2+. Consistent with reduced
activity of IDH (Qi et al. 2008), levels of NADH were also
reduced in BI-1-overexpressing cells (data not shown), and
levels of mitochondrial NADPH were increased (Supple-
mental Fig. S13).
As a direct measurement of mitochondrial respiration,
O2consumption rates were assessed by an oxymeter. BI-
1-overexpressing cells showed a marked reduction in O2
consumption (Fig. 6B) compared with control cells in which
BI-1 expression was not induced. The reduction in mito-
chondrial O2consumption in BI-1-overexpressing cells was
observed under both coupled and uncoupled (+FCCP) con-
ditions and does not appear to be due to an intrinsic defect
in the oxidative phosphorylation machinery based on anal-
ysis of respiratory control ratio (RCR) values (Fig. 6C).
To assure that reduced oxidative phosphorylation was
not due to reduced mitochondrial mass, we assessed
levels of the mitochondrial proteins AIF, Hsp60, and
Cyclophilin D (Supplemental Fig. S14). Induction of BI-1
expression for up to 72 h did not change levels of these
mitochondrial proteins. To further evaluate mitochon-
drial mass, we also stained cells with nonylacridine
orange (NAO), which binds to cardiolipin in mitochon-
drial membranes, finding that NAO staining was un-
affected by BI-1 expression (Supplemental Fig. S15).
Since mitochondria generate the vast majority of cel-
lular ATP via oxidative phosphorylation, we next mea-
sured ATP levels in control versus BI-1-overexpressing
HeLa cells. When normalized for numbers of viable cells
via trypan blue exclusion, cells overexpressing BI-1 con-
tained reduced ATP concentrations compared with con-
trols (Fig. 6D). In contrast, human H322M cells in which
BI-1 was knocked down contained elevated ATP levels
compared with cells with scrambled shRNA (Supplemen-
tal Fig. S16A). Moreover, ATP levels in Drosophila S2
cells were elevated in BI-1 siRNA-treated cells compared
with control siRNA-treated cells in both nutrient-rich
(Supplemental Fig. S16B) and nutrient-depleted (Supple-
mental Fig. S16C) cultures.
When oxygen is reduced, cells use anaerobic glycolysis
as an alternative means of energy (ATP) production,
wherein glucose is converted in lactate. HeLa cells over-
expressing BI-1 showed increased levels of lactate pro-
duction compared with control cells (Fig. 6E), suggesting
a greater reliance of glycolysis for generating ATP.
If BI-1 overexpression causes a net decline in cellular
ATP levels, we predicted that cellular sensors of ATP
ER Ca2+. HeLa cells in which BI-1 expression was
conditionally driven using a doxycycline-inducible sys-
tem were cultured with (BI-1) or without (control [C])
1 mg/mL doxycycline for 12 h. Cells were then loaded
with the cytosolic Ca2+probe Indo-1 AM (A) or the
mitochondrial Ca2+probe Rhod 2-AM (B) for 30 min at
37°C. After transfer to Ca2+-free PBS, cells were stim-
ulated with thapsigargin (5 mM) and immediately ana-
lyzed using a microplate reader. Average fluorescent
intensity was captured every 5 sec for a total time of
300 sec. We used a 405/485-nm emission ratio and 582
nm to measure Indo-1AM and Rhod-2 AM, respectively.
(C,D) BI-1-inducible HeLa cells were either transiently
transfected with control versus SERCA plasmids (C) or
treated with DMSO versus SERCA agonist compound
SB-6471 (10 mM) (D). Cells were then cultured with (to
induce BI-1) or without doxycycline for 12 h prior to
Indo1-AM loading. After transfer to Ca2+-free medium,
thapsigargin (TG; 5 mM) was added (arrow), and the
levels of cytosolic Ca2+were measured every 5 sec for
a total time of 300 sec. (E) Control and BI-1-overexpress-
ing cells (BI-1) were transfected with the ER Ca2+
cameleon plasmid YC4.3ER for 48 h prior to analysis
of ER Ca2+. (F) BI-1-inducible HeLa cells were either
transiently transfected with control versus SERCA
plasmids or treated with DMSO versus SERCA agonist
compound SB-6471 (10 mM). Cells were then cultured
with (to induce BI-1) or without doxycycline for 12 h
prior to ER cameleon transfection and ER Ca2+analysis.
Results are expressed as emission ratio YFP/CFP.
SERCA nullifies BI-1-mediated reduction of
Sano et al.
1048GENES & DEVELOPMENT
would be impacted, such as AMPK. Once activated, this
kinase phosphorylates substrates that consequently limit
anabolic processes and facilitate catabolic pathways to
provide energy. BI-1-overexpressing cells contained in-
creased levels of phospho-AMPK (indicative of kinase acti-
vation) and also showed increased phosphorylation of the
AMPK substrate acetylCoA carboxylase (Fig. 6F). These
findings confirm that BI-1 causes a net decline in cellular
ATP by impacting mitochondrial bioenergetics.
IP3R is essential for BI-1-mediated autophagy
Because IP3Rs have been reported to enhance mitochon-
drial bioenergetics (Cardenas et al. 2010), we next ex-
plored whether BI-1’s ability to modulate cellular ATP
and promote autophagy is dependent on these Ca2+
channels. Because most vertebrate genomes contain
three IP3R genes, we employed chicken DT40 lymphoma
cells in which all three IP3R isoforms (triple-knockout
[TKO] IP3R) were genetically ablated (Sugawara et al.
1997). Wild-type and TKO IP3R DT40 cells were stably
transduced with BI-1 versus control lentiviruses, and
expression of BI-1 was confirmed (Fig. 7A). Then, cells
were treated with LIs to assess autophagic flux by mea-
suring LC3 levels. Indeed, when expressed in TKO IP3R
DT40 cells, BI-1 lost its ability to stimulate LC3 flux,
whereas BI-1 retained its proautophagic activity when
expressed in wild-type DT40 cells (Fig. 7A,B). To assess
the impact of IP3R loss on mitochondrial bioenergetics,
we measured levels of ATP in wild-type and TKO IP3R
DT40 cells with or without overexpression of BI-1. When
expressed in wild-type cells, BI-1 reduced ATP levels (as
previously shown with other cell lines), whereas levels of
ATP in IP3R DT40 cells overexpressing BI-1 were not
changed or slightly increased (Fig. 7C). Altogether, these
results indicate that BI-1 reduces cellular ATP and in-
duces autophagy via a mechanism that requires IP3Rs
SERCA reverses BI-1-mediated reduction
in bioenergetics and BI-1-induced autophagy
To assess the role of altered ER Ca2+in the cellular
phenotypes mediated by BI-1, we restored ER Ca2+in BI-
1-overexpressing cells by overexpressing SERCA or stim-
ulating SERCA with chemical agonists. Then, with ER
Ca2+levels sustained at high levels (Fig. 5), we tested the
ability of BI-1 to promote autophagy and regulate cellular
bioenergetics. SERCA overexpression reduced autophagy,
which was evidenced by accumulation of p62 and re-
duced LC3-II levels (Supplemental Fig. S17A). Similarly,
the SERCA chemical agonist SB-1163 also reversed
BI-1-mediated autophagy, as evidenced by levels of the
autophagy markersLC3and p62 (Supplemental Fig. S17D).
Furthermore, SERCA overexpression (Supplemental Fig.
S17B,C) and pharmacological stimulation of SERCA
(Supplemental Fig. S18E, F) partially restored ATP levels
in BI-1-overexpressing cells while also either partially
energetics. (A) Activity of mitochondrial de-
hydrogenases was measured in control ([?]
doxy) and BI-1 overexpressing ([+] doxy) HeLa
cells. Values were normalized for the num-
ber of viable cells (trypan blue exclusion).
Background absorbance was read at 690 nm
and subtracted from readings at 440 nm
(A440nm ? A690nm). The difference be-
tween samples was significant (P < 0.05, by
unpaired t-test). (B) Endogenous oxygen con-
sumption rates in control (no doxy) and BI-1-
overexpressing (doxy) cells were monitored
by an oxymeter before (NT) and after expo-
sure to oligomycin (2 mM) and FCCP (1 mM).
Results are expressed as nanomoles of O2
per minute per 1 3 106cells. Results for
untreated control versus BI-1-expressing cells
were statistically significant (P = 0.0015). (C)
RCR in control ([?] doxy) and BI-1 over-
expressing ([+] doxy) cells was calculated by
dividing O2 consumption before and after
addition of oligomycin. The efficiency of oxi-
dative phosphorylation coupling was com-
parable in control and BI-1-overexpressing
BI-1 reduces mitochondria bio-
cells. (D) ATP levels were measured in control versus BI-1-overexpressing cells using a bioluminescence method. Data represent
relative luminescence units (RLUs) per 106viable cells (mean 6 SD; n = 3; P = 0.002. (E) Levels of extracellular lactate were
measured in control ([?] doxy) and BI-1-overexpressing ([+] doxy) cells. Results are represented as nanomoles of lactate per well
(mean 6 SD; n = 3; P = 0.021. (F) Lysates from control versus BI-1-overexpressing cells were normalized for total protein content
and subjected to immunoblot analysis to detect AMPK, phospho-AMPK, acetylCoA carboxylase (ACC), and phospho-acetylCoA
carboxylase (P-ACC). Anti-HA antibody was used to detect expression of BI-1-HA protein. Equivalent sample loading was shown
by monitoring tubulin levels.
BI-1 modulates autophagy
GENES & DEVELOPMENT 1049
or fully restoring mitochondrial dehydrogenase activity
(TCA cycle marker).
To ascertain the relevance of BI-1-mediated changes in
ER Ca2+to cytoprotection, we overexpressed SERCA in
BI-1-inducible HeLa cells and challenged these cells with
the ER stressors thapsigargin and tunicamycin. SERCA
did not modify BI-1’s ability to protect against ER stress-
induced cell death (Supplemental Fig. S18A). SERCA also
did not interfere with BI-1-mediated suppression of stress
kinase activation induced by ER stress agents (Supple-
mental Fig. S18B). Taken together, these results establish
a novel function for BI-1 whereby its ability to reduce ER
Ca2+suppresses mitochondrial ATP production to pro-
mote autophagy, independent of BI-1’s regulation of ER
stress responses and ER stress-induced cell death.
BI-1 has previously been shown to have multiple func-
tions that may contribute to its role in adaptation to cell
stress and preservation of cell survival, including (1)
interaction with and suppression of signaling by IRE1a
(Lisbona et al. 2009), an initiator of UPR signaling linked
to apoptosis, and (2) regulating ER Ca2+homeostasis,
possibly in association with anti-apoptotic Bcl-2 family
proteins that are known to regulate IP3R function in ER
membranes (Bcl-2 and Bcl-XL) (Xu et al. 2008). Addition-
ally, we demonstrate here a role for BI-1 as a modulator of
autophagy and attribute this function to its ability to
regulate ER Ca2+. Altogether, our data are consistent with
a model in which BI-1 reduces ER Ca2+levels, resulting in
diminished IP3R-mediated transport of Ca2+from ER into
mitochondria, causing a decline in mitochondrial bio-
energetics that results in reduced cellular ATP and thus
The ability of BI-1 to modulate autophagy was demon-
strated here using multiple cell lines involving both
constitutive and conditional BI-1 overexpression and us-
ing various experimental methods for BI-1 gene silenc-
ing or gene knockout. Additionally, the findings were
translated from mammalian cells (human and mouse) to
avian cells (DT40) and insect cells (Drosophila S2 cells).
The role of BI-1 in modulating autophagy was also dem-
onstrated in vivo by analysis of tissues of BI-1?/?mice
and analysis of BI-1-deficient tumor xenografts. In con-
trast to our results, Hetz and colleagues (Castillo et al.
2011) have recently reported that BI-1 inhibits autophagy
through a mechanism involving IRE1. They observed that
BI-1-mediated suppression of JNK activation downstream
from IRE1 inhibits the release of the essential autophagy
protein Beclin from Bcl-2, an event that requires JNK-
mediated phosphorylation of Bcl-2 (Pattingre et al. 2005).
Thus, it is possible that BI-1 can modulate autophagy via
two independent mechanisms having opposing effects,
including (1) suppressing IRE1-mediated JNK activation
in the context of ER stress, thus reducing autophagy, and
(2) reducing Ca2+/IP3R-dependent mitochondrial bioener-
getics, resulting in lower cellular ATP and thus stimulat-
ing autophagy under basal conditions. The relative con-
tributions of these two opposing mechanisms to net
autophagic flux may vary with cell type and cell context.
Regardless, our findings elucidate a novel mechanism of
autophagy regulation by BI-1, showing the existence of
a IP3R-dependent, IRE1-independent pathway by which
BI-1 promotes autophagy. The alterations in oxygen con-
sumption and ATP levels observed in human tumor cell
lines here, however, are inconsistent with circumstances
that would result in autophagy suppression. Our analysis
of BI-1 actions in IRE1 knockout cells also do not support
a prominent role for this UPR signaling protein, in contrast
to observations derived from IP3R knockout cells.
Recent studies have shown the existence of specific
domains in which ER and mitochondria juxtapose. These
MAMs of the ER have very specific functions that
ATP levels and induce autophagy. (A) Wild-
type (wt) and TKO IP3R DT40 cells were
stably infected with either control or BI-1
viruses. Where indicated, cells were treated
with the LIs NH4Cl (20 mM) and leupeptin
(10 mM) for either 2 h or 4 h. (B) Levels of
LC3-II were quantified by densitometry to
measure LC3 flux. (C) ATP levels were
measured in wild-type (wt) and TKO IP3R
DT40 cells stably transduced with either
GFP or BI-1 lentiviruses. Data represent
relative luminescence units (RLUs) per 106
viable cells (trypan blue exclusion) of three
independent measurements. (D) By increas-
ing ER Ca2+leakage via the IP3R, BI-1
reduces the amount of Ca2+at the micro-
domains where ER and mitochondria are in
close proximity. Consequently, low mito-
chondrial Ca2+levels reduce activity of the
TCA cycle with reduction of O2consump-
tion and ATP production. Low ATP levels
trigger a cascade of signal transduction
events that activates autophagy.
BI-1 requires IP3R to modulate
Sano et al.
1050GENES & DEVELOPMENT
permit Ca2+transfer between these two organelles
(Szabadkai et al. 2006a,b). Moreover, it was recently
reported that IP3Rs in ER membranes are required for
maintaining mitochondrial bioenergetics by mediating
the transfer of Ca2+from ER to mitochondria, thereby
supporting the activity of various dehydrogenases re-
quired for the TCA cycle (Cardenas et al. 2010). We sus-
pected a possible connection between BI-1 and IP3Rs for
three reasons. First, BI-1 causes a passive leak of Ca2+
from the ER, resulting in reduced resting levels of free
Ca2+in the ER lumen (Chae et al. 2004), a phenotype that
closely resembles the effects of anti-apoptotic Bcl-2 and
Bcl-XLproteins in ER membranes (He et al. 1997). Second,
BI-1 appears to be required for Bcl-XL-mediated regula-
tion of ER Ca2+, as demonstrated by experiments using
BI-1?/?cells (Xu et al. 2008). Third, Bcl-2 and Bcl-XL
associate with and modulate the function of IP3Rs in ER
membranes, and BI-1 appears to associate with Bcl-2 and
Bcl-XLbased on co-IP experiments (Xu and Reed 1998).
The discovery reported here that BI-1 modulates cellular
ATP levels, lactate production, and mitochondrial O2
consumption (without impacting mitochondrial mass)
points to a role for BI-1 in controlling mitochondrial
bioenergetics. Thus, BI-1, from its location in ER mem-
branes, impacts mitochondrial bioenergetics via a mech-
anism that requires IP3Rs. We therefore propose that BI-1
reduces the efficiency with which IP3Rs at MAMs trans-
fer Ca2+from ER into mitochondria. Furthermore, our
data are consistent with the concept that the downstream
consequences of this reduced efficiency of IP3R-mediated
transfer of Ca2+from ER to mitochondria are reduced
cellular ATP levels and increased basal levels of auto-
The connection between ER Ca2+and BI-1’s ability to
promote autophagy was firmly established by experimen-
tally manipulating SERCA, anER membrane ATPase that
actively pumps Ca2+into the lumen of the ER. While BI-1
causes a passive Ca2+leak from the ER, SERCA actively
pumps Ca2+into the ER. Using SERCA overexpression as
well as a pharmacological agonist of SERCA, we were
able to overcome the effects of BI-1 on ER Ca2+, showing
that when ER Ca2+is restored to normal, BI-1 fails to
modulate autophagy or cellular ATP levels. Thus, for the
first time, a specific cellular function has been attributed
to BI-1’s activity as a regulator of ER Ca2+, demonstrating
a linkage to mitochondrial bioenergetics and autophagy.
It will be interesting to explore whether Bcl-2 and Bcl-XL,
through their association with BI-1 in ER membranes,
have a similar impact on mitochondrial bioenergetics and
autophagy that is distinct from other mechanisms pre-
viously identified (Kroemer et al. 2010). While BI-1’s abil-
ity to modulate intracellular Ca2+homeostasis conceiv-
ably could impact autophagy through effects on Ca2+-
dependent kinases previously implicated in autophagy
regulation, such as CaMKKII and DAPK1 (Hoyer-Hansen
et al. 2007; Zalckvar et al. 2009), we were unable to
demonstrated an impact of either of these kinases on BI-
1’s ability to modulate autophagy.
Controversy has abounded over the question of whether
autophagy is a cell survival or cell death mechanism
(Shimizu et al. 2004; Codogno and Meijer 2005; Levine
and Kroemer 2008; Maiuri et al. 2009; Kroemer and White
2010). We found that BI-1-mediated cytoprotection does
not depend on the autophagy machinery, based on exper-
iments using Atg7?/?cells. In contrast, BI-1’s ability to
protect against cell death was determined to be IRE1a-
dependent, based on experiments using Ire1a?/?cells.
Furthermore, the role of IRE1a appears to be specific and
not a manifestation of a general perturbation in UPR
signaling, inasmuch as BI-1 retained its cytoprotective
activity in Perk?/?cells. Previously, it has been suggested
that BI-1 is an inhibitor of IRE1, a property that may
protect against ER stress-induced cell death in mamma-
lian cells by limiting downstream activation of stress
kinases that promote apoptosis (Rubio et al. 2011).
However, IRE1’s ability to activate XBP1 via its ribonu-
clease activity has been implicated in cell adaptation and
survival following ER stress (Tirasophon et al. 2000).
Together with other data implying a need for delicate
control of IRE activity (Schroder et al. 2003), our obser-
vations in yeast showing that the IRE1/XBP1 (HAC) axis
is required for BI-1-mediated protection from Bax sug-
gest that BI-1 may collaborate with IRE1 to promote
cell survival. The phenotype of BI-1 thus combines sup-
pression of cell death (apoptosis) with promotion of
autophagy and stands apart from other dual regulators
of apoptosis and autophagy, such as Bcl-2 and Bcl-XL,
which suppress both apoptosis and autophagy. Alto-
gether, the distinct and functional separable phenotypes
of BI-1 provide further evidence that BI-1 is a multifunc-
tional protein, with at least one function defined by an
ability to bind IRE1a and suppress ER stress-induced cell
death and another function defined by an ability to
modulate ER Ca2+to impact mitochondrial bioener-
getics and autophagy. The net outcome of these two
distinct and separable functions in terms of cell life and
death may vary with context, but heretofore, studies with
BI-1 have uniformly demonstrated an overall survival
Although tumor-associated elevations in BI-1 expres-
sion have been reported in many human cancers (Reimers
et al. 2008), the tumor xenograft data provide the first
functional evidence that BI-1 can be an important pro-
moter of tumorigenesis in vivo. In this regard, BI-1’s abil-
ity to promote autophagy and anaerobic glycolysis may
be an advantage for tumor cells in hypoxic microenvi-
ronments, along with its ability to prevent ER stress-
induced apoptosis that also tends to accompany harsh
microenvironments. Also, given that BI-1 hasbeen shown
to provide protection against ischemia reperfusion injury
in mice (Bailly-Maitre et al. 2006), it will be interesting
to elucidate the relative roles of the IRE1a-dependent
(ER stress) versus the IP3R-dependent (autophagy/
bioenergetics) mechanisms of BI-1 in that context. Alto-
gether, the new findings reported here suggest that BI-1
may serve an especially important role to aid cellular
resilience and ensure cellular survival under circum-
stances in which oxygen and nutrient deprivation are
encountered, such as the microenvironment of some
BI-1 modulates autophagy
GENES & DEVELOPMENT 1051
Materials and methods
Cells were homogenized in RIPA buffer (50 mM Tris-HCl at
pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM Tris-HCl,
1 mM EDTA, 1 mM PMSF, 1 mg/mL leupeptin, aprotinin, pep-
statin, 1 mM NaF, Na4VO3). Lysates were centrifuged at 10,000g
for 5 min at 4°C, and total protein content was quantified by BCA
assay (Pierce). Proteins were analyzed by SDS-PAGE and immu-
noblotting after transfer to nitrocellulose membranes (Millipore).
Antibodies used include anti-LC3 (Sigma), anti-p62 (BD Bio-
sciences), anti-HA (Roche), anti-tubulin (Roche), anti-SERCA
(BD Biosciences), anti-AMPK (Cell Signaling Technologies),
anti-p-AMPK (Cell Signaling Technologies), anti-acetylCoA car-
boxylase (Cell Signaling), and anti p-acetylCoA carboxylase (Cell
Signaling Technologies). An enhanced chemiluminescence (ECL)
method (Pierce) was used for detection.
Measurement of respiratory activity
Cultured cells were gently detached from the dish by trypsiniza-
tion, washed in PBS, harvested by centrifugation at 500g for
5 min, and immediately assessed for O2consumption. The rate
of oxygen consumption was measured polarographically with
a Clark-type oxygen electrode (Hansatech Instruments Ltd.) in
a thermostatically controlled chamber equipped with a magnetic
stirring device and a gas-tight stopper fitted with a narrow port
for additions via a Hamilton microsyringe. Aliquots of 5 3 106
viable cells per milliliter were assayed in 200 mM sucrose, 1 g/L
bovine serum albumin, 10 mM KH2PO4, 2.7 mM KCl, 1 mM
MgCl2, 20 mM HEPES, and 0.5 mM EGTA (pH 7.4) at 37°C; after
attainment of a stationary endogenous substrate-sustained respi-
ratory rate, 2 mM oligomycin was added. The rates of oxygen
consumption were corrected for 5 mM KCN-insensitive respira-
tion. The RCR was obtained by dividing the rates of oxygen
consumption achieved before and after theaddition of oligomycin.
Cellular ATP concentrations were assessed using the CellTiter-
Glo luminescent cell viability assay (Promega) as per the
manufacturer’s instructions. Luminescence was measured using
the Luminoskan Ascent (Thermo Electron Corp.) at 1-sec in-
tegration time per sample). Readings were normalized to the
number of viable cells (trypan blue exclusion).
Mitochondrial dehydrogenase activity (WST-1 assay)
Cells (in 96-well plates) were analyzed for mitochondrial
dehydrogenase activity using the WST-1 assay according to the
manufacturer’s protocol. The WST-1 substrate 4-[3-(4-iodophenyl)-
2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate was
converted to a colored formazan compound by the mitochondrial
enzymes. The color intensity is proportionally related to activity
of mitochondrial dehydrogenases. Briefly, 10 mL of WST-1 reagent
was added to 100 mL of medium. The cells were incubated with
the WST-1 reagent for 1–2 h at room temperature. The formazan
dye formed was measured at the wavelengths of 450 nm and
690 nm (background) using a FlexStation 3 microplate reader
Ca2+measurements were performed essentially as described
previously (Palmer et al. 2004). Briefly, cells were plated in
96-well plates and loaded with 50 mM Indo-1 AM and F-127
Pluronic (0.02%) for 30 min at 37°C. Cells were then stimulated
with 5 mM thapsigargin to empty ER Ca2+stores. Total fluores-
cent intensity was measured every 5 sec for a total time of 300
sec. Results were expressed as 400 nm/475 nm (calcium-free/
calcium-bound) emission ratio. For direct ER Ca2+measure-
ments, cells were plated in glass-bottomed dishes and trans-
fected with 4 mg of ER cameleon (YC4.3ER) for 48 h prior to
analysis. Cells were stimulated with 5 mM thapsigargin to empty
ER Ca2+stores and then imaged on an Inverted IX81 Olympus
wide-field and fluorescence microscope (FRET) with a cooled
charge-coupled device camera (Photmometrics, Inc.) controlled
by MetaFluor 6.1 software (Universal Imaging). Emission ratio
imaging of the cameleon was accomplished by using a 436DF20
excitation filter, a 450-nm dichroic mirror, and two emission
filters (475/40 for enhanced CFP and 535/25 for citrine). Data
represent the emission ratio YFP/CFP versus time, with each
line representing an average of not less than six cells. Mitochon-
drial Ca2+measurements were assessed by Rhod-2 AM (50 mM,
Invitrogen). Briefly, cells were loaded with the calcium-sensitive
dye Rhod-2 for 30 min at 37°C and 5% CO2, then washed with
PBS and reincubated for 30 min before analysis. Measurements
of the Rhod-2 fluorescence were performed using a microplate
reader (FlexStation 3, Molecular Devices) at 544-nm excitation
and 590-nm emission wavelengths.
Lactate levels were quantified using a L-lactate assay kit accord-
ing to the manufacturer’s instructions (BioVision). In this assay,
lactateis oxidizedbylactatedehydrogenasetogenerate aproduct
that interacts with a probe to produce a color (lmax = 450 nm),
which is proportional to the amount of L-lactate. Experiments
used 2000 cells per well of 96-well plates, which were seeded
without or with 1 mg/mL doxycycline for 24 h.
Cells were cultured for 2 h and 4 h with the LIs NH4Cl (20 mM)
and leupeptin (100 mM). Cells were then harvested, lysed,
normalized for total protein content, and subjected to immuno-
blot analysis using anti-LC3 antibody (Sigma). LC3-II levels were
quantified by densitometry and normalized for b-actin. LC3 flux
was quantified by dividing levels of LC3-II after 2 h of LI
treatment per level of LC3-II without LI.
All animal procedures were conducted in compliance with
protocols approved by the Institutional Animal Care and Use
Committee (IACUC) of Sanford-Burnham and were in accor-
dance with National Institutes of Health (NIH) guidelines. Mice
with targeted disruption of the BI-1 gene (Chae et al. 2004) were
used for experiments at 8–12 wk of age. When indicated, animals
were intraperitoneally injected with rapamycin (1 mg/kg;
Sigma), and tissues were harvested after 6 h, 12 h, 48 h, and 72
h post-injection. Before sacrificing, mice were anesthetized by
intraperitoneal injection of avertin. Tissues were dissected and
immediately frozen in liquid nitrogen.
In vivo tumor xenografts
All animal experiments were approved by the IACUC of Sanford-
Burnham. Xenograft tumors were established by subcutaneous
injection of 5 3 106H322M cells into the flanks of 6- to 8-wk-old
female nu/nu mice. Tumor growth was monitored weekly using
Sano et al.
1052 GENES & DEVELOPMENT
a Vernier caliper to calculate tumor volumes according to the
formula (length 3 width2)/2.
The data are expressed as mean 6 standard deviation (SD) from
a minimum of three determinations. Statistical significance of
differences between various samples was determined by t-test.
P < 0.05 was considered significant.
We thank Dr. Ana Maria Cuervo for help with experimental
design, data interpretation, and manuscript review; Dr. Fumihiko
Urano for providing Ire1a and PERK knockout MEFs; Dr. Noboru
Mizushima for providing the GFP-LC3 plasmid; and Dr. Kevin
Foskett for kindly providing TKO IP3R DT40 cells. We thank
Santosh Hariharan and Susanne Heynen-Genel for high-con-
tent cell imaging, Edward Monosov for transmission electron
analysis, Melanie Hanaii and Tessa Siegfried for manuscript
preparation, and Yoav Altman and Amy Cortez for technical
assistance with FACS analysis. This work was supported by
grants from the NIH (AG-15393). R.S. is supported by the Tobacco-
Related Disease Research Foundation (18FT-0179).
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