News Reader.info

ABSTRACT Apoptosis, a mechanism for programmed cell death, has key roles in human health and disease. Many signals for cellular life and death are regulated by the BCL-2 family proteins and

converge at mitochondria, where cell fate is ultimately decided. The BCL-2 family includes both pro-life (e.g. BCL-XL) and pro-death (e.g. BAX, BAK) proteins. Previously, it was thought that

a balance between these opposing proteins, like a simple ‘rheostat’, could control the sensitivity of cells to apoptotic stresses. Later, this rheostat concept had to be extended, when it

became clear that BCL-2 family proteins regulate each other through a complex network of bimolecular interactions, some transient and some relatively stable. Now, studies have shown that the

apoptotic circuitry is even more sophisticated, in that BCL-2 family interactions are spatially dynamic, even in nonapoptotic cells. For example, BAX and BCL-XL can shuttle between the

cytoplasm and the mitochondrial outer membrane (MOM). Upstream signaling pathways can regulate the cytoplasmic–MOM equilibrium of BAX and thereby adjust the sensitivity of cells to apoptotic

stimuli. Thus, we can view the MOM as the central locale of a dynamic life–death rheostat. BAX invariably forms extensive homo-oligomers after activation in membranes. However, recent

studies, showing that activated BAX monomers determine the kinetics of MOM permeabilization (MOMP), perturb the lipid bilayer and form nanometer size pores, pose questions about the role of

the oligomerization. Other lingering questions concern the molecular mechanisms of BAX redistribution between MOM and cytoplasm and the details of BAX/BAK–membrane assemblies. Future studies

need to delineate how BCL-2 family proteins regulate MOMP, in concert with auxiliary MOM proteins, in a dynamic membrane environment. Technologies aimed at elucidating the structure and

function of the full-length proteins in membranes are needed to illuminate some of these critical issues. SIMILAR CONTENT BEING VIEWED BY OTHERS MECHANISMS OF BCL-2 FAMILY PROTEINS IN

MITOCHONDRIAL APOPTOSIS Article 12 July 2023 APOPTOTIC PRIMING IS DEFINED BY THE DYNAMIC EXCHANGE OF BCL-2 PROTEINS BETWEEN MITOCHONDRIA AND CYTOSOL Article Open access 18 May 2022 LINC

COMPLEX PROTEIN NESPRIN-2 HAS PRO-APOPTOTIC ACTIVITY VIA BCL-2 FAMILY PROTEINS Article Open access 15 January 2024 FACTS * BH3-only proteins (e.g. BID, BIM and PUMA) promote MOM integration

of BAX/BAK, leading to MOMP, which typically commits the cell to death. * The cytoprotective activity of BCL-XL is exerted through interactions with BAX/BAK and BH3-only proteins. *

Non-activated BAX and BCL-XL cycle between cytosolic and MOM-associated states. In some cells, they can cause each other to retrotranslocate from the MOM back to the cytoplasm. However, in

other situations, BCL-XL stabilizes BAX association with the MOM. * MOM-resident proteins, both identified and unidentified, have been proposed to facilitate the MOM permeabilizing function

of BAX. OPEN QUESTIONS * How do BAX and BAK permeabilize the MOM? * How do the pro-survival family members, e.g. BCL-XL, protect against cell death? * How do BCL-2 family proteins function

together in the MOM? * Which MOM proteins facilitate retrotranslocation and permeabilization and by what mechanisms? RECENT RESULTS * Membrane-inserted BAX monomers control the kinetics of

permeabilization, induce the formation of lipidic pores and significantly distort phospholipid bilayers. * BAX activation in the MOM triggers the assembly of a non-BAX ‘catalyst’ complex,

which then facilitates BAX pore formation. * Some of the initial conformational events in BAX and BAK activation have been identified by structural studies. BAX and BAK can both be activated

directly by certain BH3-only proteins. For BAK, this activation involves BH3 domain binding to the ‘canonical groove’, but for BAX, activator BH3 domain binding occurs at a different

surface. * Survival signals can promote BAX retrotranslocation from the MOM to the cytoplasm (reducing the amount of BAX in the MOM); on the other hand, the loss of survival signals causes

enhanced MOM accumulation of BAX and sensitizes cells to death signals. APOPTOSIS AND BCL-2 FAMILY PROTEINS Apoptosis is an active, intracellular cell death program that is common to nearly

all higher eukaryotic cell types.1 As a counterbalance to cellular proliferation, this process helps to maintain cellular homeostasis. Consequently, its deregulation can seriously affect

health. Abnormally suppressed apoptosis has key roles in many human diseases, including tumor development and tumor cell resistance to chemotherapy. The relevance of apoptosis to human

health has propelled it to the forefront of biomedical research, with the hope that understanding its molecular mechanism will translate into new therapeutic approaches. Early discoveries

concerning the BCL-2 oncogene product, in particular that it suppresses cell death, counteracts the pro-apoptotic action of mitochondria and represents a family of anti- and pro-apoptotic

proteins,2, 3, 4, 5, 6 provided some of the first insights into the molecular basis of programmed cell death and paved the way for subsequent studies that shape our current understanding of

apoptosis. It is now well established that the BCL-2 family proteins are the central regulators of the mitochondrial cell-intrinsic apoptotic pathway. Although many kinds of stresses can

induce apoptosis via diverse signaling pathways, these signals often converge at the mitochondrial outer membrane (MOM), where cell fate is ultimately decided. BCL-2 family proteins can have

functions at non-mitochondrial sites (e.g. the endoplasmic reticulum). Nevertheless, perhaps the most prominent roles of these proteins are to promote or inhibit MOM permeabilization

(MOMP), serving as a molecular ‘rheostat’ that regulates the mitochondrial pathway to apoptosis.7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 MOMP induced by BAX and BAK allows

cytotoxic proteins to escape from the mitochondrial intermembrane space into the cytoplasm, leading to caspase activation and apoptosis. MOMP also leads to progressive mitochondrial

dysfunction, which causes energy depletion and cell death even when caspases are inactive. This means that cells can recover from MOMP only under exceptional circumstances.22, 23, 24 Even in

cases where apoptosis is initiated by ligation of death receptors, through the extrinsic pathway, the upstream signals often require amplification by the mitochondrial pathway. Thus, MOMP

is frequently the decisive event preceding cell death. BCL-2 family proteins are distinguished by four conserved BCL-2 homology (BH1–BH4) domains. In particular, the BH3 domain is invariably

present and has an essential role in regulating cell death. BCL-2 family proteins engage in a complex network of heterodimeric interactions that collectively make life and death decisions

for the cell. Anti-apoptotic family members (e.g. BCL-2 and BCL-XL) protect the cell against apoptotic stimuli, whereas pro-death proteins (BAX and BAK – one or the other is needed for

apoptosis) actively kill the cell by inducing MOMP. Their functions are regulated by a third class of BCL-2 family members, the BH3-only proteins. One such BH3-only protein, BID, provides a

key link between the extrinsic and mitochondrial pathways to cell death because it is activated when extracellular stress cues, transmitted through cell surface death receptors, activate the

protease Caspase-8, which cleaves BID into an inactive N-terminal fragment and a critical pro-death C-terminal fragment (tBID). tBID and the other BH3-only proteins BIM and PUMA have two

main functions: first, they can form stable heterodimers with anti-apoptotic family members such as BCL-XL, neutralizing their protective activity, and second, they can transiently interact

with BAX and BAK, directly activating them.10, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 Other BH3-only proteins (e.g. BAD) can only suppress anti-apoptotic family members.

The anti-apoptotic family members act in two ways: to antagonize the multidomain proteins BAX and BAK and to sequester the BH3-only proteins. WHAT REGULATES THE INTERPLAY OF SOLUBLE

CYTOPLASMIC BCL-2 FAMILY PROTEINS WITH MEMBRANES? Structural and biochemical studies have identified some of the early events in BAX and BAK occurring upon interaction with BH3 domain

peptides.35, 36, 38, 39 However, interactions among full-length BCL-2 family proteins normally take place in the membrane environment. This is obviously true for BAK, which is permanently

membrane-resident. Nevertheless, some experimental alterations of the BAK molecule increase its solubility or decrease its MOM-association, even causing it to be inducibly MOM-translocated,

much like BAX.40 With regard to BAX, experiments in protein-free, cardiolipin-containing liposomes showed that tBID becomes recruited to the membrane before BAX,41 suggesting that initially,

MOM-associated tBID might promote BAX accumulation in the MOM by evoking a conformational change needed for membrane integration. The MOM protein Mtch2 has been proposed as a tBID-binding

receptor in the MOM.42 However, a recent study43 has shown that Mtch2 accelerates the separation of the N- and C-terminal fragments of Caspase-8-cleaved BID and promotes a complex process of

tBID integration in the membrane. This would then trigger BAX recruitment to the MOM and activation. However, activation by tBID and other BH3-only proteins appears not to be the only

trigger for BAX translocation. Recent studies in epithelial cells44 confirmed other studies in fibroblasts45, 46 showing that, under nonapoptotic conditions, BAX exists in equilibrium

between cytoplasmic and MOM-associated forms. A recent study by Schellenberg _et al._44 now shows that in epithelial cells, detachment from the extracellular matrix causes a loss of

adhesion-dependent survival signaling; somehow, this reduces the rate of BAX membrane dissociation, thereby shifting the equilibrium in favor of MOM localization. However, if cells are

allowed to reattach before they become committed to apoptosis, MOM-associated BAX can move back to the cytoplasm. This reversibility of MOM localization suggests that BAX can be stably

associated with mitochondria even in a non-activated state. Together, the retrotranslocation studies show that at least one form of BAX recruitment to the MOM does not appear to depend on

full apoptotic activation by BH3-only proteins. How BAX reversibly associates with the MOM is still not understood. As the BAX S184V mutant displays a lower rate of dissociation from the

MOM, Schellenberg _et al._44 suggest that the C-terminal _α_-helix of wt BAX can interact weakly with the membrane. _In vitro_, BAX does interact transiently with liposomes, causing it to

display the 6A7 epitope.47 This membrane-induced BAX conformational change is not inhibited by BCL-XL. Mixing BAX alone with liposomes does not lead to BAX oligomerization or even to

measurable BAX membrane association, as measured by a float-up assay. For stable BAX integration, full activation (e.g. by tBID) is required. In principle, membrane integration, which would

involve at least the hydrophobic C terminus and potentially other regions of this protein, is likely irreversible, as the energy barrier for removal of a transmembrane helix from the

membrane to water would be very high. Within cells, the MOM-association equilibrium could instead involve a reversible interaction of BAX with resident MOM proteins, especially as

adhesion-dependent signaling alters this equilibrium. We can well imagine that BAX (retro-)translocation and activation in whole cells involves multiple steps and still-unknown mechanisms.

Anoikis in mammary epithelial cells involves an hours-long interval between BAX translocation to mitochondria and BAX activation. In this setting, instead of the previously reported ability

of BCL-XL and BAX to retrotranslocate each other,45 BCL-XL stabilizes BAX association with the MOM. Perhaps this reflects co-integration of these two proteins, as in the ‘embedded together’

scenario previously described by Andrews _et al._14 Similarly, Llambi _et al._48 found that when BAX-induced MOMP is blocked by BCL-XL, both proteins become membrane-integrated. We do not

understand what determines co-integration _versus_ co-retrotranslocation. In particular, the structural details of how these proteins interact in the membrane environment are yet unknown.

However, one recent study dealt with the contribution of the C-terminal helix of BCL-XL, which is necessary for the ability of BCL-XL to retrotranslocate BAX.46 Interestingly, replacing the

C terminus of BCL-XL with the corresponding portion of BAX retained the property of the chimeric molecule to interact reversibly with the MOM, but eliminated the ability to retrotranslocate

BAX. MOLECULAR BASIS FOR BCL-2 FAMILY PROTEIN FUNCTION The molecular structures of BCL-XL,49 BAX50 and BID,51, 52 all determined in solution by NMR, provided the initial framework for

understanding the functions and interactions of the BCL-2 family proteins. Despite their opposing functions, all three proteins adopt a common fold, with two central, more hydrophobic

_α_-helices forming a hairpin surrounded by six to eight more polar _α_-helices (Figure 1). This structure appears to be shared by all other pro- and anti-apoptotic BCL-2 family members.53,

54 The prototypical BCL-2 fold also resembles that of the pore-forming domains of bacterial toxins, suggestive of a common pore-forming function in which the two central helices (_α_5-_α_6

in BAX) are likely important for membrane insertion.55, 56, 57 The BH1, BH2 and BH3 domains form a hydrophobic groove on the protein surface that is conserved across both anti- and

pro-apoptotic BCL-2 family members and is crucial for mediating their interactions. In BCL-XL, this canonical groove (binding cleft) serves as the binding site for the BH3 domains of its

pro-apoptotic BCL-2 partners, including BAX/BAK and BID.58 This interaction produces a stable complex that is thought to protect against cell death by effectively sequestering the death

agonists. In the cytosolic form of BAX, however, the groove is normally occupied by BAX’s own hydrophobic C-terminal helix (_α_9), and certain BH3-only proteins (BID, BIM and perhaps PUMA)

appear to interact with a different BAX site, diametrically opposed to the canonical BH3-binding groove.35 This interaction is weak, but it is needed to trigger BAX activation. BAK lacks

this alternative site and appears to be activated by binding the BH3 domain of either BID or BIM to its canonical groove.38, 39, 59 Recent structural studies show that truncated BAXΔC and

BAKΔC (lacking the hydrophobic C-terminal helix) can bind activator BH3 peptides through their respective BH3-binding clefts.39, 60 BAXΔC treated with the detergent CHAPS and BID, BIM or BAX

BH3 peptides forms a domain-swapped dimer60 similar to that formed by BCL-XLΔC exposed to high pH or heat.61, 62 Dimers induced by the detergent octylmaltoside yielded indistinguishable

structures without the bound peptide. In these domain-swapped dimers, the _α_5–_α_6 helices of each monomer form one long extended helix and interact with the _α_6–_α_5 helices of the second

monomer, forming a planar surface that is proposed to engage the membrane. A mutant form of BAK, rendered soluble by mutagenesis of its C terminus, also binds BH3 peptides through its

surface cleft,38 as does a highly truncated form of BAX spanning only helices _α_2 to _α_5.60 BH3 ligand association with the BAX or BAK surface cleft is distinct from its interaction with a

diametrically opposed BH3 interaction site observed by Gavathiotis _et al._35 for intact, monomeric BAX in solution, suggesting that BAX and BAK may each be activated by direct interaction

with a BH3 ligand at distinct molecular sites, and that activation likely involves multiple steps.63 All of these studies suggest very interesting hypotheses about BCL-2 protein function.

However, because they were performed with extensively truncated proteins, in the presence of detergents and far removed from the natural environment of the phospholipid bilayer membrane –

conditions that can produce distortions in many integral membrane and membrane-associated proteins64 – it is unclear where and how these structures fit into the apoptosis pathways.

Cell-based and biophysical studies focused on BAK and BAX activation indicate that the BH3–groove interaction is essential for BAK65 and BAX66 oligomerization. However, the structures of the

domain-swapped dimers appear at odds with data on the interfaces in BAX oligomers formed during apoptosis66, 67, 68 and incompatible with a model in which helices _α_5 and _α_6 insert into

the membrane during permeabilization. The similarity of the BAXΔC structures with pro-survival proteins, together with the facts that BAX inserts in membranes can form membrane pores as a

monomer and is likely to unfold on the membrane (see below), indicate that swapped-dimer structures may not be directly involved in the activation pathway. However, they may represent some

off-pathway conformations, possibly related to the reversible retrotranslocation process. The latter would be an attractive possibility because the blockage of helices _α_5 and _α_6 through

the domain-swapping mechanism would prevent insertion of these helices into the membrane and thus prevent the subsequent, irreversible MOMP. Structural studies on full-length proteins in the

presence of a fully functional membrane bilayer environment will be needed to obtain a full picture of BAX/BAK activation. HOW DOES THE BCL-2 FAMILY REGULATE MOMP? For MOMP to occur, BAX

and BAK require direct activation by a ‘direct activator’ BH3-only protein, for example, BID, BIM or PUMA.34, 35, 36, 38, 39, 59, 69, 70 Anti-apoptotic BCL-2 family proteins can block MOMP

both by sequestering BH3-only proteins and by neutralizing BAX/BAK. This inhibitory cytoprotective function can in turn be neutralized by an excess of ‘indirect activator’ BH3-only proteins

(e.g. BAD), which bind the anti-apoptotic BCL-2 proteins, thereby liberating the pro-apoptotic direct activator BH3-only proteins. Some chemotherapeutic drugs currently under development

(e.g. ABT-263, Navitoclax) act in a similar manner to antagonize the interactions of BCL-XL and other anti-apoptotic relatives with their pro-apoptotic partners, thereby promoting

apoptosis.71, 72 After BAX activation via a transient hit-and-run interaction with a direct activator BH3-only protein such as tBID, BAX typically translocates from its soluble cytoplasmic

state to a MOM-integrated state. BAK is constitutively membrane-resident, but also becomes activated by BH3-only proteins in a similar manner. Activation of BAX and BAK leads to complete

membrane insertion of these proteins and then to MOMP, which generally commits the cell to death. The early stages of BAX and BAK activation have been analyzed elegantly by structure-guided

mutational analysis.35, 36, 38, 39 However, as the structures of membrane-inserted BAX and BAK are not known, the exact conformational changes in these proteins associated with MOMP activity

are still unclear. Indeed, the limited structural information that is available for the membrane-associated states of BCL-XL, BAX and tBID50, 73, 74, 75, 76, 77, 78 provide only a glimpse

of how BCL-2 proteins interact with membranes. tBID has been shown to adopt a stable helical conformation in lipids, with its helices parallel to the membrane surface and no transmembrane

helix insertion.75, 79 BAX-derived peptides corresponding to _α_5–_α_6 readily insert into membranes and mediate the formation of protein-lipidic pores.80, 81, 82, 83 The conformation of

BCL-XL in lipid bilayers may involve membrane embedding of the BCL-XL helical hairpin (_α_5-_α_6) as well as helix _α_1.73, 74 However, these results were obtained using truncated BCL-XL

lacking the hydrophobic C terminus (BCL-XLΔC), or using detergents to either solubilize or activate a conformational change, manipulations that can significantly distort protein structure

and function.64 Indeed, a number of recent reports have demonstrated the importance of studying membrane-associated proteins, including BCL-2 family proteins, in a lipid membrane

environment.84, 85 BAX MONOMERS ARE A DRIVING FORCE FOR MEMBRANE PERTURBATION Major conformational rearrangements and extensive homo-oligomerization of BAX and BAK are widely assumed to be

necessary for pore formation and MOMP.50 However, a detailed spatiotemporal molecular mechanism for these events remains elusive. Until recently, it has not been possible experimentally to

separate BAX/BAK oligomerization from the pore-formation activity involved in MOMP, and so researchers have assumed that oligomerization is a requirement. However, two independent

investigations have now converged on the notion that integrated BAX monomers are sufficient to perturb the lipid bilayer. In recent electron cryomicroscopy (cryo-EM) studies, Volkmann and

co-workers78 dissected the structural correlates of BAX membrane insertion, in a situation where oligomerization is physically impossible. This was accomplished through the use of

nanometer-scale phospholipid bilayer islands (nanodiscs), each capable of accommodating only a single BAX molecule.78 The three-dimensional reconstructions of these membrane assemblies

(Figure 2) demonstrate that in the presence of a BID BH3 peptide, an individual BAX molecule can insert into the nanodisc membrane, perturb the lipid bilayer and form a pore of ∼3.5 nm in

diameter. While the precise structure of this BAX pore will have to be determined, its monomeric state is dictated by the small, discrete size of the lipid bilayer nanodiscs, which can only

accommodate a single membrane-inserted BAX. Native gels, fluorescence and immunoblotting data all confirmed that BAX was monomeric in the nanodisc under the conditions used for imaging.

Interestingly, the three-dimensional structure of this full-length BAX-membrane assembly contained no discernible, compact, extra globular density that would account for a folded BAX monomer

globular structure associated with the nanodiscs (Figure 2). This is in stark contrast to other recent nanodisc studies where integrated proteins such as integrin receptors, anthrax toxin

or SNARE proteins are clearly visible.86, 87, 88 This observation suggests that BAX is unfolded on top of the nanodisc surface (Figure 3), presumably with its amphiphilic portions inserted

into the nanodiscs in a similar way to the ‘umbrella model’ proposed for colicin, where the outer helices of the globular structure rearrange to form the ‘umbrella’ resting on the membrane

surface, while the central hydrophobic helical hairpin (corresponding to _α_5–_α_6 in BAX) inserts across the membrane to form the ‘umbrella handle’.89, 90, 91 Such unfolding is consistent

with the finding that a substantial structural reorganization of BAX occurs during BH3-triggered activation.36 In contrast to these small BAX monomer-induced pores, Bax-dependent

permeabilization of the MOM involves a much greater permeability of the membrane, allowing the efflux of macromolecules such as Smac/DIABLO dimers or very large dextran molecules.10 How this

permeabilization occurs is a key question. Judging from the sizes of the released macromolecules, up to 2000 kD, we can infer that MOMP involves a large-scale rearrangement of the membrane.

Paradoxically, however, traditional EM methods did not reveal any alterations in the MOM.10 This suggested the possibility that MOMP arises not from the formation of fixed proteinaceous

pores but rather from pores that are lipidic (and not easily preserved during embedding for EM). Indeed, when Bax-induced pores in small unilamellar vesicles92 and in bacteria93 were later

imaged using gentler cryo-EM techniques, they were seen to be large and heterogeneous (25–100 nm), much like those produced by bacterial toxins. Similar large Bax-induced pores have now been

imaged in purified MOMs by cryo-EM (Newmeyer and Kuwana, personal communication). These and other observations80, 82, 94 argue that MOMP is the result of lipidic pore formation. Indeed, the

pores produced in nanodiscs by single Bax molecules are already lipidic in nature. The number of helices that can conceivably insert into the membrane from a single BAX molecule (four) is

too small to allow complete protein lining of a 3.5-nm diameter pore78 (Figure 3). The formation of lipidic pores induced by BAX monomers in the initial stages of MOMP is an attractive

alternative to models where the initial pores are protein-lined and formed by ordered BAX oligomers. The minimal protein requirement of monomer-induced pores potentially favors faster

induction of the pro-death signaling cascade, similar to the efficient process implemented by bacterial toxins. The joining of the initial monomer-induced lipidic pores at later stages of

MOMP to increase pore size can be achieved with far less conformational adaption than the joining of protein-lined pores would require. Independent evidence from biochemical kinetic analysis

now implies that Bax monomers are important for the formation even of large protein-conducting pores. Newmeyer and co-workers95 showed that MOMP kinetics are linearly dependent on BAX

concentration and show neither cooperativity nor saturability relative to BAX (Figures 4 and 5). This implies that BAX monomers, not oligomers, drive the kinetics of pore formation.

Together, the results of the nanodisc cryo-EM and kinetic experiments argue that monomeric membrane-inserted BAX is the key functional unit responsible for initiating MOM pore formation, MOM

destabilization and MOMP, leading to apoptosis. How do the small membrane perturbations produced by individual Bax monomers lead to the formation of supramolecular pores? The mechanism is

still unclear. One possibility is that an abundance of integrated BAX monomers in a local membrane region could produce enough curvature stress to destabilize the membrane. Several reports

have argued that curvature stress is the ultimate mechanism promoting the formation of protein-conducting membrane pores, and quite possibly the process can involve a cooperation between

protein and lipid components to sculpt the membrane.94, 96, 97, 98, 99, 100 Indeed, in cryo-EM images, Bax-induced pores in liposomes92 and MOM vesicles (Kuwana and Newmeyer, personal

communication) are frequently adjoined by negatively curved membrane regions. This strongly suggests a pore formation or enlargement mechanism involving curvature stress. An alternative

model was proposed by Martinez _et al._101 after observing a BAX-dependent stepwise opening of high-conductance membrane channels. Possibly, integrated BAX monomers could cluster together in

a quantum fashion to form ever-larger pores ringed by BAX molecules. These models need not be mutually exclusive. Cryo-EM imaging of extended lipid bilayer membranes sufficiently large to

accommodate several BAX molecules, together with additional structure determination approaches, will ultimately be needed to determine the membrane-associated structures of membrane-embedded

BAX oligomers at a high resolution. Imaging of colloidal gold-labeled BAX molecules in the MOM, if feasible, might also provide information about the organization of BAX molecules around

the lipidic pores. How BAX oligomerization might have a role in this process is still unclear. Based on the universal finding so far that BAX/BAK mutants blocking oligomerization also block

MOMP, it is tempting to conclude that oligomerization is required for pore formation. One common model is that the pores are enlarged through a process of Bax autoactivation, in which BAX

oligomers at the pore rim catalyze the integration of free BAX monomers and their addition to the ends of existing oligomers. However, to explain the linear response of MOMP kinetics to BAX

concentrations, the effect of oligomerization, if any, would have to be non-rate-limiting, that is, very rapid.95 This is formally possible. Nevertheless, a requirement for oligomerization

has not been proven. Although oligomerization is an inevitable byproduct of _α_-helix insertion, it might not contribute to membrane destabilization, but perhaps only to the autoactivation

of free Bax monomers. Oligomers could have a function different from pore formation, for example, to regulate mitochondrial dynamics. Alternatively, oligomers could be inert or even

inhibitory to pore formation. In this way, oligomers could act as a brake to limit the runaway amplification of a sub-threshold apoptotic signal. If so, cell death would require the

sustained production of integrated BAX/BAK monomers to promote pore formation and enlargement. As mentioned, BAX by itself can form pores in liposome membranes, upon activation by a

BH3-domain peptide or a BH3-only protein. However, to what extent this intrinsic activity explains MOM permeabilization in apoptotic cells remains unclear. Highlighting this uncertainty, the

recent study by Kushnareva _et al._95 showed that native MOMs are more responsive to low concentrations of BAX than liposomes and display more complex, biphasic permeabilization kinetics

(Figure 5). This enhanced BAX response requires the participation of heat-labile MOM proteins. Furthermore, these authors found that, in order to fit the measured kinetic curves, it was

necessary to consider a two-stage reaction scheme. In the first stage, activated BAX triggers the assembly of a non-Bax oligomeric complex. In the second stage, this complex catalyzes the

BAX-dependent formation of membrane pores. Interestingly, the catalyst formation reaction exhibited a membrane phase-like transition near 28 °C, suggesting that it involves a localized

membrane-remodeling event. The behavior of this catalyst complex is reminiscent of the membrane-distorting activities of Drp1 and Bif-1, two proteins previously proposed to have roles in

MOMP.99, 102, 103, 104 However, Kushnareva _et al._95 found no evidence supporting a role for these proteins in Bax-induced permeabilization of isolated MOM vesicles. Chemical compounds

(mDivi-1 analogs) that had been shown to inhibit Drp1’s yeast ortholog, Dnm1, also inhibit MOMP in mammalian cells,105 and some mDivi-1 analogs blocked BAX-induced pore formation in rat

liver and Xenopus MOM vesicles. Kushnareva _et al._95 were unable to detect Drp1 in their MOM preparations, suggesting that the catalyst protein whose existence was inferred by their studies

is different from Drp1. Although Bif-1 was weakly detectable in rat liver MOMs, it was below the limits of immunoblot detection in Xenopus egg MOM vesicles, which display the same type of

MOMP kinetics as rat liver MOM vesicles. Naturally, these results do not exclude the possibility that Drp1 or Bif-1 participate in MOMP within cells, as these proteins could be recruited

from the cytoplasm. However, the cell-free studies argue that Drp1 and Bif-1 are not essential for MOMP and that another unidentified MOM-resident protein catalyzes BAX-induced MOMP. In

general, the auxiliary roles of non-BCL-2 family proteins in MOMP remain to be clarified. Nevertheless, the simplest hypothesis is that such proteins facilitate BAX’s intrinsic pore-forming

function. HOW DOES BCL-XL PROTECT AGAINST CELL DEATH? BCL-XL protects cells both by interacting with BAX to prevent its membrane integration and by interacting with BID and other BH3-only

proteins, to sequester their pro-death activity. The C terminus of BCL-XL contains a clear MOM-targeting motif and shares sequence homology with those of BAX, BAK and other MOM-targeted

BCL-2 proteins. The lipid bilayer membrane strongly influences BCL-2 protein–protein interactions,20, 48, 84, 106 and several lines of evidence show that the hydrophobic C terminus of BCL-XL

is important for regulating both the interaction of BCL-XL with its partners and its membrane integration. However, most structural studies have focused on truncated forms of BCL-XL lacking

the hydrophobic C terminus, to promote solubility, and little is known about how BCL-XL interacts with the membrane or with membrane-associated BAX and BID. Solid-state NMR studies suggest

an ‘umbrella’ model of BCL-XL membrane integration,74 as proposed for colicins.89, 90, 91 However, the studies with BCL-XL were performed with truncated protein, and the presence of the C

terminus is likely to have a significant effect on the membrane-associated conformation of this protein. The structure of BAX is the only one determined for an intact, multidomain BCL-2

protein including the hydrophobic C terminus.50 In solution, this membrane-targeting module folds into BAX’s BH3-binding cleft, suggesting that it may act as a regulatory switch for

intermolecular interactions. Recent structural studies with moderately truncated forms of anti-apoptotic BCL-W indicate that it also may adopt a similar conformation.107, 108 Thus, the C

terminus of BCL-XL is also likely to interact with the BH3-binding cleft, with important consequences for the interactions of BCL-XL with its partners and with membranes. Recently, BCL-XL

and BAX have been reported to cycle between cytosolic and MOM-associated states, and each protein can cause the other to retrotranslocate from the MOM back to the cytoplasm.45, 46 The

mechanism of retrotranslocation is still unknown. The simplest hypothesis is that the mutual membrane repulsion of BCL-XL and BAX results from the intrinsic interactions of these proteins in

the membrane. Although the presence of an activating BH3-only protein would be required for the full integration of BAX in the membrane, even the unactivated BAX molecule can interact

weakly with the membrane, perhaps through its C terminus.47, 109 If so, retrotranslocation may then result from competitive displacement of this weak membrane interaction of the Bax C

terminus by interaction with the hydrophobic groove of a BCL-XL molecule (which would be exposed upon the transient interaction of this BCL-XL molecule with the membrane). Afterwards, the

resulting BAX–BCL-XL heterodimer would dissociate from the membrane and then further dissociate into soluble monomers. Similarly, retrotranslocation of BCL-XL could involve a similar

displacement of its C-terminal weak membrane interaction by the hydrophobic groove of Bax. In the presence of a BH3-only protein such as tBID, the situation is different, as BAX and BCL-XL

can become irreversibly membrane-integrated. In this situation, how BCL-XL acts is still unclear, as in some cases BCL-XL can block the membrane integration of BAX, while in other cases,

BCL-XL and BAX can both be membrane-integrated. For example, in the nanodisc cryo-EM study, Volkmann _et al._78 found that BCL-XL abolished the integration of BAX into nanodisc membranes in

the presence of BID BH3 peptides. This agrees with other work10, 34, 95, 110, 111, 112 showing that BCL-XL or BCL-2 can block membrane recruitment and integration of BAX, even in the

presence of a BAX-activating signal. In other studies, membrane integration of both BCL-XL and BAX (i.e. the ‘embedded together’ scenario14) was observed.44, 48 Perhaps co-integration

requires a balanced stoichiometry of BCL-XL and BAX.113 Alternatively, it could require the participation of a MOM protein that facilitates insertion of BAX and BCL-XL, similar to the

recently reported role of Mtch2 in facilitating the membrane integration of tBID.43 OUTLOOK Understanding the process of BAX/BAK-dependent membrane pore formation remains a Holy Grail of the

apoptosis field. Recent publications significantly advance our understanding of the mitochondrion-dependent intrinsic pathway to apoptosis, but also emphasize the need for high-resolution

structural studies aimed at determining the molecular conformations and interactions of key BCL-2 family proteins without truncations and within a membrane environment. This kind of detailed

molecular understanding is needed to define the intrinsic activities of this protein family and also to facilitate analysis of the roles of cofactor proteins, once these are identified. The

evidence is overwhelming that the membrane environment is critical for preserving the structural and functional integrity of both integral and membrane-associated proteins. This is entirely

consistent with Anfinsen’s hypothesis that protein conformation ‘is determined by the totality of inter-atomic interactions and hence by the amino acid sequence in a given environment’.109

For membrane-associated proteins such as the BCL-2 family proteins, the ‘given environment’ of the phospholipid bilayer is essential for preserving native structure and function.

ABBREVIATIONS * MOM: mitochondrial outer membrane * MOMP: MOM permeabilization REFERENCES * Kerr JF, Wyllie AH, Currie AR . Apoptosis: a basic biological phenomenon with wide-ranging

implications in tissue kinetics. _Br J Cancer_ 1972; 26: 239–257. CAS  PubMed  PubMed Central  Google Scholar  * Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM . Cloning of the

chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. _Science_ 1984; 226: 1097–1099. CAS  PubMed  Google Scholar  * Vaux DL, Cory S, Adams JM . Bcl-2 gene

promotes haemopoietic cell survival and cooperates with c-Myc to immortalize pre-B cells. _Nature_ 1988; 335: 440–442. CAS  PubMed  Google Scholar  * 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: 597–608. CAS  PubMed  Google Scholar  *

Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ . Bcl-2 functions in an antioxidant pathway to prevent apoptosis. _Cell_ 1993; 75: 241–251. CAS  PubMed  Google Scholar  *

Newmeyer DD, Farschon DM, Reed JC . Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. _Cell_ 1994; 79:

353–364. CAS  PubMed  Google Scholar  * Korsmeyer SJ . Regulators of cell death. _Trends Genet_ 1995; 11: 101–105. CAS  PubMed  Google Scholar  * Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer

DD . The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. _Science_ 1997; 275: 1132–1136. CAS  PubMed  Google Scholar  * Yang J, Liu X, Bhalla K,

Kim CN, Ibrado AM, Cai J _et al_. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. _Science_ 1997; 275: 1129–1132. CAS  PubMed  Google Scholar  * Kuwana

T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R _et al_. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. _Cell_ 2002; 111:

331–342. CAS  PubMed  Google Scholar  * Kuwana T, Newmeyer DD . Bcl-2-family proteins and the role of mitochondria in apoptosis. _Curr Opin Cell Biol_ 2003; 15: 691–699. CAS  PubMed  Google

Scholar  * Danial NN, Korsmeyer SJ . Cell death: critical control points. _Cell_ 2004; 116: 205–219. CAS  PubMed  Google Scholar  * Adams JM, Cory S . Bcl-2-regulated apoptosis: mechanism

and therapeutic potential. _Curr Opin Immunol_ 2007; 19: 488–496. CAS  PubMed  PubMed Central  Google Scholar  * Leber B, Lin J, Andrews DW . Embedded together: the life and death

consequences of interaction of the Bcl-2 family with membranes. _Apoptosis_ 2007; 12: 897–911. CAS  PubMed  PubMed Central  Google Scholar  * Chipuk JE, Green DR . How do BCL-2 proteins

induce mitochondrial outer membrane permeabilization? _Trends Cell Biol_ 2008; 18: 157–164. CAS  PubMed  PubMed Central  Google Scholar  * Degterev A, Yuan J . Expansion and evolution of

cell death programmes. _Nature reviews_ 2008; 9: 378–390. CAS  PubMed  Google Scholar  * Reed JC . Bcl-2-family proteins and hematologic malignancies: history and future prospects. _Blood_

2008; 111: 3322–3330. CAS  PubMed  PubMed Central  Google Scholar  * Youle RJ, Strasser A . The BCL-2 protein family: opposing activities that mediate cell death. _Nat Rev Mol Cell Biol_

2008; 9: 47–59. CAS  PubMed  Google Scholar  * Yao Y, Marassi FM . BAX and BAK caught in the act. _Mol Cell_ 2009; 36: 353–354. CAS  PubMed  PubMed Central  Google Scholar  * Leber B, Lin J,

Andrews DW . Still embedded together binding to membranes regulates Bcl-2 protein interactions. _Oncogene_ 2010; 29: 5221–5230. CAS  PubMed  PubMed Central  Google Scholar  * Martinou JC,

Youle RJ . Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. _Dev Cell_ 2011; 21: 92–101. CAS  PubMed  PubMed Central  Google Scholar  * Colell A, Ricci JE, Tait S,

Milasta S, Maurer U, Bouchier-Hayes L _et al_. GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation. _Cell_ 2007; 129: 983–997.

CAS  PubMed  Google Scholar  * Lartigue L, Kushnareva Y, Seong Y, Lin H, Faustin B, Newmeyer DD . Caspase-independent mitochondrial cell death results from loss of respiration, not cytotoxic

protein release. _Mol Biol Cell_ 2009; 20: 4871–4884. CAS  PubMed  PubMed Central  Google Scholar  * Tait SW, Parsons MJ, Llambi F, Bouchier-Hayes L, Connell S, Munoz-Pinedo C _et al_.

Resistance to caspase-independent cell death requires persistence of intact mitochondria. _Dev Cell_ 2010; 18: 802–813. CAS  PubMed  PubMed Central  Google Scholar  * Wang K, Yin XM, Chao

DT, Milliman CL, Korsmeyer SJ . BID: a novel BH3 domain-only death agonist. _Genes Dev_ 1996; 10: 2859–2869. CAS  PubMed  Google Scholar  * Gross A, Yin XM, Wang K, Wei MC, Jockel J,

Milliman C _et al_. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. _J Biol

Chem_ 1999; 274: 1156–1163. CAS  PubMed  Google Scholar  * Li H, Zhu H, Xu CJ, Yuan J . Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. _Cell_

1998; 94: 491–501. CAS  PubMed  Google Scholar  * Luo X, Budihardjo I, Zou H, Slaughter C, Wang X . Bid a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in

response to activation of cell surface death receptors. _Cell_ 1998; 94: 481–490. CAS  PubMed  Google Scholar  * Eskes R, Desagher S, Antonsson B, Martinou JC . Bid induces the

oligomerization and insertion of Bax into the outer mitochondrial membrane. _Mol Cell Biol_ 2000; 20: 929–935. CAS  PubMed  PubMed Central  Google Scholar  * Korsmeyer SJ, Wei MC, Saito M,

Weiler S, Oh KJ, Schlesinger PH . Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. _Cell Death Differ_ 2000; 7:

1166–1173. CAS  PubMed  Google Scholar  * Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T _et al_. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and

BAK-mediated mitochondrial apoptosis. _Mol Cell_ 2001; 8: 705–711. CAS  PubMed  Google Scholar  * Scorrano L, Ashiya M, Buttle K, Weiler S, Oakes SA, Mannella CA _et al_. A distinct pathway

remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. _Dev Cell_ 2002; 2: 55–67. CAS  PubMed  Google Scholar  * Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds

MG _et al_. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. _Mol Cell_ 2005; 17: 393–403. CAS  PubMed  Google Scholar

  * Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR _et al_. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane

permeabilization both directly and indirectly. _Mol Cell_ 2005; 17: 525–535. CAS  PubMed  Google Scholar  * Gavathiotis E, Suzuki M, Davis ML, Pitter K, Bird GH, Katz SG _et al_. BAX

activation is initiated at a novel interaction site. _Nature_ 2008; 455: 1076–1081. CAS  PubMed  PubMed Central  Google Scholar  * Gavathiotis E, Reyna DE, Davis ML, Bird GH, Walensky LD .

BH3-triggered structural reorganization drives the activation of proapoptotic BAX. _Mol Cell_ 2010; 40: 481–492. CAS  PubMed  PubMed Central  Google Scholar  * Du H, Wolf J, Schafer B,

Moldoveanu T, Chipuk JE, Kuwana T . BH3 domains other than Bim and Bid can directly activate Bax/Bak. _J Biol Chem_ 2011; 286: 491–501. CAS  PubMed  Google Scholar  * Leshchiner ES, Braun

CR, Bird GH, Walensky LD . Direct activation of full-length proapoptotic BAK. _Proc Natl Acad Sci USA_ 2013; 110: E986–E995. CAS  PubMed  PubMed Central  Google Scholar  * Moldoveanu T,

Grace CR, Llambi F, Nourse A, Fitzgerald P, Gehring K _et al_. BID-induced structural changes in BAK promote apoptosis. _Nat Struct Mol Biol_ 2013; 20: 589–597. CAS  PubMed  PubMed Central 

Google Scholar  * Ferrer PE, Frederick P, Gulbis JM, Dewson G, Kluck RM . Translocation of a Bak C-terminus mutant from cytosol to mitochondria to mediate cytochrome C release: implications

for Bak and Bax apoptotic function. _PLoS One_ 2012; 7: e31510. CAS  PubMed  PubMed Central  Google Scholar  * Lovell JF, Billen LP, Bindner S, Shamas-Din A, Fradin C, Leber B _et al_.

Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. _Cell_ 2008; 135: 1074–1084. CAS  PubMed  Google Scholar  * Zaltsman Y,

Shachnai L, Yivgi-Ohana N, Schwarz M, Maryanovich M, Houtkooper RH _et al_. MTCH2/MIMP is a major facilitator of tBID recruitment to mitochondria. _Nat Cell Biol_ 2010; 12: 553–562. CAS 

PubMed  PubMed Central  Google Scholar  * Shamas-Din A, Bindner S, Zhu W, Zaltsman Y, Campbell C, Gross A _et al_. tBid undergoes multiple conformational changes at the membrane required for

Bax activation. _J Biol Chem_ 2013; 288: 22111–22127. CAS  PubMed  PubMed Central  Google Scholar  * Schellenberg B, Wang P, Keeble JA, Rodriguez-Enriquez R, Walker S, Owens TW _et al_. Bax

exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming. _Mol Cell_ 2013; 49: 959–971. CAS  PubMed  PubMed Central  Google Scholar  * Edlich F,

Banerjee S, Suzuki M, Cleland MM, Arnoult D, Wang C _et al_. Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol. _Cell_ 2011; 145: 104–116. CAS  PubMed  PubMed Central 

Google Scholar  * Todt F, Cakir Z, Reichenbach F, Youle RJ, Edlich F . The C-terminal helix of Bcl-x(L) mediates Bax retrotranslocation from the mitochondria. _Cell Death Differ_ 2013; 20:

333–342. CAS  PubMed  Google Scholar  * Yethon JA, Epand RF, Leber B, Epand RM, Andrews DW . Interaction with a membrane surface triggers a reversible conformational change in Bax normally

associated with induction of apoptosis. _J Biol Chem_ 2003; 278: 48935–48941. CAS  PubMed  Google Scholar  * Llambi F, Moldoveanu T, Tait SW, Bouchier-Hayes L, Temirov J, McCormick LL _et

al_. A unified model of mammalian BCL-2 protein family interactions at the mitochondria. _Mol Cell_ 2011; 44: 517–531. CAS  PubMed  PubMed Central  Google Scholar  * Muchmore SW, Sattler M,

Liang H, Meadows RP, Harlan JE, Yoon HS _et al_. X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. _Nature_ 1996; 381: 335–341. CAS  PubMed  Google Scholar  *

Suzuki M, Youle RJ, Tjandra N . Structure of Bax: coregulation of dimer formation and intracellular localization. _Cell_ 2000; 103: 645–654. CAS  PubMed  Google Scholar  * Chou JJ, Li H,

Salvesen GS, Yuan J, Wagner G . Solution structure of BID, an intracellular amplifier of apoptotic signaling. _Cell_ 1999; 96: 615–624. CAS  PubMed  Google Scholar  * McDonnell JM, Fushman

D, Milliman CL, Korsmeyer SJ, Cowburn D . Solution structure of the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists. _Cell_ 1999; 96: 625–634. CAS 

PubMed  Google Scholar  * Fesik SW . Insights into programmed cell death through structural biology. _Cell_ 2000; 103: 273–282. CAS  PubMed  Google Scholar  * Petros AM, Olejniczak ET, Fesik

SW . Structural biology of the Bcl-2 family of proteins. _Biochim Biophys Acta_ 2004; 1644: 83–94. CAS  PubMed  Google Scholar  * Minn AJ, Velez P, Schendel SL, Liang H, Muchmore SW, Fesik

SW _et al_. Bcl-x(L) forms an ion channel in synthetic lipid membranes. _Nature_ 1997; 385: 353–357. CAS  PubMed  Google Scholar  * Sobko AA, Kotova EA, Antonenko YN, Zakharov SD, Cramer WA

. Effect of lipids with different spontaneous curvature on the channel activity of colicin E1: evidence in favor of a toroidal pore. _FEBS Lett_ 2004; 576: 205–210. CAS  PubMed  Google

Scholar  * Annis MG, Soucie EL, Dlugosz PJ, Cruz-Aguado JA, Penn LZ, Leber B _et al_. Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. _EMBO J_

2005; 24: 2096–2103. CAS  PubMed  PubMed Central  Google Scholar  * Sattler M, Liang H, Nettesheim D, Meadows RP, Harlan JE, Eberstadt M _et al_. Structure of Bcl-xL-Bak peptide complex:

recognition between regulators of apoptosis. _Science_ 1997; 275: 983–986. CAS  PubMed  Google Scholar  * Dai H, Smith A, Meng XW, Schneider PA, Pang YP, Kaufmann SH . Transient binding of

an activator BH3 domain to the Bak BH3-binding groove initiates Bak oligomerization. _J Cell Biol_ 2011; 194: 39–48. CAS  PubMed  PubMed Central  Google Scholar  * Czabotar PE, Westphal D,

Dewson G, Ma S, Hockings C, Fairlie WD _et al_. Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis. _Cell_ 2013; 152: 519–531.

CAS  PubMed  Google Scholar  * O'Neill JW, Manion MK, Maguire B, Hockenbery DM . BCL-XL dimerization by three-dimensional domain swapping. _J Mol Biol_ 2006; 356: 367–381. CAS  PubMed 

Google Scholar  * Denisov AY, Sprules T, Fraser J, Kozlov G, Gehring K . Heat-induced dimerization of BCL-xL through alpha-helix swapping. _Biochemistry_ 2007; 46: 734–740. CAS  PubMed 

Google Scholar  * Walensky LD . Direct BAKtivation. _Nat Struct Mol Biol_ 2013; 20: 536–538. CAS  PubMed  Google Scholar  * Zhou HX, Cross TA . Influences of membrane mimetic environments on

membrane protein structures. _Annu Rev Biophys_ 2013; 42: 361–392. CAS  PubMed  PubMed Central  Google Scholar  * Dewson G, Kratina T, Sim HW, Puthalakath H, Adams JM, Colman PM _et al_. To

trigger apoptosis, Bak exposes its BH3 domain and homodimerizes via BH3:groove interactions. _Mol Cell_ 2008; 30: 369–380. CAS  PubMed  Google Scholar  * Bleicken S, Classen M, Padmavathi

PV, Ishikawa T, Zeth K, Steinhoff HJ _et al_. Molecular details of Bax activation, oligomerization, and membrane insertion. _J Biol Chem_ 2010; 285: 6636–6647. CAS  PubMed  Google Scholar  *

Zhang Z, Zhu W, Lapolla SM, Miao Y, Shao Y, Falcone M _et al_. Bax forms an oligomer via separate, yet interdependent, surfaces. _J Biol Chem_ 2010; 285: 17614–17627. CAS  PubMed  PubMed

Central  Google Scholar  * Dewson G, Ma S, Frederick P, Hockings C, Tan I, Kratina T _et al_. Bax dimerizes via a symmetric BH3:groove interface during apoptosis. _Cell Death Differ_ 2012;

19: 661–670. CAS  PubMed  Google Scholar  * Ren D, Tu HC, Kim H, Wang GX, Bean GR, Takeuchi O _et al_. BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell

death program. _Science_ 2010; 330: 1390–1393. CAS  PubMed  PubMed Central  Google Scholar  * Gavathiotis E, Walensky LD . Tracking BAX once its trigger is pulled. _Cell Cycle_ 2011; 10:

868–870. CAS  PubMed  PubMed Central  Google Scholar  * Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA _et al_. An inhibitor of Bcl-2 family proteins induces

regression of solid tumours. _Nature_ 2005; 435: 677–681. CAS  PubMed  Google Scholar  * Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S _et al_. ABT-263: a potent and orally

bioavailable Bcl-2 family inhibitor. _Cancer Res_ 2008; 68: 3421–3428. CAS  PubMed  Google Scholar  * Losonczi JA, Olejniczak ET, Betz SF, Harlan JE, Mack J, Fesik SW . NMR studies of the

anti-apoptotic protein Bcl-xL in micelles. _Biochemistry_ 2000; 39: 11024–11033. CAS  PubMed  Google Scholar  * Franzin CM, Choi J, Zhai D, Reed JC, Marassi FM . Structural studies of

apoptosis and ion transport regulatory proteins in membranes. _Magn Reson Chem_ 2004; 42: 172–179. CAS  PubMed  PubMed Central  Google Scholar  * Gong XM, Choi J, Franzin CM, Zhai D, Reed

JC, Marassi FM . Conformation of membrane-associated proapoptotic tBid. _J Biol Chem_ 2004; 279: 28954–28960. CAS  PubMed  Google Scholar  * Malia TJ, Wagner G . NMR structural investigation

of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl-xL. _Biochemistry_ 2007; 46: 514–525. CAS  PubMed  Google Scholar  * Denisov AY, Chen G, Sprules

T, Moldoveanu T, Beauparlant P, Gehring K . Structural model of the BCL-w-BID peptide complex and its interactions with phospholipid micelles. _Biochemistry_ 2006; 45: 2250–2256. CAS  PubMed

  Google Scholar  * Xu XP, Zhai D, Kim E, Swift M, Reed JC, Volkmann N _et al_. Three-dimensional Structure of Bax-mediated Pores in Membrane Bilayers. _Cell Death Dis_ 2013; 4: e683. CAS 

PubMed  PubMed Central  Google Scholar  * Oh KJ, Barbuto S, Meyer N, Kim RS, Collier RJ, Korsmeyer SJ . Conformational changes in BID, a pro-apoptotic BCL-2 family member, upon membrane

binding. A site-directed spin labeling study. _J Biol Chem_ 2005; 280: 753–767. CAS  PubMed  Google Scholar  * Epand RF, Martinou JC, Montessuit S, Epand RM . Transbilayer lipid diffusion

promoted by Bax: implications for apoptosis. _Biochemistry_ 2003; 42: 14576–14582. CAS  PubMed  Google Scholar  * Garcia-Saez AJ, Coraiola M, Serra MD, Mingarro I, Muller P, Salgado J .

Peptides corresponding to helices 5 and 6 of Bax can independently form large lipid pores. _FEBS J_ 2006; 273: 971–981. CAS  PubMed  Google Scholar  * Qian S, Wang W, Yang L, Huang HW .

Structure of transmembrane pore induced by Bax-derived peptide: evidence for lipidic pores. _Proc Natl Acad Sci USA_ 2008; 105: 17379–17383. CAS  PubMed  PubMed Central  Google Scholar  *

Fuertes G, Garcia-Saez AJ, Esteban-Martin S, Gimenez D, Sanchez-Munoz OL, Schwille P _et al_. Pores formed by Baxalpha5 relax to a smaller size and keep at equilibrium. _Biophys J_ 2010; 99:

2917–2925. CAS  PubMed  PubMed Central  Google Scholar  * Bogner C, Leber B, Andrews DW . Apoptosis: embedded in membranes. _Curr Opin Cell Biol_ 2010; 22: 845–851. CAS  PubMed  Google

Scholar  * Cross TA, Sharma M, Yi M, Zhou HX . Influence of solubilizing environments on membrane protein structures. _Trends Biochem Sci_ 2011; 36: 117–125. CAS  PubMed  Google Scholar  *

Katayama H, Wang J, Tama F, Chollet L, Gogol EP, Collier RJ _et al_. Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles. _Proc Natl Acad

Sci USA_ 2010; 107: 3453–3457. CAS  PubMed  PubMed Central  Google Scholar  * Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA _et al_. Recreation of the terminal events in

physiological integrin activation. _J Cell Biol_ 2010; 188: 157–173. CAS  PubMed  PubMed Central  Google Scholar  * Shi L, Shen QT, Kiel A, Wang J, Wang HW, Melia TJ _et al_. SNARE proteins:

one to fuse and three to keep the nascent fusion pore open. _Science_ 2012; 335: 1355–1359. CAS  PubMed  PubMed Central  Google Scholar  * Parker MW, Tucker AD, Tsernoglou D, Pattus F .

Insights into membrane insertion based on studies of colicins. _Trends Biochem Sci_ 1990; 15: 126–129. CAS  PubMed  Google Scholar  * Song HY, Cohen FS, Cramer WA . Membrane topography of

ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin. _J Bacteriol_ 1991; 173: 2927–2934. CAS  PubMed  PubMed Central  Google Scholar  * Shin YK,

Levinthal C, Levinthal F, Hubbell WL . Colicin E1 binding to membranes: time-resolved studies of spin-labeled mutants. _Science_ 1993; 259: 960–963. CAS  PubMed  Google Scholar  * Schafer B,

Quispe J, Choudhary V, Chipuk JE, Ajero TG, Du H _et al_. Mitochondrial outer membrane proteins assist Bid in Bax-mediated lipidic pore formation. _Mol Biol Cell_ 2009; 20: 2276–2285. CAS 

PubMed  PubMed Central  Google Scholar  * Pang X, Moussa SH, Targy NM, Bose JL, George NM, Gries C _et al_. Active Bax and Bak are functional holins. _Genes Dev_ 2011; 25: 2278–2290. CAS 

PubMed  PubMed Central  Google Scholar  * Terrones O, Antonsson B, Yamaguchi H, Wang HG, Liu J, Lee RM _et al_. Lipidic pore formation by the concerted action of proapoptotic BAX and tBID.

_J Biol Chem_ 2004; 279: 30081–30091. CAS  PubMed  Google Scholar  * Kushnareva Y, Andreyev AY, Kuwana T, Newmeyer DD . Bax activation initiates the assembly of a multimeric catalyst that

facilitates Bax pore formation in mitochondrial outer membranes. _PLoS Biol_ 2012; 10: e1001394. CAS  PubMed  PubMed Central  Google Scholar  * Campelo F, Fabrikant G, McMahon HT, Kozlov MM

. Modeling membrane shaping by proteins: focus on EHD2 and N-BAR domains. _FEBS Lett_ 2010; 584: 1830–1839. CAS  PubMed  Google Scholar  * Kozlov MM, McMahon HT, Chernomordik LV .

Protein-driven membrane stresses in fusion and fission. _Trends Biochem Sci_ 2010; 35: 699–706. CAS  PubMed  PubMed Central  Google Scholar  * McMahon HT, Kozlov MM, Martens S . Membrane

curvature in synaptic vesicle fusion and beyond. _Cell_ 2010; 140: 601–605. CAS  PubMed  Google Scholar  * Montessuit S, Somasekharan SP, Terrones O, Lucken-Ardjomande S, Herzig S,

Schwarzenbacher R _et al_. Membrane remodeling induced by the dynamin-related protein Drp1 stimulates Bax oligomerization. _Cell_ 2010; 142: 889–901. CAS  PubMed  PubMed Central  Google

Scholar  * Landeta O, Landajuela A, Gil D, Taneva S, Di Primo C, Sot B _et al_. Reconstitution of proapoptotic BAK function in liposomes reveals a dual role for mitochondrial lipids in the

BAK-driven membrane permeabilization process. _J Biol Chem_ 2011; 286: 8213–8230. CAS  PubMed  PubMed Central  Google Scholar  * Martinez-Caballero S, Dejean LM, Kinnally MS, Oh KJ, Mannella

CA, Kinnally KW . Assembly of the mitochondrial apoptosis-induced channel, MAC. _J Biol Chem_ 2009; 284: 12235–12245. CAS  PubMed  PubMed Central  Google Scholar  * Lee YJ, Jeong SY,

Karbowski M, Smith CL, Youle RJ . Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis. _Mol Biol Cell_ 2004; 15: 5001–5011. CAS  PubMed 

PubMed Central  Google Scholar  * Takahashi Y, Karbowski M, Yamaguchi H, Kazi A, Wu J, Sebti SM _et al_. Loss of Bif-1 suppresses Bax/Bak conformational change and mitochondrial apoptosis.

_Mol Cell Biol_ 2005; 25: 9369–9382. CAS  PubMed  PubMed Central  Google Scholar  * Etxebarria A, Terrones O, Yamaguchi H, Landajuela A, Landeta O, Antonsson B _et al_. Endophilin B1/Bif-1

stimulates BAX activation independently from its capacity to produce large scale membrane morphological rearrangements. _J Biol Chem_ 2009; 284: 4200–4212. CAS  PubMed  Google Scholar  *

Cassidy-Stone A, Chipuk JE, Ingerman E, Song C, Yoo C, Kuwana T _et al_. Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer

membrane permeabilization. _Dev Cell_ 2008; 14: 193–204. CAS  PubMed  PubMed Central  Google Scholar  * Garcia-Saez AJ, Ries J, Orzaez M, Perez-Paya E, Schwille P . Membrane promotes tBID

interaction with BCL(XL). _Nat Struct Mol Biol_ 2009; 16: 1178–1185. CAS  PubMed  Google Scholar  * Denisov AY, Madiraju MS, Chen G, Khadir A, Beauparlant P, Attardo G _et al_. Solution

structure of human BCL-w: modulation of ligand binding by the C-terminal helix. _J Biol Chem_ 2003; 278: 21124–21128. CAS  PubMed  Google Scholar  * Hinds MG, Lackmann M, Skea GL, Harrison

PJ, Huang DC, Day CL . The structure of Bcl-w reveals a role for the C-terminal residues in modulating biological activity. _EMBO J_ 2003; 22: 1497–1507. CAS  PubMed  PubMed Central  Google

Scholar  * Anfinsen CB . Principles that govern the folding of protein chains. _Science_ 1973; 181: 223–230. CAS  PubMed  Google Scholar  * Murphy KM, Streips UN, Lock RB . Bax membrane

insertion during Fas(CD95)-induced apoptosis precedes cytochrome c release and is inhibited by Bcl-2. _Oncogene_ 1999; 18: 5991–5999. CAS  PubMed  Google Scholar  * Murphy KM, Ranganathan V,

Farnsworth ML, Kavallaris M, Lock RB . Bcl-2 inhibits Bax translocation from cytosol to mitochondria during drug-induced apoptosis of human tumor cells. _Cell Death Differ_ 2000; 7:

102–111. CAS  PubMed  Google Scholar  * Murphy KM, Streips UN, Lock RB . Bcl-2 inhibits a Fas-induced conformational change in the Bax N terminus and Bax mitochondrial translocation. _J Biol

Chem_ 2000; 275: 17225–17228. CAS  PubMed  Google Scholar  * Billen LP, Kokoski CL, Lovell JF, Leber B, Andrews DW . Bcl-XL inhibits membrane permeabilization by competing with Bax. _PLoS

Biol_ 2008; 6: e147. PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by National Institutes of Health (NIH) grants P01-GM098412 (DH, NV),

P01-AI074805 and R01-GM100265 (FMM) and R01-GM62289 (DDN). AUTHOR INFORMATION Author notes * N Volkmann, F M Marassi, D D Newmeyer and D Hanein: All authors contributed equally to this

review. AUTHORS AND AFFILIATIONS * Bioinformatics and Systems Biology Program, Sanford Burnham Medical Research Institute, La Jolla, 92037, CA, USA N Volkmann & D Hanein * Apoptosis and

Cell Death Research Program, Sanford Burnham Medical Research Institute, La Jolla, 92037, CA, USA F M Marassi * Division of Immune Regulation, La Jolla Institute for Allergy and Immunology,

La Jolla, CA, USA D D Newmeyer Authors * N Volkmann View author publications You can also search for this author inPubMed Google Scholar * F M Marassi View author publications You can also

search for this author inPubMed Google Scholar * D D Newmeyer View author publications You can also search for this author inPubMed Google Scholar * D Hanein View author publications You can

also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to D Hanein. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest.

ADDITIONAL INFORMATION Edited by C Borner RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Volkmann, N., Marassi, F., Newmeyer, D. _et al._ The rheostat

in the membrane: BCL-2 family proteins and apoptosis. _Cell Death Differ_ 21, 206–215 (2014). https://doi.org/10.1038/cdd.2013.153 Download citation * Received: 03 July 2013 * Revised: 22

August 2013 * Accepted: 17 September 2013 * Published: 25 October 2013 * Issue Date: February 2014 * DOI: https://doi.org/10.1038/cdd.2013.153 SHARE THIS ARTICLE Anyone you share the

following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer

Nature SharedIt content-sharing initiative KEYWORDS * BCL-XL * BAX * BID * mitochondria * structure * membrane