Preservation of Mitochondrial Structure and Function after Bid- or Bax-Mediated Cytochrome c Release

Proapoptotic members of the Bcl-2 protein family, including Bid and Bax, can activate apoptosis by directly interacting with mitochondria to cause cytochrome c translocation from the intermembrane space into the cytoplasm, thereby triggering Apaf-1–mediated caspase activation. Under some circumstances, when caspase activation is blocked, cells can recover from cytochrome c translocation; this suggests that apoptotic mitochondria may not always suffer catastrophic damage arising from the process of cytochrome c release. We now show that recombinant Bid and Bax cause complete cytochrome c loss from isolated mitochondria in vitro, but preserve the ultrastructure and protein import function of mitochondria, which depend on inner membrane polarization. We also demonstrate that, if caspases are inhibited, mitochondrial protein import function is retained in UV-irradiated or staurosporine-treated cells, despite the complete translocation of cytochrome c. Thus, Bid and Bax act only on the outer membrane, and lesions in the inner membrane occurring during apoptosis are shown to be secondary caspase-dependent events

The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release

Bax Crystal Structures Reveal How BH3 Domains Activate Bax and Nucleate Its Oligomerization to Induce Apoptosis

SummaryIn stressed cells, apoptosis ensues when Bcl-2 family members Bax or Bak oligomerize and permeabilize the mitochondrial outer membrane. Certain BH3-only relatives can directly activate them to mediate this pivotal, poorly understood step. To clarify the conformational changes that induce Bax oligomerization, we determined crystal structures of BaxΔC21 treated with detergents and BH3 peptides. The peptides bound the Bax canonical surface groove but, unlike their complexes with prosurvival relatives, dissociated Bax into two domains. The structures define the sequence signature of activator BH3 domains and reveal how they can activate Bax via its groove by favoring release of its BH3 domain. Furthermore, Bax helices α2–α5 alone adopted a symmetric homodimer structure, supporting the proposal that two Bax molecules insert their BH3 domain into each other’s surface groove to nucleate oligomerization. A planar lipophilic surface on this homodimer may engage the membrane. Our results thus define critical Bax transitions toward apoptosis

Disordered clusters of Bak dimers rupture mitochondria during apoptosis

During apoptosis, Bak and Bax undergo major conformational change and form symmetric dimers that coalesce to perforate the mitochondrial outer membrane via an unknown mechanism. We have employed cysteine labelling and linkage analysis to the full length of Bak in mitochondria. This comprehensive survey showed that in each Bak dimer the N-termini are fully solvent-exposed and mobile, the core is highly structured, and the C-termini are flexible but restrained by their contact with the membrane. Dimer-dimer interactions were more labile than the BH3:groove interaction within dimers, suggesting there is no extensive protein interface between dimers. In addition, linkage in the mobile Bak N-terminus (V61C) specifically quantified association between dimers, allowing mathematical simulations of dimer arrangement. Together, our data show that Bak dimers form disordered clusters to generate lipidic pores. These findings provide a molecular explanation for the observed structural heterogeneity of the apoptotic pore

Translocation of a Bak C-Terminus Mutant from Cytosol to Mitochondria to Mediate Cytochrome c Release: Implications for Bak and Bax Apoptotic Function

One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear.To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1.Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax

Mechanisms by which Bak and Bax permeabilise mitochondria during apoptosis

Mitochondrial outer membrane permeabilisation (MOMP) is the point of no return in many forms of apoptotic cell death. The killing effect of MOMP is twofold; it both initiates a proteolytic cascade of pro-apoptotic enzymes and damages mitochondrial function. Accordingly, prevention of MOMP can rescue cells from death. It is clear that either Bak or Bax, which are Bcl-2 family members, are required for MOMP to occur; however, the pore complexes that are formed by Bak and Bax remain poorly defined in terms of their composition, size, number and structure, as well as the mechanism by which they are regulated by other Bcl-2 family members. We recently reported that a key step leading to Bak homo-oligomerisation following an apoptotic stimulus involves transient exposure of the Bak BH3 domain before it binds to the hydrophobic groove of another activated Bak molecule to form a novel symmetric dimer. To form the higher-order oligomers that probably constitute the apoptotic pore complex, Bak dimers then interact via regions away from the BH3 domain and groove. The BH3:groove interaction within Bak homodimers supports a general model to explain the associations between Bcl-2 family members. In this Commentary, we discuss the implications of these findings for the regulation of apoptosis by Bcl-2 family proteins

Basic residues in the C-segment are necessary for Bak stability and function.

<p>(<b>A</b>) C-terminal sequence of Bak variants indicating which basic residues in the C-segment that were substituted with serine. (<b>B</b>) Substitution of basic residues in the C-segment reduces Bak proapoptotic function. <i>bak^−/−bax^−/−</i> MEFs expressing Bak, BakRRS, BakRSS or BakSSS were left untreated, or treated with UV or etoposide for 24 h. Percentage cell death is expressed as the mean ± SEM from three independent experiments. Statistical significance for treatment when compared to Bak; *p<0.05, **p<0.01. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for β-actin as a loading control. (<b>C</b>) Substitution of basic residues in the C-segment destabilizes Bak. Cells from (B) were incubated with cycloheximide for up to 24 h and cell lysates immunoblotted for Bak, and for β-actin as a loading control. Note that due to low expression of BakSSS, 4-fold total protein was loaded onto the gels. (<b>D</b>) Substitution of basic residues does not prevent targeting to membranes. Cells from (B) were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and for the cytosolic marker HSP70. Results are representative of two or more independent experiments.</p

Bak regulation is independent of initial subcellular localization.

<p>(<b>A</b>) Schematic of Bak-mediated apoptosis initiated by Noxa-Mcl-1 signalling in MEFs. The four prosurvival proteins expressed in MEF (Mcl-1, Bcl-x_L, Bcl-w and Bcl-2) are depicted, together with their preferential binding of BH3-only proteins (Noxa, Bim and Bad) and of Bak and Bax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Willis1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Chen1" target="_blank">[46]</a>. The Noxa-Mcl-1-Bak pathway to apoptosis is indicated (<i>bold</i>). (<b>B</b>) Expression of Bim_S^NOXA preferentially mediates apoptosis via either Bak or Bak/BaxCS. <i>bak^−/−bax^−/−</i>MEFs expressing the indicated Bak and Bax variants were retrovirally infected with Bim_S or with Bim_S containing the Bad or Noxa BH3 domains (Bim_S^BAD and Bim_S^NOXA). Percentage cell death at 36 h (normalized to the efficiency of infection) is expressed as the mean ± SEM of three independent experiments. Statistical significance for treatment when compared to Bak; **p<0.01.</p

A TM:groove interaction forms in cytosolic Bax and in cytosolic Bak/BaxCS.

<p>(<b>A</b>) Schematic of Bax converting from a TM:groove to a TM:membrane conformation during apoptosis. Bax is shown as surface representation (<i>orange</i>) with the hydrophobic surface groove highlighted (<i>white</i>). The Bax TM domain is represented (<i>blue</i>) and the C-segment (KKMG) has the sidechains shown. Residues proposed to interact during TM:groove interaction are highlighted (S184 and D98; <i>red</i>). Images were generated in Pymol using the RCSB Protein Data Bank file 1F16 for Bax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Suzuki1" target="_blank">[38]</a>. (<b>B</b>) Mutations in both the TM domain and the groove alter mitochondrial targeting. Cytosolic and membrane fractions from <i>bak^−/−bax^−/−</i> MEFs expressing the indicated Bax or Bak/BaxCS cysteine mutants were immunoblotted for Bax, Bak/BaxCS, or HSP70. (<b>C</b>) A TM:groove interaction forms in cytosolic Bax and in cytosolic Bak/BaxCS. Cytosolic and membrane fractions from cells in (B) were incubated with oxidant (CuPhe) and electrophoresed under non-reducing conditions (<i>upper</i>) or reducing conditions (+2ME, <i>lower</i>) and immunoblotted for Bax or Bak/BaxCS. Intramolecular cysteine linkage (D98C:S184C in Bax; S117C:Q202C in Bak/BaxCS) results in faster migration under non-reducing conditions (<i>X-link</i>). (<b>D</b>) Etoposide treatment decreases TM:groove interaction in membrane-associated Bak/BaxCS. Cells expressing the Bak/BaxCS S117C/Q202C variant were incubated with or without etoposide for 24 h. Cytosolic and membrane fractions were incubated with CuPhe, electrophoresed under non-reducing conditions, and immunoblotted for Bak. (<b>E</b>) tBid treatment decreases the TM:groove interaction in membrane-associated Bak/BaxCS. Cells in (D) were permeabilized and incubated with or without tBid (100 nM). Cytosolic and membrane fractions were separated and then incubated with CuPhe, electrophoresed under non-reducing conditions, and immunoblotted for Bak. (<b>F</b>) The TM domain of Bak/BaxCS inserts into membranes following tBid. Permeabilized cells were treated with or without tBid as in (E), then incubated with or without IASD. Membrane fractions were electrophoresed under reducing conditions and immunoblotted for Bak. Results are representative of two or more independent experiments.</p