Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC
oligomerization: release of cytochrome c, AIF and Smac/Diablo
Varda Shoshan-Barmatz⁎, Nurit Keinan, Salah Abu-Hamad, Dalia Tyomkin, Lior Aram
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
a b s t r a c t a r t i c l ei n f o
Received 3 December 2009
Received in revised form 9 February 2010
Accepted 2 March 2010
Available online 6 March 2010
Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue
for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-
dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the
cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by
VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and
subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or
cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF.
Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the
release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells
expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not
only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is
associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells
using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H2O2or selenite augmented
the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis
machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1
N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-
apoptotic proteins, such as HK and Bcl2.
© 2010 Elsevier B.V. All rights reserved.
Located in the outer mitochondrial membrane (OMM), the
voltage-dependent anion channel (VDAC) not only assumes a crucial
position in the cell, controlling metabolic cross-talk between the
mitochondrion and the rest of the cell, thus regulating the metabolic
and energetic functions of the mitochondria [1,2], but is also a key
player in mitochondria-mediated apoptosis [3,4]. Apoptosis is a highly
regulated process that plays a protective role in physiological con-
ditions, with dysregulation of apoptosis having been implicated in
many diseases. During apoptosis, coordinated morphological and bio-
chemical changes occur within the nucleus, cytoplasm, organelles and
the plasma membrane . Mitochondria-mediated apoptosis can
be triggered by both external and internal stimuli and results in
the release of a large number of apoptogenic proteins from the
intermembrane space (IMS) to the cytosol , including cytochrome c
(Cyto c) , second mitochondrial activator of caspases (Smac/
Diablo)  and HtrA2/Omi . Smac/Diablo and Omi/HtrA2 
potentiate caspase activation by binding inhibitor of apoptosis
proteins (IAPs) and blocking their caspase inhibitory activities .
Other proteins released include apoptosis-inducing factor (AIF) and
endonuclease G (EndoG). AIF induces chromatin condensation and
large-scale DNA fragmentation (50 kbp) when released into the
cytosol . Since these mitochondrial apoptogenic proteins are
confined within the IMS, their release, following an apoptotic stimu-
lus, requires permeabilization of the OMM [13–15]. Hence, VDAC,
acting as an OMM channel, might mediate the release of mitochon-
drial proteins, such as Cyto c and AIF. Indeed, VDAC has been impli-
cated in apoptosis-relevant events, due to it serving as the target
for members of the pro- and anti-apoptotic Bcl2-family of proteins
[16–18] and due to its function in the release of apoptotic proteins
from the IMS [3,4]. Recently, we have shown that cells expressing N-
terminally truncated VDAC1 do not release cytochrome c and are
resistant to apoptosis .
Biochimica et Biophysica Acta 1797 (2010) 1281–1291
Abbreviations: AIF, apoptosis-inducing factor; ANT, adenine nucleotide translocase;
AzRu, Azido ruthenium; BRET2, bioluminescence resonance energy transfer; Cyto c,
cytochrome c; DBC, DeepBlueC coelentrazine; DFDNB, 1,5-difluoro-2,4-dinitrobenzene;
DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; EGS, ethylene glycol-bis (succi-
nimidylsuccinate); HK-I, hexokinase-I; OMM and IMM, outer and inner mitochondrial
membranes; PDL, poly-D-lysine; STS, staurosporine; RuR, Ruthenium Red; Smac/
Diablo, Second mitochondria activator of caspases; VDAC, voltage-dependent anion
⁎ Corresponding author. Department of Life Sciences, Ben-Gurion University of the
Negev, Beer-Sheva 84105, Israel. Fax: +972 8 6472992.
0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbabio
When considering models of VDAC-mediated protein release, one
should consider the molecular sizes of the released proteins (12 to
100 kDa). The inner diameter of the VDAC pore is 1.5 nm , large
enough to accommodate nucleotides and small molecules but too
small to allow the passage of a folded protein, like Cyto c (diameter
3.1 nm) . The protein-conducting channel could thus reside with-
in a VDAC1 homo-oligomer or hetero-oligomers containing VDAC1
and pro-apoptotic proteins [2,3,21,22]. We have demonstrated that
VDAC can exist as oligomers and proposed that oligomeric VDAC1
mediates the release of Cyto c [2,3,22–24]. Atomic Force Microscopy
(AFM) imaging allowed revealed the supra-molecular organization of
VDAC in the outer membrane of mitochondria isolated from yeast or
potato tubes [25,26]. No such studies are available for mammalian
mitochondrial VDAC. Meanwhile, NMR-based studies suggest the
existence of VDAC1 dimers [19,27,28], while recent analysis of crystal
packing in mVDAC1  revealed a strong anti-parallel dimer that
further assembled into hexamers, mimicking the native six pore
packing observed in EM  and AFM images of the OMM [25,26].
The most convincing finding connecting VDAC oligomerization to
apoptosis to date was presented in our recent study demonstrating
that apoptosis induction by various stimuli, including cisplatin, eto-
poside, staurosporine, As2O3, curcumin, UV irradiation and H2O2, is
accompanied by an up to 20-fold increase in VDAC oligomerization
. Thus, substantial evidence showing the formation of higher
order VDAC-containing complexes and the enhancement of supra-
molecular assembly of VDAC in cultured cells upon apoptosis in-
duction support the involvement of VDAC oligomerization in Cyto c
release and, thus, in apoptosis.
Alternative models for the release of Cyto c and AIF also exist. These
after the opening of the permeability transition pore (PTP) , or Bax/
of pro-apoptotic proteins soluble in the IMS, such as Cyto c [32,33].
Indeed, accumulating evidence suggests that multiple pathways and
mechanisms of Cyto c release can co-exist within a single model of cell
Despite the fact that numerous studies suggest the existence of
multimeric VDAC complexes [2,3,16,22,25,26], visualization and
monitoring of the dynamics of complex assembly remain a challenge.
Recently, we have demonstrated that Cyto c release and apoptosis are
coupled to VDAC oligomerization, as visualized using chemical cross-
linking and, in living cells, using Bioluminescence Resonance Energy
Transfer (BRET2) [23,36]. In this study, we further explored the
relationship between VDAC oligomerization, Cyto c and AIF release,
and apoptosis, as well as the function of the N-terminal region of
VDAC1 in the release of apoptotic proteins, apoptosis induction and
regulation by anti-apoptotic proteins.
2. Materials and methods
Poly-D-lysine (PDL), tetracycline, staurosporine (STS) and sodium
selenite, 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS),
H2O2, 1,5-difluoro-2,4-dinitrobenzene (DFDNB) and cytochalasin B
were purchased from Sigma (St. Louis, MO). EGS (ethylene glycol bis
[succinimidylsuccinate]) was obtained from Pierce Chemical (Rock-
ford, IL). An annexin V-FITC kit was purchased from Bender
MedSystem (Burlingame, CA). Metefectene was purchased from
Biotex (Munich, Germany). DMEM (Dulbecco's Modified Eagle
Medium) growth media and the supplements, fetal calf serum
(FCS), L-glutamine and penicillin-streptomycin were purchased
from Biological Industries (Beit Haemek, Israel). Blasticidin was
purchased from InvivoGen (San Diego, CA). Digitonin came from
Calbiochem-Novobiochem (Nottingham, UK). Coelentrazine (Deep-
BlueC, DBC) was obtained from Bioline (Taunton, MA). Monoclonal
anti-VDAC antibodies directed against the N-terminal region of 31HL
human porin came from Calbiochem-Novobiochem (Nottingham,
UK). Monoclonal anti-actin antibodies were obtained from MP
Biomedicals (Aurora, Ohio), while anti-Smac (2D12) antibodies
were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Polyclonal anti-AIF antibodies raised to recombinant AIF amino acid
residues 121–613 came from R&D Systems (Minneapolis, MN).
Monoclonal anti-Cyto c antibodies were obtained from BD Biosciences
Pharmingen (San Jose, CA). Alexa Fluor-488-conjugated goat anti-
mouse and anti-rabbit antibodies and 4',6-diiamino-2-phenylindole
dihydrochloride (DAPI) were from Molecular Probes (Carlsbad, CA).
Horseradish peroxidase (HRP)-conjugated anti-mouse antibodies
(1:10,000) were obtained from Promega (Madison, WI). Horseradish
peroxidase (HRP)-conjugated anti-rabbit antibodies (1:20,000) were
obtained from Zymed (San Francisco, CA).
DNA encoding murine (m)VDAC1 (obtained from W.J. Craigen,
University of Houston) or N-terminally truncated mVDAC1 (Δ(26)
mVDAC1) was cloned into the pcDNA4/TO vector to allow for
tetracycline-regulated expression, as described previously .
Plasmids encoding the fusion proteins, rVDAC1-GFP2 and rVDAC1-
Rluc were constructed using the BRET2 plasmids (Perkin Elmer,
Waltham, MA). The rVDAC1 gene was cloned into BamHI and HindIII
sites of the BRET2 plasmids encoding luciferase (RLuc) or GFP2 (N2
TATGGCTGTGCACCCACGT-ATGCC and reverse primer: GGATCCGCCG-
CCGCCGGAGCCGCCGCCGCCTGCTTGAAA-TTC. The reverse primer was
designed to include a double linker sequence encoding three glycines
and one serine ((GGGS)2) connecting the rVDAC1 and RLuc or GFP2
genes, to allow flexibility of the region .
Plasmids encoding shRNA against human VDAC1 (hVDAC1) for
specific silencing of endogenous human (h)VDAC1 were generated
using a shRNA-expressing vector. The hVDAC1-shRNA-encoding se-
quence was created using the two complimentary oligonucleotides
indicated below, each containing the 19 nucleotide target sequence of
hVDAC1 (337–355), followed by a short spacer and an anti-sense
sequence directed against the target: oligonucleotide 1, AGCTTAA-
and oligonucleotide 2, GATCCACACTAGGCACCGAGATTATTCAAGAGA-
TAATCTCGG TGCCTAGTGTTTTTTA. The hVDAC1-shRNA-encoding se-
quence was cloned into the BglII and HindIII sites of the pSUPERretro
plasmid (OligoEngine, Seattle, WA), containing a puromycin-
resistance gene. Transcription of this sequence under the control
of the H1 RNA promoter of RNA Polymerase III produces a hairpin
2.3. Tissue culture
T-REx-293 cells (HEK cells stably containing the pcDNA4/TO
regulatory vector containing the tetracycline repressor; Invitrogen)
and T-REx-293 cells stably expressing hVDAC1-shRNA, showing low
(10–20%) endogenous hVDAC1 expression (referred to as T-REx-
pS10), were grown at 37 °C under an atmosphere of 95% air and 5%
CO2 in DMEM supplemented with 10% FCS, 2 mM L-glutamine,
1000 U/ml penicillin, 1 mg/ml streptomycin, 5 μg/ml blasticidin
and 0.5 μg/ml puromycin (only for T-REx-pS10 cells). MCF7 and
HeLa cell line were grown under the same conditions as were T-REx-
293 cells, except that blasticidin was not added.
2.4. Cell transfection
T-REx-293 cells at ∼40% confluency were transiently transfected
(using metafecene) with plasmids pcDNA4/TO encoding rVDAC1,
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