REVIEW

Central roles of apoptotic proteins in mitochondrial function

SM Kilbride and JHM Prehn
Mitochondria have been classically characterized as organelles with responsibility for cellular energy production in the form of ATP,
but they are also the organelles through which apoptotic signaling occurs. Cell stress stimuli can result in outer membrane
permeabilization, after which mitochondria release numerous proteins involved in apoptotic signaling, including cytochrome c,
apoptosis-inducing factor, endonuclease G, Smac/DIABLO and Omi/HtrA2. Cell fate is determined by signaling through apoptotic
proteins within the Bcl-2 (B-cell lymphoma 2) protein family, which converges on mitochondria. Many cancerous cells display
abnormal levels of Bcl-2 protein family member expression that results in defective apoptotic signaling. Alterations in bioenergetic
function also contribute to cancer as well as numerous other disorders. Recent evidence indicates that several pro-apoptotic
proteins localized within mitochondria, as well as proteins within the Bcl-2 protein family, can inuence mitochondrial bioenergetic
function. This review focuses on the emerging roles of these proteins in the control of mitochondrial activity.
Oncogene (2013) 32, 27032711; doi:10.1038/onc.2012.348; published online 6 August 2012
Keywords: mitochondria; bioenergetics; apoptosis
INTRODUCTION
Mitochondria are vital organelles required by most normal cell
types, canonically dened as being the primary contributor to the
intracellular adenosine triphosphate (ATP) pool by the process of
oxidative phosphorylation (OXPHOS). They are structurally char-
acterized by four compartments, a mitochondrial matrix, a
convoluted inner membrane, an intermembrane space and an
outer membrane (Figure 1). The enzymes of the tricarboxylic acid
cycle, located within the mitochondrial matrix, generate the
substrates required to fuel OXPHOS of adenosine diphosphate
(ADP) by the enzymes of the electron transport chain (ETC),
located on the mitochondrial inner membrane. The enzymes of
tricarboxylic acid cycle, located within the mitochondrial matrix,
are fueled by acetyl coenzyme A. The pyruvate dehydrogenase
complex converts pyruvate derived from glycolysis into acetyl
coenzyme A, and mitochondria also derive acetyl coenzyme A
from fatty acids by b-oxidation within the mitochondrial matrix.
Abnormalities in metabolic pathways are common in malignant
cell types, with most cancer cells using aerobic glycolysis for ATP
production to a much greater extent than nontransformed cells.
Although this phenomenon, termed the Warburg effect, is not
fully understood, it has been proposed that the adaptation
facilitates nutrient incorporation into the biomass (such as
nucleotides, amino acids and lipids) required for cell division.
1
Indeed, it was recently demonstrated that reversal of the enzymes
of the tricarboxylic acid cycle occurs under hypoxic conditions
2
or
where ETC enzyme activity is defective,
3
promoting fatty acid
synthesis in tumor cells.
Intracellular ATP is generated most efciently by OXPHOS.
OXPHOS uses a proton circuit, whereby the ETC enzymes pump
protons out of the matrix, creating an electrochemical gradient or
proton motive force, the electrical component of which is termed
the mitochondrial membrane potential (Dcm). Protons then
re-enter the mitochondrial matrix through the ATP synthase, a
rotary nanomotor that uses the proton motive force to combine a
phosphate group and an ADP molecule into ATP within in the
matrix.
4
The Dcm can also be affected by proton leak through the
mitochondrial inner membrane into the matrix or by the activity of
uncoupling proteins, which act to uncouple the processes of
oxygen consumption and ATP production from each other.
5
The
proton motive force also enables mitochondria to maintain
intracellular ion homeostasis, with particularly important roles in
Ca
2
and Na

sequestering.
6
Among the molecules that govern
mitochondrial ion transport are the outer membrane voltage-
dependent anion channel (VDAC), and the inner membrane Ca
2
uniporter, Ca
2
/H

exchanger and Na

/H

exchanger. VDAC
also governs transport of metabolites of up to 5 kDa (including
ATP and ADP) across the mitochondrial outer membrane,
7
whereas exchange of ATP and ADP between the matrix and the
intermembrane space is carried out by the adenine nucleotide
translocator (ANT).
Mitochondria are also the primary site of intracellular reactive
oxygen species (ROS) production, as an unavoidable generation of
ROS occurs during the process of electron transfer by ETC
enzymes.
8
ROS generated include both oxygen-containing
molecules that extract electrons indiscriminately to ll their
outer orbital shell (termed free radicals) and molecules that are
unstable in the presence of transition metals (such as H
2
O
2
). The
most commonly generated within mitochondria include
superoxide (O
2

), hydrogen peroxide (H
2
O
2
) and hydroxyl radicals
(OH). ROS generation within mitochondria occurs when electrons
are transferred directly to oxygen by the redox intermediaries, in
particular complex I, which releases ROS into the mitochondrial
matrix, and complex III, which releases ROS into both the matrix
and the intermembrane space.
9
Other sites of signicant
mitochondrial ROS production include the reduced ubiquinol
pool within the ETC
10
and monoamine oxidase, an enzyme tightly
associated with mitochondrial outer membrane and found in
most mammalian tissues, which generates H
2
O
2
when catalyz-
ing deamination of monoamies such as 5-hydroxytryptamine
Department of Physiology and Medical Physics, Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin 2, Ireland. Correspondence: Professor JHM Prehn,
Department of Physiology and Medical Physics, Centre for Systems Medicine, Royal College of Surgeons in Ireland, 123 St Stephens Green, Dublin 2, Ireland.
E-mail: prehn@rcsi.ie
Received 5 April 2012; revised 22 June 2012; accepted 26 June 2012; published online 6 August 2012
Oncogene (2013) 32, 27032711
& 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13
www.nature.com/onc
(or serotonin), histamine, dopamine, noradrenaline and adrenaline.
11
Although excessive ROS production is known to cause oxidative
damage and has been shown to induce cell death,
12
there is an
increasing body of evidence suggesting that low level of ROS
production is involved in survival and proliferation signaling in
cancer cells.
13,14
In recent years, mitochondrial biology has enjoyed a resurgence
toward the forefront of biomedical research elds. This is because
of the discovery that mitochondria are also critically important in
programmed cell death signaling pathways. It has emerged that
apoptotic signaling proteins converge on the mitochondria, and
following exposure to excessive levels of cell stress, mitochondria
can initiate or amplify cell death signaling, committing the fate of
the cell to suicide in a process termed the intrinsic apoptotic
pathway or mitochondrial apoptosis.
1517
Among the many cell
stress signals known to trigger the intrinsic apoptotic pathway are
DNA damage, withdrawal of trophic support and endoplasmic
reticulum (ER) stress. These stress pathways are activated by
current chemotherapeutics as well as radiation therapy. Defects in
the mitochondrial apoptotic pathway have the potential to confer
a survival advantage to cancer cells, contributing to the resistance
of cancer cells to chemotherapy and radiotherapy.
18
The signals that exert the greatest control over the mitochon-
drial apoptosis pathway are proteins within the Bcl-2 (B-cell
lymphoma 2) family.
15,19
Two multidomain proteins within this
family, Bax (Bcl-2 associated protein X) and Bak, are pro-apoptotic
proteins that have been shown to be required for the activation of
this pathway.
20
Activated Bax and Bak homo-oligomerize on the
mitochondrial outer membrane when activated, resulting in
mitochondrial outer membrane permeabilization (MOMP), after
which pro-apoptotic factors are released. Monomeric activated
Bax and Bak can be sequestered by a number of multidomain anti-
apoptotic proteins within the Bcl-2 family, which are localized to
mitochondria and the ER. Among these are Bcl-2 itself, Bcl-x
L
(B-cell lymphoma-extra large), Mcl-1 (myeloid cell leukemia-1),
Bcl-w and B-1.
21
The anti-apoptotic function of these proteins
may be antagonized by a third set of proteins within the Bcl-2
family, the BH3-only proteins. These proteins all possess a BH-3
(Bcl-2 homology 3) region, which is required for their pro-
apoptotic function. BH3-only proteins can be activated by
transcription or posttranslational modication in response to
damage. At least two BH3-only proteins, Bim and Bid, and possibly
other BH3-only proteins such as PUMA (p53 upregulated
modulator of apoptosis), can also interact with anti-apoptotic
Bcl-2 family members and can also directly activate Bax and Bak.
Other BH-3-only proteins such as Bmf, Bad, Noxa and Hrk pre-
ferably interact with anti-apoptotic Bcl-2 family members with
various and distinctive afnities.
16,22
Increased mitochondrial
susceptibility to BH3 proteins in tumor cells is thought to be the
foremost reason as to why they selectively respond to cytotoxic
chemotherapy.
23
Thus, treatment with BH3 mimetics may prove to
be a fruitful eld for the development of novel chemotherapeutics
with less toxic side effects on noncancerous tissue. Furthermore,
chemosensitivity in multiple myeloma, acute myeloid leukemia
and acute, lymphocytic leukemia and ovarian cancer was found to
correlate with pretreatment mitochondrial susceptibility to BH3
peptides Puma and Bmf,
23
suggesting that the balance of Bcl-2
family members in malignant cells is a key determinant of clinical
outcome in these types of cancer.
Another subject of scrutiny in the eld of cancer research in
recent years is signaling through AMP-activated protein kinase
(AMPK), an evolutionary conserved intracellular sensor of bioener-
getic stress. AMPK is activated by an increase in the AMP/ATP ratio
and when activated promotes catabolic processes such as fatty
acid oxidation and glucose uptake.
24,25
The downstream effects of
AMPK activation have not been fully elucidated, and although
recent reports suggest that AMPK activation promotes tumor cell
survival under conditions of energy stress,
2628
prolonged AMPK
activation has also been shown to induce expression of pro-
apoptotic BH3 proteins.
29,30
There is evidence indicating that
pharmacological activation of AMPK may have therapeutic
potential in cancer, primarily the observation by multiple groups
that the incidence of cancer is reduced in patients treated with
metformin, an antidiabetic drug known to activate AMPK.
31,32
TCA
Cycle
Outer membrane
Intermembrane space
Inner membrane
Matrix
OXPHOS
NADH,
succinate
MOMP
ATP
ADP + Pi Ca
2+
Na
+
Ca
2+ m
m
Proapoptotic
factors
Bcl-2 family
signalling
Survival
ROS
Ca
2+
MPT
Swelling
m
Proapoptotic
factors
H
2
O,
Solutes
Rupture
O
2
Death
Figure 1. Mitochondrial processes involved in cell survival (left) and death signaling (right). The enzymes of the tricarboxylic acid (TCA) cycle
are located within the mitochondrial matrix, and fuel OXPHOS of ADP to generate ATP. OXPHOS is carried out by the enzymes of the ETC
(complexes IV). Complexes I, III and IV generate the Dcm by producing an electochemical gradient of protons across the mitochondrial inner
membrane. The energy of Dcm is harnessed by complex V of the chain to combine ADP and inoganic phosphate (Pi) to produce ATP. ATP/ADP
exchange between mitochondria and the cytoplasm occurs through the VDAC on the outer membrane, and the ANT catalyzes exchange
between the matrix and intermembrane space. Pi is symported into the matrix with H

in a process driven by Dcm. The Dcm also drives

Ca
2
ion uptake by the calcium uniporter, and antiport of Na

and Ca
2
occurs via the Na

/Ca
2
exchanger. Mitochondria can also amplify
apoptotic signaling, which is controlled by the Bcl-2 family proteins. Signaling by pro- and anti-apoptotic members of this family converge on
the mitochondrial outer membrane, and can induce pore formation involving Bax and Bak oligomerization, resulting in MOMP. Following
MOMP, pro-apoptotic factors such as cyt c, endoG, AIF, Smac/DIABLO and Omi cross the outer membrane through the pore, inducing
apoptosis by caspase activation and DNA fragmentation. The loss of cyt c from the intermembrane space under these circumstances is
sufcient to diminish the capacity of OXPHOS to the extent that Dcm collapses. ROS generated by the process of OXPHOS can contribute to
cell death, as can excess intramitochondrial Ca
2
, by inducing mPT. mPT pore formation allows inux of H
2
O and solutes from the cytoplasm
into the matrix, resulting in mitochondrial swelling and rupture of the outer membrane, releasing all intramitochondrial contents, including
the aforementioned pro-apoptotic factors.
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2704
Oncogene (2013) 2703 2711 & 2013 Macmillan Publishers Limited
Under certain circumstances, mitochondria may undergo a
distinct form of permeabilization, termed mitochondrial perme-
ability transition (mPT), which also leads to cell death.
33
Prolonged elevated cytoplasmic Ca
2
concentration can result
in excessive Ca
2
inux into the mitochondrial matrix to cause
formation of a complex involving the cyclophilin D (cypD), VDAC
and the ANT (Figure 1). The complex forms a pore that spans the
inner and outer membranes, allowing inux of H
2
O, ions and small
molecules of up to 1500 kDa in size, causing swelling of the matrix
that eventually leads to rupture of both inner and outer
membranes and release of mitochondrial pro-apoptotic factors.
ROS formation, misfolding of proteins within the mitochondrial
matrix and ER stress have been implicated in mPT-induced cell
death, but the process has been most closely associated with
excessive inux of extracellular Ca
2 34
and ischemia/reperfusion
injury.
35
However, the mechanisms underlying the initiation
of pore formation, as well as the exact structure of the pore,
remain incompletely dened. mPT can still occur in the absence
of ANT
36
and cypD knockout mice show no developmental
abnormalities.
37
Dissociation of cypD from ANT is induced by
sirtuin-3, a histone deacetylase localized to the mitochondrial
matrix.
38
Interestingly, inactivation of cypD by sirtuin-3-mediated
deacetylation also triggers detachment of the glycolytic enzyme
hexokinase II from mitochondria.
38
Binding of hexokinase II to VDAC
is increased in many cancer cells, and is thought to contribute to the
accelerated rate of glycolysis.
39
Indeed, increased hexokinase
localization at mitochondria has been suggested to protect
against cell death and has thus been associated with cancer cell
resistance to cytotoxicity.
40
Although both pro- and anti-apoptotic
Bcl-2 family proteins have also been shown to interact with mPT
components,
4143
mPT-induced cell death is generally considered to
be necrotic rather than apoptotic.
37,44
Following MOMP or mPT, a number of pro-apoptotic factors
that are normally conned to the mitochondrial intermembrane or
intracristae space are released, including cytochrome c (cyt c),
apoptosis-inducing factor (AIF), endonuclease G (EndoG), Smac/
DIABLO and Omi/HtrA2. In this review we discuss recent emerging
evidences that these two functions of mitochondria
bioenergetics and cell death executionare interlinked. We
describe the roles that cyt c, AIF, EndoG, Smac/DIABLO and
Omi/HtrA2, as well as proteins of the Bcl-2 family, may play in the
physiological regulation of mitochondrial bioenergetics, ROS
production and ion homeostasis, thus highlighting their important
biological activities in addition to their role in cell death execution.
CYTOCHROME C
Cyt c is the only protein involved in apoptosis that was originally
identied for its role in mitochondrial bioenergetics. The presence
of cyt c within the mitochondrial intermembrane space is essential
for synthesis of ATP by OXPHOS. Complexes I (NADH:ubiquinone
oxidoreductase) and II (succinate dehydrogenase) of the ETC are
fueled by the respective substrates NADH and FADH
2
, which are
generated by the tricarboxylic acid cycle within the mitochondrial
matrix. Electrons donated by these fuels are passed to complex III
(cytochrome bc
1
complex) via ubiquinone. Cyt c accepts a single
electron from complex III and carries it to complex IV (cyt c
oxidase), in the rate-limiting step of the ETC. Using four electrons,
complex IV reduces oxygen to water, and the ux of electrons
through complexes I, III and IV of the chain drives the pumping of
protons across the mitochondrial inner membrane by these
complexes.
45
The activity of ATP synthase (complex V) is driven by
proton inux into the matrix, and thus requires an intact Dcm to
function normally. The importance of cyt c in shuttling electrons
from complex III to complex IV during respiration is emphasized
by the observation that cyt c knockout mice die in utero by
midgestation.
46
Along with this classically dened vital role in
respiration, the presence of cyt c has also recently been shown to
be required for normal expression and assembly of mitochondrial
ETC complexes I and IV.
47
Following the occurrence of MOMP, cyt c is released from the
intermembrane space and binds to Apaf-1 and dATP.
4850
This
initiates formation of the apoptosome complex, which activates
caspase 9 and triggers the caspase cascade (Figure 2). The
requirement for cyt c for apoptosis was examined by generating
mice that express a mutant form of cyt c that could retain normal
electron transfer function but could not activate Apaf-1.
51
These
mice display embryonic lethality and brain developmental defects
to a similar extent as those observed in mice lacking Apaf-1,
52
or
caspase 9,
53
indicating that the role of cyt c in apoptosis is crucial
in tissue development and homeostasis. After MOMP, the level of
cyt c that is available to the mitochondrial respiration machinery is
severely reduced and electron transport through the complexes is
impaired, resulting in a collapse in the proton gradient across the
mitochondrial inner membrane.
5457
Under these conditions, the
contribution of mitochondria to intracellular ion homeostasis is
lost and ATP synthase activity often reverses in order to maintain
mitochondrial integrity, so that mitochondria may consume rather
than generate ATP.
57,58
MOMP-induced cyt c release therefore also
omi
SMAC
Endo G
Apoptosome
formation
AIF
IAPs
DNAfragmentation
Caspase
cascade
Nucleus
Oligomerization
Activators
(e.g. Bim, Bid)
Bcl-2
Matrix
Cytoplasm
IMS
Protein
Degradation
Bax
Cell
stress
Bak
Bcl-x
L
cyt c
MOMP mPT
VDAC
CypD
ANT
Oxidative stress
Ca
2+
overload
HK
Proapoptotic
factors,
Ca
2+

Cell
death
Figure 2. Proteins involved in mitochondrial cell death. (Left)
Signaling leading to MOMP. The pro-apoptotic activity of BH3-only
proteins is sequestered by anti-apoptotic proteins such as Bcl-2 and
Bcl-x
L
, which also interact with Bak on the mitochondrial outer
membrane to prevent activation. Various cell stress signals can lead
to upregulation of BH-3-only proteins, including the Bax/Bak
activators Bim and Bid. Activated Bax is translocated to the
mitochondrial outer membrane and oligomerizes with Bax causing
pore opening and allowing release of multiple proteins normally
localized to the mitochondrial intermembrane space (IMS) or
associated with the inner membrane. Upon release, EndoG and
AIF translocate to the nucleus and initiate DNA fragmentation and
chromatin condensation. Smac/DIABLO and Omi/HtrA2 act within
the cytoplasm by inhibiting IAPs, thus disinhibiting caspases,
leading to protein degradation and apoptosis. (Right) The mPT
occurs by formation of a pore that spans both mitochondrial
membranes. Oxidative stress or Ca
2
overload within the mito-
chondrial matrix can lead to opening of the pore, a process
involving interaction between numerous proteins including cypD,
the VDAC, the ANT and Bax. Hexokinase (HK) is also known to
interact with the VDAC. Following mPT-induced outer membrane
rupture, release of mitochondrial pro-apoptotic factors and Ca
2
may contribute to cell death; however, the mechanisms are not yet
fully understood.
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2705
& 2013 Macmillan Publishers Limited Oncogene (2013) 2703 2711
induces a bioenergetic crisis that may play a role in caspase-
independent cell death.
17
However, recent studies showed that
under conditions of high glycolytic activity and/or sufcient
remnant cyt c available for respiration, mitochondria within cancer
cells are capable of maintaining Dcm with ATP synthase working
in the forward mode, with Dcm being maintained at pre-MOMP
levels.
54,59
APOPTOSIS-INDUCING FACTOR
AIF was identied in 1996 as a protein normally localized within
the mitochondrial intermembrane space that is released following
mitochondrial permeability transition.
60,61
AIF possesses three
domains: an amino-terminal presequence that is removed upon
import into the intermembrane space of mitochondria, a spacer
sequence and an oxidoreductase domain.
62
Upon initiation of
MOMP, AIF translocates to the nucleus where it acts as a death
effector, causing chromatin condensation and DNA fragmentation.
63
Although the nuclease targets of AIF remain undiscovered, there
is convincing evidence to suggest that following localization to
the nucleus, cells subsequently undergo caspase-independent
apoptosis.
64
Homozygous AIF knockout mice do not survive past embryonic
day 12, and abnormal cell death occurs as early as embryonic day
9.
65
Thus, tissue-specic AIF knockout mice have been developed
to investigate the role of AIF in mitochondrial physiology.
Inactivated AIF in cardiac and skeletal muscle resulted
in decreased ETC complex I activity related to a reduction in
protein expression, was related to severe dilated cardio-
myopathy and skeletal muscle atrophy.
66
Harlequin (Hq) mice
display an 80% decrease in AIF expression and were generated by
a retroviral insertion into the rst intron of the AIF gene.
67
Both
Hq/Y males and Hq/Hq females survive until adulthood but display
progressive degeneration of terminally differentiated cerebellar
and retinal neurons, resulting in ataxia and blindness.
67
Expression
of a mitochondrially anchored version of AIF was sufcient to
rescue mitochondrial defects and enhance survival of AIF
knockout neurons.
68
Taken together, these data indicate that
the presence of AIF is essential for OXPHOS in cells that rely
heavily on respiration. In HeLa cells, the expression of complex I
subunits was diminished following AIF ablation, and the
associated 4050% decrease in complex I activity resulted in a
reduction in the rate of OXPHOS and consequent increase in
dependence on glycolysis.
63
Although the role of AIF in OXPHOS is
still not completely understood, AIF is known to bind avin
adenine dinucleotide and has NADH oxidase activity.
69
Sensitivity to oxidative stress is increased in AIF-decient
neurons and cardiomyocytes,
67,70
and a role for AIF as an
antioxidant that protects the ETC from oxidative stress has also
been proposed.
71
However, ROS production in isolated
mitochondria from Hq mice oxidizing complex I substrates was
no different from mitochondria isolated from wild type mice,
72
and the mechanism by which AIF ameliorates the damaging
effects of oxidative stress remains unclear.
ENDONUCLEASE G
Although encoded entirely by nuclear DNA, EndoG normally
resides within mitochondria, and there is evidence to indicate that
it is localized within the intermembrane space
73
and also binds to
the inner membrane.
74
A 5-kDa section of the 32-kDa inactive pro-
peptide targets the protein to mitochondria, and is subsequently
cleaved off. The mature 27-kDa EndoG is released following
MOMP and can initiate oligonucleosomal DNA fragmentation
upon translocation to the nucleus.
75
EndoG-induced apoptosis can
occur independently of caspase activity
76
and depletion of EndoG
can protect from a range of injury stimuli both in vivo
77
and
in vitro.
7880
A recent study elegantly identied a novel role for EndoG as a
regulator of mitochondrial mass and function in cardiac tissue.
81
EndoG was shown to control the expression of mitochondrial
mass, with diminished levels of both mitochondrial DNA (mtDNA)
and protein in isolated heart mitochondria from Endog
/
mice
compared with wild-type (WT) mice, whereas overexpression of
EndoG increased mitochondrial protein and mass in vitro.
In addition, respiration rates were decreased in isolated heart
mitochondria from Endog
/
mice compared with WT, fueled by
either complex I- or complex II-related substrates, and the rate of
ROS production was increased. Among the genes identied to be
regulated by EndoG were those controlling ETC enzyme
expression, including subunits of complexes I (ND1 and ND2), IV
(COX2) and V (ATPase6). EndoG has also been shown to cleave RNA
and is capable of generating RNA primers required by DNA
polymerase-g to initiate replication of mtDNA.
82
Although
expressed in many cancer cells, the role of EndoG in cancer
metabolism has been underexplored and warrants further
investigation.
Omi STRESS-REGULATED ENDOPROTEASE/HTRA2 (Omi)
Omi is a serine protease normally located within the mitochondrial
intermembrane space. The 49-kDa mtDNA-encoded proenzyme
includes a mitochondrial localizing sequence that is cleaved off
within the intermembrane space to expose an inhibitor of
apoptosis protein (IAP)-binding motif.
83
Following MOMP, Omi is
released into the cytoplasm and contributes apoptosis, by
cleaving both IAPs
84
and cytoskeletal proteins.
85
Thus, Omi
contributes to apoptosis in both caspase-dependent and
-independent mechanisms.
A number of links between Omi function and mitochondrial
dysfunction-related chronic neurodegeneration have been identi-
ed. Mnd2 (motor neuron degeneration 2) mice, which exhibit
early-onset neurodegeneration associated with parkinsonism and
juvenile lethality, were found to contain a loss-of-function
mutation in omi,
86
indicating that omi is required for protection
against stress stimuli in striatal neurons. In addition, mnd2 mice
displayed reduced body weight, and organs including heart,
thymus and spleen were found to be reduced in size.
86
Targeted
deletion of omi in mice conrmed these ndings.
87
Mutation
screening of the omi gene in Parkinsons disease patients
uncovered two loss-of-function mutations in single nucleotides
(A141S and G399S), which were related to disease pathogenesis.
88
Overexpression of the mutated enzymes in cancer cells caused
changes to mitochondrial morphology, depolarization of Dcm
and sensitized the cells to staurosporine-induced apoptosis.
88
Challenging mitochondria isolated from mnd2 mouse embryonic
broblasts with Ca
2
revealed that susceptibility to permeabilization
was increased in the absence of active Omi compared with
mitochondria isolated from WT mouse embryonic broblasts.
86
These data suggest that Omi plays a role in mitochondrial Ca
2
sequestering and mitochondrial dysfunction. In contrast, ETC
enzyme activities were not reduced in omi-decient cells.
87
Thus,
the exact function of Omi in normal mitochondrial physiology
remains unclear.
Smac/DIABLO
Second mitochondria-derived activator of caspase (Smac) was
discovered as a protein normally localized to mitochondria and
released during apoptosis, promoting caspase activation by binding
to and sequestering the activity of IAPs, thus relieving them of their
caspase-binding partners, and sensitizing cells to apoptosis.
89
The
human ortholog, named direct IAP-binding protein with a low pI
(DIABLO), was simultaneously and independently identied.
90
Immature Smac/DIABLO is encoded entirely within the nuclear
genome and contains a mitochondrial-localizing sequence, the
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2706
Oncogene (2013) 2703 2711 & 2013 Macmillan Publishers Limited
cleavage of which is required to expose the IAP-binding domain.
89
The mature 23-kDa protein resides within the intermembrane
space and is only released upon induction of MOMP, where it
homodimerizes and inhibits various IAPs, including X-linked IAP
and cellular IAPs 1 and 2.
91
Smac/DIABLO-decient mice were
shown to be viable and mature normally with no obvious
histological abnormalities alterations, and Smac/DIABLO-decient
cells responded normally to multiple apoptotic stimuli.
92
However,
a recent study has identied a heterozygous Smac/DIABLO
mutation that was related to a progressive hearing loss
disorder.
93
Overexpression of the mutant Smac/DIABLO
increased mitochondrial sensitivity to Ca
2
, as measured by
depolarization of Dcm in intact cells in the presence of the Ca
2
ionophore A23187. Smac is retained as a dimer within
mitochondria in X-linked IAP-overexpressing breast and colon
cancer cells during the process of MOMP,
94
suggesting that the
protective effects of X-linked IAP may also be associated with
preservation of mitochondrial function.
Bax
Bax plays a central role in the execution of mitochondrial
apoptosis. In healthy cells, the majority of Bax molecules are
located within the cytosol, with only a small fraction localized to
mitochondria.
95
Upon induction of apoptosis, Bax undergoes a
conformational activation whereby it translocates to mitochondria
and aggregates, contributing to pore formation.
96
Bax may also
mediate mPT-induced cell death, as double knockout of Bax and
Bak protected against treatment with the Ca
2
ionophore
ionomycin in mouse embryonic broblasts.
97
Interestingly,
reconstitution of an oligomerization-decient Bax mutant was
sufcient to restore mPT-induced cell death, but not MOMP-
mediated apoptosis, suggesting that Bax oligomerization is not
required for mPT.
97
Bax has been shown to have roles in normal cell physiology,
including Ca
2
homeostasis
98
and mitochondrial dynamics.
99
Results in a recent study indicate that Bax contributes to
mitochondrial ATP production in non-apoptotic cells. Although
only 7% of total endogenous Bax was found to be associated with
mitochondria in colorectal cancer-derived bax
/
HCT 116 cells,
both ATP level and the oxygen consumption rate were shown to
be reduced in corresponding bax
/
cells.
100
Similar results were
found in nontransformed primary hepatocytes from bax knockout
mice compared with those from WT animals. Interestingly, citrate
synthase activity was also found to be reduced in bax-decient
cells, and the rate of glycolysis was increased, conrming that the
bioenergetic deciencies were of mitochondrial origin. Moreover,
reintroduction of Bax into knockout cells was sufcient to reverse
the bioenergetic deciencies. The authors concluded that such a
role for Bax in mitochondrial bioenergetics is supported by the
observation that complete mutational inactivation (or deletion) of
Bax is rarely found in cancers, whereas mutations that prevent the
pro-apoptotic activity of Bax are relatively frequent.
100
Bcl-2
The bcl-2 was initially identied as a gene excessively transcribed
in human B-cell follicular lymphoma.
101
The Bcl-2 protein can
prevent initiation of mitochondrial apoptosis by binding both Bax
and Bak at the mitochondrial outer membrane, inhibiting Bax/Bak
oligomerization, and can also interact with VDAC, thus inhibiting
pore formation.
102
Bcl-2 also has anti-apoptotic properties due to
its ability to antagonize BH3-only activators Bim and Bid and
sensitizers including Puma, Bad and Bik.
16,21
Bcl-2 is thought to
localize to the membranes of multiple organelles in nonapoptotic
cells, including both mitochondrial inner and outer membranes,
the ER membrane and the nuclear outer membrane.
103
The anti-apoptotic properties of Bcl-2 have been extensively
studied, but recent evidence indicates that Bcl-2 can also directly
mediate the rate of OXPHOS by interaction with mitochondrial
ETC complex IV.
14,104106
Overexpression of Bcl-2 increased O
2

within mitochondria, interpreted as an increase in the rate of

electron ow through the respiratory chain, and both isolated
mitochondrial respiration rate and complex IV activity were also
found to be increased in Bcl-2-overexpressing tumor cells.
104
The
Va subunit of complex IV was found to physically interact with
Bcl-2, and the extent of localization of this subunit to the
mitochondrial inner membrane was shown to correlate to Bcl-2
expression in cancer cell lines, offering convincing evidence that
Bcl-2 controls mitochondrial bioenergetic function by inuencing
the activity of the rate-limiting ETC enzyme, complex IV.
105
Gene
silencing of Bcl-2 expression diminished O
2

generation and
reduced inner membrane localization of both Va and Vb complex
IV subunits. However, Bcl-2 did not affect the expression levels of
either subunits, indicating that the effects of Bcl-2 on respiration
rate and ROS production are due to the role of Bcl-2 in complex IV
assembly.
105
The abilities of Bcl-2 to regulate the rates of OXPHOS
and ROS production have been implicated in the promotion of
survival and oncogenic signaling in cancer cells.
14
Bcl-X
L
The Bcl-x
L
protein is encoded by the bcl-x gene, which can be
alternatively spliced to produce two mRNA isoforms, Bcl-x
L
and Bcl-x
S
.
107
Expression of bcl-x is required for embryonic
development, as evidenced by the observation that bcl-x
knockout mice do not survive past embryonic day 13.
108
Histological analysis revealed excessive neuronal and liver
hematopoietic cell apoptosis in the knockout embryos,
implicating bcl-x in the development of these systems.
108
It was
initially observed that Bcl-x
L
was similar in size and structure to
Bcl-2, and accordingly was also found to have anti-apoptotic
properties, with overexpression protecting against growth factor
withdrawal to a similar extent as Bcl-2 overexpression.
107
Numerous Bcl-x
L
-binding partners have also been identied
with roles in mitochondrial metabolite exchange
109
and
dynamics
110
as well as autophagy signaling
111
and neuronal
synaptic transmission.
112
Using a Bcl-x
L
conditional knockout
model in primary neurons, two research groups recently jointly
demonstrated a direct involvement of Bcl-x
L
in mitochondrial
bioenergetics.
113,114
Mitochondrial membrane potential was
found to be hyperpolarized in bcl-x-decient neurons, NAD(P)H
levels were increased and ROS production was decreased.
114
In
contrast to the reported localization of endogenous Bcl-x
L
as
cytosolic homodimers in non-apoptotic HeLa cells,
115
the majority
of neuronal Bcl-x
L
was found to be localized within mitochondria.
Furthermore, 450% of the mitochondrial fraction was shown to
be localized to the mitochondrial inner membrane and/or matrix
by electron microscopy.
114
This study also reported that Bcl-x
L
directly interacts with the b-subunit of mitochondrial ETC complex V
(ATP synthase) to stabilize Dcm, as bcl-x deciency and inhibition
of Bcl-x
L
with the chemotherapeutic agent ABT-737 both caused
rapid uctuations of Dcm in neurons.
114
In a separate study, Bcl-x
L
overexpression was found to increase ATP levels in primary
neurons, whereas knockdown of Bcl-x
L
or inhibition with ABT-737
depleted ATP without affecting cell death.
113
Interestingly, the
level of lactate production in cells overexpressing Bcl-x
L
was
reduced compared with control neurons, suggesting a reduction
in glycolytic rate in cells overexpressing Bcl-x
L
. This indicates that
the Bcl-x
L
-induced increase in ATP occurred as a result of
enhanced mitochondrial activity. However, oxygen consumption
was unexpectedly found to be decreased in Bcl-x
L
-overexpressing
neurons, and knockdown or inhibition of Bcl-x
L
increased oxygen
uptake compared with control neurons.
113
Together, these data
suggest that depletion of endogenous Bcl-x
L
partially uncouples
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2707
& 2013 Macmillan Publishers Limited Oncogene (2013) 2703 2711
OXPHOS, and it was concluded that Bcl-x
L
functions to bolster
mitochondrial energy capacity by preventing energy wastage by
futile ion ux across in mitochondrial inner membrane.
114
Mcl-1
Mcl-1 is an anti-apoptotic Bcl-2 family member that is required for
the survival of a variety of adult cell types, such as hematopoietic
stem cells,
116
lymphocytes
117
and neurons.
118
Mcl-1 is essential for
development of the embryo, and Mcl-1 knockout mouse embryos
die peri-implantation.
119
Amplication of MCL-1 has been found to
occur in numerous cancer types, including lung, breast,
gastrointestinal and sarcomas.
120
The level of Mcl-1 expression
at diagnosis was found to be related to disease severity in multiple
myeloma, with the highest amounts corresponding to shortest
event-free survival times
121
and Mcl-1 also contributes to
glucocorticoid resistance in acute lymphoblastic leukemia.
122
Interestingly, although Mcl-1 deletion was found to be lethal
peri-implantation, there was no difference in the percentage of
apoptotic cells in Mcl-1-null blastocysts compared with WT,
hinting at the existence of an essential function for Mcl-1 other
than the canonical anti-apoptotic role.
119
A recent study
demonstrated that Mcl-1 contains a mitochondrial targeting
sequence, and the protein undergoes proteolytic cleavage
giving rise to different Mcl-1 species that are localized to
subcompartments within mitochondria.
123
A 40-K relative-
molecular-mass (M
r
) form is localized to the outer leaet of the
mitochondrial outer membrane and binds pro-apoptotic BH3-only
proteins such as bim, whereas the 36-K M
r
form associates with
the mitochondrial inner membrane and has no ability to prevent
apoptosis.
123
Enzyme activities of ETC complexes I, II and IV were
all reduced in mouse liver mitochondria with MCL-1 deletion,
suggesting that Mcl-1 is required for normal respiration.
123
Changes in mitochondrial morphology and fusion were found
upon MCL-1 deletion, and the mtDNA content was reduced,
leading to decreased expression of the subunits of complex IV
encoded by mtDNA (Cox 1 and Cox 2).
123
MCL-1 deletion also
disrupted the organization of ETC enzymes into supercomplexes
and diminished ATP synthase oligomerization, an important
process in mitochondrial cristae formation.
123,124
Thus, the 36-K
M
r
form of Mcl-1 required for mitochondrial function may
represent a novel therapeutic target in numerous types of cancer.
CONCLUSION
It is widely accepted that mitochondria originated from an ancient
eubacterial ancestor, which was incorporated into eukaryotic cells.
Since this time, the mitochondrial genome is known to have
undergone a streamlining process whereby the current coding
capacity is vastly reduced compared with that of their closest
eubacterial relatives.
125
In this respect, it is perhaps unsurprising
that nuclear-encoded proteins full some of the bioenergetic
requirements of mitochondria. Undoubtedly, eukaryotic cells are
constantly subjected to a similar level of reductive evolution,
leading to the expression of proteins with multiple functions.
Indeed, in attempting to understand various aspects of cell
physiology, researchers attribute labels (such as bioenergetics
and apoptosis) to envisage intracellular phenomena. However,
evolution has no obligation to obey such labels. Hence, most of
the proteins involved in mitochondrial apoptosis signaling have
been discovered because of their functions in cell death, and often
bear names to reect these functions, but it is becoming
increasingly evident that someif not mostof these proteins
also have important roles in mitochondrial bioenergetics in non-
apoptotic cells (Figure 3). Indeed, these proteins have also been
shown to contribute to vital functions related to other intracellular
processes.
126
Many of the roles have become evident because of
the physiological consequences of gene mutation or deletion,
resulting in various pathological phenotypes where specic tissue
types were affected. This highlights not only the differences in
contribution of mitochondrial bioenergetics to cell viability
between tissue types, but also the heterogeneity of the
mitochondria themselves. Predictably, the effects of gene
deciency in mitochondrial apoptosis proteins primarily affect
excitable tissues that have a high energy demand and rely on
OXPHOS as the primary means of energy metabolism.
Importantly, irregularities in energy metabolism are also a
common feature of a wide range of cancer types, with many forms
of tumor cells capable of adapting to conditions of hypoxia and
ischemia. Increased expression levels of anti-apoptotic proteins
including Bcl-2, Mcl-1 and Bcl-x
L
are also commonly reported in
various types of cancer.
127,128
Such increased expression can
prevent mitochondrial amplication of apoptosis signaling, but
may also facilitate mitochondrial bioenergetic function, thus
promoting survival by at least both of these mechanisms. In
many cases, the details of how apoptotic proteins affect
mitochondrial physiology in cancer cells remain to be
elucidated, and the likelihood that further functions of
apoptotic proteins in non-apoptotic cellular bioenergetic
function will be discovered cannot be disregarded.
CONFLICT OF INTEREST
The authors declare no conict of interest.
ACKNOWLEDGEMENTS
This work was funded by Health Research Board (RP/2008/14) and Science
Foundation Ireland (08/IN1/1949) grants to JHMP.
Inner
membrane
I
II
III IV
V
2H
+
2e
-
2e
-
m
Matrix
Intermembrane
space
Ca
2+
EndoG
AIF
EndoG
Smac
cyt c
Bcl-x
L
omi
Bcl-2
H
2
O
O
2
+ 2H
+
2H
+
4H
+
4H
+
ADP + Pi
Bax
ATP
Mcl-1
Figure 3. Sites of interaction of apoptotic proteins with mitochondrial
bioenergetics. During OXPHOS, electrons are passed along the
respiratory chain enzyme complexes IIV, and are accepted by O
2
.
Electron ux through complexes I, III and IV allow them to pump
protons out, creating an electrochemical gradient across the inner
membrane, thus generating Dcm. Protons re-enter through complex V,
generating ATP. Mitochondria rely on Dcm to import Ca
2
into the
matrix, particularly important in excitable cells. Conversely, an
increase in Ca
2
within the matrix can increase the rate of
mitochondrial energy metabolism. AIF has NADH oxidase activity
and controls expression of complex I subunits in neuronal
mitochondria. Although there is no known physical interaction
between EndoG and ETC enzymes, EndoG regulates mitochondrial
mass and expression of subunits from complexes I, IV and V in
cardiomyocytes. Cytochrome c is required by the respiratory chain of
all mitochondria to carry electrons from complex III to complex IV.
Mcl-1 is required for normal expression of complex IV subunits Cox 1
and Cox 2, and Bcl-2 directly interacts with the Va subunit of complex
IV, facilitating assembly and electron ow. Bcl-x
L
directly interacts
with the b-subunit of mitochondrial ETC complex V to regulate ion
ux across the inner membrane. The presence of Bax within
mitochondria can positively inuence the rate of OXPHOS by
unknown means. Omi contributes to Ca
2
sequestering capacity in
striatal neuron mitochondria among others, as does Smac in inner ear
cell mitochondria, although the exact mechanisms of interaction of
both with the mitochondrial bioenergetic machinery remain unclear.
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2708
Oncogene (2013) 2703 2711 & 2013 Macmillan Publishers Limited
REFERENCES
1 Vender Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg
effect: the metabolic requirements of cell proliferation. Science 2009; 324:
10291033.
2 Metallo CM, Gameiro PA, Bell EL, Mattaini KR, Yang J, Hiller K et al. Reductive
glutamine metabolism by IDH1 mediates lipogenesis under hypoxia. Nature
2011; 481: 380384.
3 Mullen AR, Wheaton WW, Jin ES, Chen PH, Sullivan LB, Cheng T et al. Reductive
carboxylation supports growth in tumour cells with defective mitochondria.
Nature 2011; 481: 385388.
4 Stock D, Leslie AG, Walker JE. Molecular architecture of the rotary motor in ATP
synthase. Science 1999; 286: 17001705.
5 Andrews ZB, Diano S, Horvath TL. Mitochondrial uncoupling proteins in the CNS:
in support of function and survival. Nat Rev Neurosci 2005; 6: 829840.
6 Nicholls DG, Budd SL. Mitochondria and neuronal survival. Physiol Rev 2000; 80:
315360.
7 Lemasters JJ, Holmuhamedov E. Voltage-dependent anion channel (VDAC) as
mitochondrial governator--thinking outside the box. Biochim Biophys Acta 2006;
1762: 181190.
8 Cadenas E, Davies KJ. Mitochondrial free radical generation, oxidative stress, and
aging. Free Radic Biol Med 2000; 29: 222230.
9 Han D, Williams E, Cadenas E. Mitochondrial respiratory chain-dependent
generation of superoxide anion and its release into the intermembrane space.
Biochem J 2001; 353: 411416.
10 Turrens JF, Alexandre A, Lehninger AL. Ubisemiquinone is the electron donor for
superoxide formation by complex III of heart mitochondria. Arch Biochem
Biophys 1985; 237: 408414.
11 Youdim MB, Edmondson D, Tipton KF. The therapeutic potential of monoamine
oxidase inhibitors. Nat Rev Neurosci 2006; 7: 295309.
12 Mammucari C, Rizzuto R. Signaling pathways in mitochondrial dysfunction and
aging. Mech Ageing Dev 2010; 131: 536543.
13 Clement MV, Pervaiz S. Intracellular superoxide and hydrogen peroxide con-
centrations: a critical balance that determines survival or death. Redox Rep 2001;
6: 211214.
14 Krishna S, Low IC, Pervaiz S. Regulation of mitochondrial metabolism:
yet another facet in the biology of the oncoprotein Bcl-2. Biochem J 2011; 435:
545551.
15 Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate
cell death. Nat Rev Mol Cell Biol 2008; 9: 4759.
16 Tait SW, Green DR. Mitochondria and cell death: outer membrane permeabili-
zation and beyond. Nat Rev Mol Cell Biol 2010; 11: 621632.
17 Kroemer G, Martin SJ. Caspase-independent cell death. Nat Med 2005; 11:
725730.
18 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;
144: 646674.
19 Adams JM, Cory S. The Bcl-2 apoptotic switch in cancer development and
therapy. Oncogene 2007; 26: 13241337.
20 Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ et al.
Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction
and death. Science 2001; 292: 727730.
21 Brunelle JK, Letai A. Control of mitochondrial apoptosis by the Bcl-2 family. J Cell
Sci 2009; 122: 437441.
22 Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ. Distinct
BH3 domains either sensitize or activate mitochondrial apoptosis, serving as
prototype cancer therapeutics. Cancer Cell 2002; 2: 183192.
23 Ni Chonghaile T, Sarosiek KA, Vo TT, Ryan JA, Tammareddi A, Moore Vdel G et al.
Pretreatment mitochondrial priming correlates with clinical response to
cytotoxic chemotherapy. Science 2012; 334: 11291133.
24 Weisova P, Davila D, Tuffy LP, Ward MW, Concannon CG, Prehn JH. Role of
5-adenosine monophosphate-activated protein kinase in cell survival and death
responses in neurons. Antioxid Redox Signal 2010; 14: 18631876.
25 Hardie DG. AMP-activated protein kinase: an energy sensor that regulates all
aspects of cell function. Genes Dev 2011; 25: 18951908.
26 Jeon SM, Chandel NS, Hay N. AMPK regulates NADPH homeostasis to promote
tumour cell survival during energy stress. Nature 2012; 485: 661665.
27 Kato K, Ogura T, Kishimoto A, Minegishi Y, Nakajima N, Miyazaki M et al. Critical
roles of AMP-activated protein kinase in constitutive tolerance of cancer cells to
nutrient deprivation and tumor formation. Oncogene 2002; 21: 60826090.
28 Laderoute KR, Amin K, Calaoagan JM, Knapp M, Le T, Orduna J et al. 5-AMP-
activated protein kinase (AMPK) is induced by low-oxygen and glucose
deprivation conditions found in solid-tumor microenvironments. Mol Cell Biol
2006; 26: 53365347.
29 Concannon CG, Tuffy LP, Weisova P, Bonner HP, Davila D, Bonner C et al. AMP
kinase-mediated activation of the BH3-only protein Bim couples energy
depletion to stress-induced apoptosis. J Cell Biol 2010; 189: 8394.
30 Kilbride SM, Farrelly AM, Bonner C, Ward MW, Nyhan KC, Concannon CG et al.
AMP-activated protein kinase mediates apoptosis in response to bioenergetic
stress through activation of the pro-apoptotic Bcl-2 homology domain-3-only
protein BMF. J Biol Chem 2010; 285: 3619936206.
31 Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, Morris AD. Metformin and
reduced risk of cancer in diabetic patients. BMJ 2005; 330: 13041305.
32 Decensi A, Puntoni M, Goodwin P, Cazzaniga M, Gennari A, Bonanni B et al.
Metformin and cancer risk in diabetic patients: a systematic review and meta-
analysis. Cancer Prev Res 2010; 3: 14511461.
33 Zamzami N, Larochette N, Kroemer G. Mitochondrial permeability transition in
apoptosis and necrosis. Cell Death Differ 2005; 12(Suppl 2): 14781480.
34 Dong XX, Wang Y, Qin ZH. Molecular mechanisms of excitotoxicity and their
relevance to pathogenesis of neurodegenerative diseases. Acta Pharmacol Sin
2009; 30: 379387.
35 Baines CP, Kaiser RA, Purcell NH, Blair NS, Osinska H, Hambleton MA et al. Loss of
cyclophilin D reveals a critical role for mitochondrial permeability transition in
cell death. Nature 2005; 434: 658662.
36 Kokoszka JE, Waymire KG, Levy SE, Sligh JE, Cai J, Jones DP et al. The ADP/ATP
translocator is not essential for the mitochondrial permeability transition pore.
Nature 2004; 427: 461465.
37 Nakagawa T, Shimizu S, Watanabe T, Yamaguchi O, Otsu K, Yamagata H et al.
Cyclophilin D-dependent mitochondrial permeability transition regulates some
necrotic but not apoptotic cell death. Nature 2005; 434: 652658.
38 Shulga N, Wilson-Smith R, Pastorino JG. Sirtuin-3 deacetylation of cyclophilin D
induces dissociation of hexokinase II from the mitochondria. J Cell Sci 2010; 123:
894902.
39 Pedersen PL. Warburg, me and Hexokinase 2: Multiple discoveries of key
molecular events underlying one of cancers most common phenotypes, the
Warburg Effect, i.e., elevated glycolysis in the presence of oxygen. J Bioenerg
Biomembr 2007; 39: 211222.
40 Abu-Hamad S, Zaid H, Israelson A, Nahon E, Shoshan-Barmatz V. Hexokinase-I
protection against apoptotic cell death is mediated via interaction with the
voltage-dependent anion channel-1: mapping the site of binding. J Biol Chem
2008; 283: 1348213490.
41 Belzacq AS, Vieira HL, Verrier F, Vandecasteele G, Cohen I, Prevost MC et al. Bcl-2
and Bax modulate adenine nucleotide translocase activity. Cancer Res 2003; 63:
541546.
42 Marzo I, Brenner C, Zamzami N, Susin SA, Beutner G, Brdiczka D et al. The
permeability transition pore complex: a target for apoptosis regulation by cas-
pases and bcl-2-related proteins. J Exp Med 1998; 187: 12611271.
43 Shimizu S, Eguchi Y, Kamiike W, Funahashi Y, Mignon A, Lacronique V et al. Bcl-2
prevents apoptotic mitochondrial dysfunction by regulating proton ux. Proc
Natl Acad Sci USA 1998; 95: 14551459.
44 Tsujimoto Y, Nakagawa T, Shimizu S. Mitochondrial membrane permeability
transition and cell death. Biochim Biophys Acta 2006; 1757: 12971300.
45 Hosler JP, Ferguson-Miller S, Mills DA. Energy transduction: proton transfer
through the respiratory complexes. Annu Rev Biochem 2006; 75: 165187.
46 Li K, Li Y, Shelton JM, Richardson JA, Spencer E, Chen ZJ et al. Cytochrome c
deciency causes embryonic lethality and attenuates stress-induced apoptosis.
Cell 2000; 101: 389399.
47 Vempati UD, Han X, Moraes CT. Lack of cytochrome c in mouse broblasts
disrupts assembly/stability of respiratory complexes I and IV. J Biol Chem 2009;
284: 43834391.
48 Garrido C, Galluzzi L, Brunet M, Puig PE, Didelot C, Kroemer G. Mechanisms of
cytochrome c release from mitochondria. Cell Death Differ 2006; 13: 14231433.
49 Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in
cell-free extracts: requirement for dATP and cytochrome c. Cell 1996; 86: 147157.
50 Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homo-
logous to C. elegans CED-4, participates in cytochrome c-dependent activation
of caspase-3. Cell 1997; 90: 405413.
51 Hao Z, Duncan GS, Chang CC, Elia A, Fang M, Wakeham A et al. Specic ablation
of the apoptotic functions of cytochrome C reveals a differential requirement for
cytochrome C and Apaf-1 in apoptosis. Cell 2005; 121: 579591.
52 Cecconi F, Alvarez-Bolado G, Meyer BI, Roth KA, Gruss P. Apaf1 (CED-4 homolog)
regulates programmed cell death in mammalian development. Cell 1998; 94:
727737.
53 Kuida K, Haydar TF, Kuan CY, Gu Y, Taya C, Karasuyama H et al. Reduced
apoptosis and cytochrome c-mediated caspase activation in mice lacking cas-
pase 9. Cell 1998; 94: 325337.
54 Waterhouse NJ, Sedelies KA, Sutton VR, Pinkoski MJ, Thia KY, Johnstone R et al.
Functional dissociation of DeltaPsim and cytochrome c release denes the
contribution of mitochondria upstream of caspase activation during granzyme
B-induced apoptosis. Cell Death Differ 2006; 13: 607618.
55 Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR.
Cytochrome c maintains mitochondrial transmembrane potential and ATP
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2709
& 2013 Macmillan Publishers Limited Oncogene (2013) 2703 2711
generation after outer mitochondrial membrane permeabilization during the
apoptotic process. J Cell Biol 2001; 153: 319328.
56 Mootha VK, Wei MC, Buttle KF, Scorrano L, Panoutsakopoulou V, Mannella CA
et al. A reversible component of mitochondrial respiratory dysfunction in
apoptosis can be rescued by exogenous cytochrome c. EMBO J 2001; 20:
661671.
57 Dussmann H, Rehm M, Kogel D, Prehn JH. Outer mitochondrial membrane
permeabilization during apoptosis triggers caspase-independent mitochondrial
and caspase-dependent plasma membrane potential depolarization: a single-
cell analysis. J Cell Sci 2003; 116: 525536.
58 Rego AC, Vesce S, Nicholls DG. The mechanism of mitochondrial membrane
potential retention following release of cytochrome c in apoptotic GT1-7 neural
cells. Cell Death Differ 2001; 8: 9951003.
59 Huber HJ, Dussmann H, Kilbride SM, Rehm M, Prehn JH. Glucose metabolism
determines resistance of cancer cells to bioenergetic crisis after cytochrome-c
release. Mol Syst Biol 2011; 7: 470.
60 Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A et al. Bcl-2
inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 1996;
184: 13311341.
61 Zamzami N, Susin SA, Marchetti P, Hirsch T, Gomez-Monterrey I, Castedo M et al.
Mitochondrial control of nuclear apoptosis. J Exp Med 1996; 183: 15331544.
62 Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM et al. Molecular
characterization of mitochondrial apoptosis-inducing factor. Nature 1999; 397:
441446.
63 Vahsen N, Cande C, Briere JJ, Benit P, Joza N, Larochette N et al. AIF deciency
compromises oxidative phosphorylation. EMBO J 2004; 23: 46794689.
64 Cande C, Vahsen N, Garrido C, Kroemer G. Apoptosis-inducing factor (AIF):
caspase-independent after all. Cell Death Differ 2004; 11: 591595.
65 Brown D, Yu BD, Joza N, Benit P, Meneses J, Firpo M et al. Loss of Aif function
causes cell death in the mouse embryo, but the temporal progression of
patterning is normal. Proc Natl Acad Sci USA 2006; 103: 99189923.
66 Joza N, Oudit GY, Brown D, Benit P, Kassiri Z, Vahsen N et al. Muscle-specic
loss of apoptosis-inducing factor leads to mitochondrial dysfunction, skeletal
muscle atrophy, and dilated cardiomyopathy. Mol Cell Biol 2005; 25:
1026110272.
67 Klein JA, Longo-Guess CM, Rossmann MP, Seburn KL, Hurd RE, Frankel WN et al.
The harlequin mouse mutation downregulates apoptosis-inducing factor. Nature
2002; 419: 367374.
68 Cheung EC, Joza N, Steenaart NA, McClellan KA, Neuspiel M, McNamara S et al.
Dissociating the dual roles of apoptosis-inducing factor in maintaining
mitochondrial structure and apoptosis. EMBO J 2006; 25: 40614073.
69 Miramar MD, Costantini P, Ravagnan L, Saraiva LM, Haouzi D, Brothers G et al.
NADH oxidase activity of mitochondrial apoptosis-inducing factor. J Biol Chem
2001; 276: 1639116398.
70 van Empel VP, Bertrand AT, van der Nagel R, Kostin S, Doevendans PA, Crijns HJ
et al. Downregulation of apoptosis-inducing factor in harlequin mutant mice
sensitizes the myocardium to oxidative stress-related cell death and pressure
overload-induced decompensation. Circ Res 2005; 96: e92e101.
71 Krantic S, Mechawar N, Reix S, Quirion R. Apoptosis-inducing factor: a matter of
neuron life and death. Prog Neurobiol 2007; 81: 179196.
72 Chinta SJ, Rane A, Yadava N, Andersen JK, Nicholls DG, Polster BM. Reactive
oxygen species regulation by AIF- and complex I-depleted brain mitochondria.
Free Radic Biol Med 2009; 46: 939947.
73 Ohsato T, Ishihara N, Muta T, Umeda S, Ikeda S, Mihara K et al. Mammalian
mitochondrial endonuclease G. Digestion of R-loops and localization in
intermembrane space. Eur J Biochem 2002; 269: 57655770.
74 Uren RT, Dewson G, Bonzon C, Lithgow T, Newmeyer DD, Kluck RM. Mito-
chondrial release of pro-apoptotic proteins: electrostatic interactions can hold
cytochrome c but not Smac/DIABLO to mitochondrial membranes. J Biol Chem
2005; 280: 22662274.
75 Li LY, Luo X, Wang X. Endonuclease G is an apoptotic DNase when released from
mitochondria. Nature 2001; 412: 9599.
76 Zanna C, Ghelli A, Porcelli AM, Martinuzzi A, Carelli V, Rugolo M. Caspase-
independent death of Lebers hereditary optic neuropathy cybrids is driven by
energetic failure and mediated by AIF and Endonuclease G. Apoptosis 2005; 10:
9971007.
77 Wu Y, Dong M, Toepfer NJ, Fan Y, Xu M, Zhang J. Role of endonuclease G in
neuronal excitotoxicity in mice. Neurosci Lett 2004; 364: 203207.
78 Basnakian AG, Apostolov EO, Yin X, Abiri SO, Stewart AG, Singh AB et al.
Endonuclease G promotes cell death of non-invasive human breast cancer cells.
Exp Cell Res 2006; 312: 41394149.
79 Schafer P, Scholz SR, Gimadutdinow O, Cymerman IA, Bujnicki JM, Ruiz-Carrillo A
et al. Structural and functional characterization of mitochondrial EndoG, a sugar
non-specic nuclease which plays an important role during apoptosis. J Mol Biol
2004; 338: 217228.
80 Ishihara Y, Shimamoto N. Involvement of endonuclease G in nucleosomal DNA
fragmentation under sustained endogenous oxidative stress. J Biol Chem 2006;
281: 67266733.
81 McDermott-Roe C, Ye J, Ahmed R, Sun XM, Seran A, Ware J et al. Endonuclease
G is a novel determinant of cardiac hypertrophy and mitochondrial function.
Nature 2011; 478: 114118.
82 Cote J, Ruiz-Carrillo A. Primers for mitochondrial DNA replication generated by
endonuclease G. Science 1993; 261: 765769.
83 Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, Takahashi R. A serine pro-
tease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing
cell death. Mol Cell 2001; 8: 613621.
84 Yang QH, Church-Hajduk R, Ren J, Newton ML, Du C. Omi/HtrA2 catalytic clea-
vage of inhibitor of apoptosis (IAP) irreversibly inactivates IAPs and facilitates
caspase activity in apoptosis. Genes Dev 2003; 17: 14871496.
85 Vande Walle L, Van Damme P, Lamkan M, Saelens X, Vandekerckhove J,
Gevaert K et al. Proteome-wide identication of HtrA2/Omi substrates.
J Proteome Res 2007; 6: 10061015.
86 Jones JM, Datta P, Srinivasula SM, Ji W, Gupta S, Zhang Z et al. Loss of Omi
mitochondrial protease activity causes the neuromuscular disorder of mnd2
mutant mice. Nature 2003; 425: 721727.
87 Martins LM, Morrison A, Klupsch K, Fedele V, Moisoi N, Teismann P et al.
Neuroprotective role of the Reaper-related serine protease HtrA2/Omi revealed
by targeted deletion in mice. Mol Cell Biol 2004; 24: 98489862.
88 Strauss KM, Martins LM, Plun-Favreau H, Marx FP, Kautzmann S, Berg D et al. Loss
of function mutations in the gene encoding Omi/HtrA2 in Parkinsons disease.
Hum Mol Genet 2005; 14: 20992111.
89 Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes
cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell
2000; 102: 3342.
90 Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE et al. Identi-
cation of DIABLO, a mammalian protein that promotes apoptosis by binding to
and antagonizing IAP proteins. Cell 2000; 102: 4353.
91 Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X et al. Structural basis of IAP
recognition by Smac/DIABLO. Nature 2000; 408: 10081012.
92 Okada H, Suh WK, Jin J, Woo M, Du C, Elia A et al. Generation and character-
ization of Smac/DIABLO-decient mice. Mol Cell Biol 2002; 22: 35093517.
93 Cheng J, Zhu Y, He S, Lu Y, Chen J, Han B et al. Functional mutation of SMAC/
DIABLO, encoding a mitochondrial proapoptotic protein, causes human pro-
gressive hearing loss DFNA64. Am J Hum Genet 2011; 89: 5666.
94 Flanagan L, Sebastia J, Tuffy LP, Spring A, Lichawska A, Devocelle M et al. XIAP
impairs Smac release from the mitochondria during apoptosis. Cell Death Dis
2010; 1: e49.
95 Hsu YT, Wolter KG, Youle RJ. Cytosol-to-membrane redistribution of Bax and Bcl-
X(L) during apoptosis. Proc Natl Acad Sci USA 1997; 94: 36683672.
96 Burge D, Youle L, McIntosh N. An audit of transfers for neonatal surgical care in
England in 2007. Arch Dis Child Fetal Neonatal Ed 2009; 94: F290F293.
97 Whelan RS, Konstantinidis K, Wei AC, Chen Y, Reyna DE, Jha S et al. Bax regulates
primary necrosis through mitochondrial dynamics. Proc Natl Acad Sci USA 2012;
109: 65666571.
98 Jones RG, Bui T, White C, Madesh M, Krawczyk CM, Lindsten T et al. The
proapoptotic factors Bax and Bak regulate T Cell proliferation through
control of endoplasmic reticulum Ca(2 ) homeostasis. Immunity 2007; 27:
268280.
99 Karbowski J, Cronin CJ, Seah A, Mendel JE, Cleary D, Sternberg PW. Conservation
rules, their breakdown, and optimality in Caenorhabditis sinusoidal locomotion.
J Theor Biol 2006; 242: 652669.
100 Boohaker RJ, Zhang G, Carlson AL, Nemec KN, Khaled AR. BAX supports the
mitochondrial network, promoting bioenergetics in nonapoptotic cells. Am J
Physiol Cell Physiol 2011; 300: C1466C1478.
101 Pegoraro L, Palumbo A, Erikson J, Falda M, Giovanazzo B, Emanuel BS et al.
A 14;18 and an 8;14 chromosome translocation in a cell line derived from an
acute B-cell leukemia. Proc Natl Acad Sci USA 1984; 81: 71667170.
102 Chipuk JE, Bouchier-Hayes L, Green DR. Mitochondrial outer membrane per-
meabilization during apoptosis: the innocent bystander scenario. Cell Death
Differ 2006; 13: 13961402.
103 Akao Y, Otsuki Y, Kataoka S, Ito Y, Tsujimoto Y. Multiple subcellular localization of
bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane,
and mitochondrial membranes. Cancer Res 1994; 54: 24682471.
104 Chen ZX, Pervaiz S. Bcl-2 induces pro-oxidant state by engaging mitochondrial
respiration in tumor cells. Cell Death Differ 2007; 14: 16171627.
105 Chen ZX, Pervaiz S. Involvement of cytochrome c oxidase subunits Va and Vb in
the regulation of cancer cell metabolism by Bcl-2. Cell Death Differ 2010; 17:
408420.
106 Low IC, Kang J, Pervaiz S. Bcl-2: a prime regulator of mitochondrial redox
metabolism in cancer cells. Antioxid Redox Signal 2011; 15: 29752987.
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2710
Oncogene (2013) 2703 2711 & 2013 Macmillan Publishers Limited
107 Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA et al.
bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic
cell death. Cell 1993; 74: 597608.
108 Motoyama N, Wang F, Roth KA, Sawa H, Nakayama K, Nakayama K et al. Massive
cell death of immature hematopoietic cells and neurons in Bcl-x-decient mice.
Science 1995; 267: 15061510.
109 Vander Heiden MG, Li XX, Gottleib E, Hill RB, Thompson CB, Colombini M. Bcl-xL
promotes the open conguration of the voltage-dependent anion channel and
metabolite passage through the outer mitochondrial membrane. J Biol Chem
2001; 276: 1941419419.
110 Karbowski M, Norris KL, Cleland MM, Jeong SY, Youle RJ. Role of Bax and Bak in
mitochondrial morphogenesis. Nature 2006; 443: 658662.
111 Levine B, Sinha S, Kroemer G. Bcl-2 family members: dual regulators of apoptosis
and autophagy. Autophagy 2008; 4: 600606.
112 Hickman JA, Hardwick JM, Kaczmarek LK, Jonas EA. Bcl-xL inhibitor ABT-737 reveals
a dual role for Bcl-xL in synaptic transmission. J Neurophysiol 2008; 99: 15151522.
113 Alavian KN, Li H, Collis L, Bonanni L, Zeng L, Sacchetti S et al. Bcl-xL regulates
metabolic efciency of neurons through interaction with the mitochondrial
F1FO ATP synthase. Nat Cell Biol 2011; 13: 12241233.
114 Chen YB, Aon MA, Hsu YT, Soane L, Teng X, McCaffery JM et al. Bcl-xL regulates
mitochondrial energetics by stabilizing the inner membrane potential. J Cell Biol
2011; 195: 263276.
115 Jeong SY, Gaume B, Lee YJ, Hsu YT, Ryu SW, Yoon SH et al. Bcl-x(L) sequesters its
C-terminal membrane anchor in soluble, cytosolic homodimers. EMBO J 2004;
23: 21462155.
116 Opferman JT, Iwasaki H, Ong CC, Suh H, Mizuno S, Akashi K et al. Obligate role of
anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science 2005;
307: 11011104.
117 Opferman JT, Letai A, Beard C, Sorcinelli MD, Ong CC, Korsmeyer SJ. Develop-
ment and maintenance of B and T lymphocytes requires antiapoptotic MCL-1.
Nature 2003; 426: 671676.
118 Arbour N, Vanderluit JL, Le Grand JN, Jahani-Asl A, Ruzhynsky VA, Cheung EC
et al. Mcl-1 is a key regulator of apoptosis during CNS development and after
DNA damage. J Neurosci 2008; 28: 60686078.
119 Rinkenberger JL, Horning S, Klocke B, Roth K, Korsmeyer SJ. Mcl-1
deciency results in peri-implantation embryonic lethality. Genes Dev 2000; 14:
2327.
120 Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J et al. The
landscape of somatic copy-number alteration across human cancers. Nature
2010; 463: 899905.
121 Wuilleme-Toumi S, Robillard N, Gomez P, Moreau P, Le Gouill S, Avet-Loiseau H
et al. Mcl-1 is overexpressed in multiple myeloma and associated with relapse
and shorter survival. Leukemia 2005; 19: 12481252.
122 Wei G, Twomey D, Lamb J, Schlis K, Agarwal J, Stam RW et al. Gene
expression-based chemical genomics identies rapamycin as a modulator
of MCL1 and glucocorticoid resistance. Cancer Cell 2006; 10:
331342.
123 Perciavalle RM, Stewart DP, Koss B, Lynch J, Milasta S, Bathina M et al. Anti-
apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochon-
drial fusion to respiration. Nat Cell Biol 2012; 14: 575583.
124 Velours J, Dautant A, Salin B, Sagot I, Brethes D. Mitochondrial F1F0-ATP
synthase and organellar internal architecture. Int J Biochem Cell Biol 2009; 41:
17831789.
125 Gray MW, Burger G, Lang BF. The origin and early evolution of mitochondria.
Genome Biol 2001; 2: REVIEWS1018.
126 Galluzzi L, Joza N, Tasdemir E, Maiuri MC, Hengartner M, Abrams JM et al. No
death without life: vital functions of apoptotic effectors. Cell Death Differ 2008;
15: 11131123.
127 Yip KW, Reed JC. Bcl-2 family proteins and cancer. Oncogene 2008; 27:
63986406.
128 Tsujimoto Y, Cossman J, Jaffe E, Croce CM. Involvement of the bcl-2 gene in
human follicular lymphoma. Science 1985; 228: 14401443.
Roles of apoptotic proteins in mitochondrial activity
SM Kilbride and JHM Prehn
2711
& 2013 Macmillan Publishers Limited Oncogene (2013) 2703 2711