Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size

Bleicken, Stephanie; Landeta, Olatz; Landajuela, Ane; Basáñez, Gorka; García‐Sáez, Ana J.

doi:10.1074/jbc.m113.512087

Cited by 134 publications

(153 citation statements)

References 62 publications

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“…Strikingly, the large number of coexisting species present in the membrane suggests that oligomers of different stoichiometry simultaneously contribute to membrane permeabilization. This may be a strategy of this kind of toxins to kill cells in a broad range of conditions (35) and is in line with a flexible pore structure, as recently shown for Bax and Bak (44). Importantly, our findings change the paradigm of how we understand membrane permeabilization by ␣-PFTs, so far focused on a unique oligomeric state as the predominant form of the toxin on the target membrane.…”

Section: Discussionmentioning

confidence: 81%

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging

Subburaj

Ros²,

Hermann

et al. 2015

Journal of Biological Chemistry

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Background:Equinatoxin II is a model ␣-pore-forming toxin that kills cells by porating the host plasma membrane. Results: On the membrane, equinatoxin II does not adopt a unique oligomeric state, but assembles into multiple coexisting species related to toxicity. Conclusion: Toxicity of Equinatoxin II depends on its assembly mechanism. Significance: A new molecular mechanism is proposed for ␣-pore-forming toxins action.

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Section: Discussionmentioning

confidence: 81%

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging

Subburaj

Ros²,

Hermann

et al. 2015

Journal of Biological Chemistry

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“…Linkage at Bak α9:α9 could link BH3:groove dimers to the higher-order oligomers that are associated with pore formation. 26,27 The dimers can also be linked via cysteine residues placed in α6, 18,24,25 and more recently in α3 or α5, 6,21 suggesting a variety of dimer arrangements in the high-order complexes.…”

Section: Discussionmentioning

confidence: 99%

“…7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation. 26,27 However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells, 28 whereas the Bax C terminus is either exposed 29 or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.…”

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confidence: 99%

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

et al. 2015

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Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore. Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins. 1-3 Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices. 4,5 They are activated by BH3-only proteins binding to the α2-α5 surface groove, 6-12 or for Bax, to the α1/α6 'rear pocket'. 13 Binding triggers dissociation of the latch domain (α6-α8) from the core domain (α2-α5), together with exposure of N-terminal epitopes and the BH3 domain. 6,7,14-16 The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers. 6,7,14,17,18 In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer. 19 In Bax, the α5 and α6 helices may insert into the MOM, 20 although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface. 7,[21][22][23] The dimers can be linked via cysteine residues placed in α6, 18,24,25 and more recently via cysteine residues in either α3 or α5, 6,21 allowing detection of the higherorder oligomers associated with pore formation. 26,27 However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the...

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“…The percentage of internalization of Rho-10-kDa dextran to the lumen of GUV was measured with ImageJ software as described previously (27). Protein concentrations were 300 nM for BAK g / BAK, 400 nM for MCL1 g /MCL1, and 15 nM for cBID r /cBID.…”

Section: Methodsmentioning

confidence: 99%

“…In SUV/LUV-type liposomes, we used 55:35:10 PC/PE/PI (mol/ mol/mol) (0% CL) as a "template" lipid composition in which increasing proportions of PC were substituted by CL to yield the following liposome compositions: 51:35:10:4 PC/PE/ PI/CL (mol/mol/mol/mol) (4% CL), 40:35:10:15 PC/PE/ PI/CL (mol/mol/mol/mol) (15% CL), 25:35:10:30 PC/ PE/PI/CL (mol/mol/mol/mol) (30% CL), and CL (100% CL). GUV-type vesicles were prepared as described previously (27). Briefly, around 4 l of the lipid mixture stock (1 mM) in chloroform was spread on the platinum wires of the electroformation chamber.…”

Section: Methodsmentioning

confidence: 99%

Minimalist Model Systems Reveal Similarities and Differences between Membrane Interaction Modes of MCL1 and BAK

Landeta

Landajuela

García‐Sáez

et al. 2015

Journal of Biological Chemistry

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Background: BCL2 family protein interactions with and at mitochondrial membranes are poorly understood. Results: Fluorescence-based studies applied to minimalist model systems provide new insight into membrane activities of MCL1 and BAK under apoptotic-like conditions. Conclusion: Membrane interaction modes of MCL1 and BAK share particular features but also display important differences. Significance: BCL2 family protein function can be modulated at the mitochondrial membrane level through manifold mechanisms.

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Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size

Cited by 134 publications

References 62 publications

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging

Toxicity of an α-Pore-forming Toxin Depends on the Assembly Mechanism on the Target Membrane as Revealed by Single Molecule Imaging

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Minimalist Model Systems Reveal Similarities and Differences between Membrane Interaction Modes of MCL1 and BAK

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