Differential retrotranslocation of mitochondrial Bax and Bak

Todt, Franziska; Cakir, Zeynep; Reichenbach, Frank; Emschermann, Fréderic; Lauterwasser, Joachim; Kaiser, Andrea; Ichim, Gabriel; Tait, Stephen W.G.; Frank, Stephan; Langer, Harald F.; Edlich, Frank

doi:10.15252/embj.201488806

Cited by 150 publications

(186 citation statements)

References 48 publications

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“…Alternatively, because both BAK and BAX interact with VDAC2 at the MOM (35-37), binding of BH3-only proteins to the α6 site in BAK or BAX may be an important step in dissociating them from VDAC2 during apoptosis. Recent evidence that the distinct subcellular localization of BAK and BAX is a consequence of their relative rates of retrotranslocation to the cytosol argues against qualitatively different modes of BAK and BAX activation (13,15). Although our linkage and blocking data support a two-site mechanism of BAK activation, it remains possible that labeling the BAK α6 indirectly impairs a key event in BAK activation induced by groove binding that precedes homodimerization.…”

Section: Discussionmentioning

confidence: 53%

“…Interaction of BH3-only proteins at this noncanonical site is not thought to be necessary for BAK activity because BAK is constitutively anchored in the mitochondrial outer membrane (MOM) via its transmembrane domain (12). However, recent reports that both BAX (13,14) and BAK (15) are constantly "retrotranslocated" from the mitochondria to the cytosol suggest conserved mechanisms of activation for BAK and BAX.…”

mentioning

confidence: 99%

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BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove

Tan

Stephen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain.AK and BAX are the pivotal effectors of intrinsic apoptosis, with one or the other being required for mitochondrial damage and cell death (1, 2). They are activated by interaction with BCL-2 homology 3 (BH3)-only proteins including BID and BIM (3). Structures show that peptides based on the BH3 domains of activator BH3-only proteins can bind directly to BAK and BAX via a hydrophobic groove (comprising α-helices 2-5) that is also shared with their prosurvival homologs (4-8). BAX has been proposed to have an additional activation site, distinct from the hydrophobic groove, at the rear of the molecule comprising α-helices 1 and 6 (9). Binding of stapled BH3 peptides at this site induced the dissociation of the BAX C-terminal transmembrane domain from the hydrophobic groove to facilitate mitochondrial localization (10, 11). Interaction of BH3-only proteins at this noncanonical site is not thought to be necessary for BAK activity because BAK is constitutively anchored in the mitochondrial outer membrane (MOM) via its transmembrane domain (12). However, recent reports that both BAX (13, 14) and BAK (15) are constantly "retrotranslocated" from the mitochondria to the cytosol suggest conserved mechanisms of activation for BAK and BAX.Characterizing the interaction of BH3-only proteins with fulllength BAK at mitochondria has proven difficult because the bound BH3-only protein dissociates during consequent BAK conformation changes, including exposure of the N-terminus (amino terminus) and and dissociation of their α2-5 helices ("core") from their α6-8 helices ("latch") (6, 7) to facilitate selfassociation. Additionally, structural studies have been largely confined to truncated protein or peptides in the absence of a membrane where interactions between BH3-only proteins and BAK occur.Once activated BAK and BAX self-associate to form pores that damage the MOM to release cytochrome c and...

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Section: Discussionmentioning

confidence: 53%

mentioning

confidence: 99%

BAK α6 permits activation by BH3-only proteins and homooligomerization via the canonical hydrophobic groove

Tan

Stephen

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Interestingly, Bak was also found to be retrotranslocated by Bcl-xL, albeit at a much slower rate (Todt et al 2015). Although the mechanism of retrotranslocation is not well understood, it is safe to suggest that the different abilities to be retrotranslocated by the anti-apoptotic Bcl-2 family proteins likely determine the differential subcellular localization of Bax and Bak before apoptotic stimulation.…”

Section: Discussionmentioning

confidence: 99%

Inactivation of prosurvival Bcl-2 proteins activates Bax/Bak through the outer mitochondrial membrane

et al. 2016

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The mechanism of Bax/Bak activation remains a central question in mitochondria-dependent apoptotic signaling. While it is established that all proapoptotic Bcl-2 homology 3 (BH3)-only proteins bind and neutralize the antiapoptotic Bcl-2 family proteins, how this neutralization leads to Bax/Bak activation has been actively debated. Here, genome editing was used to generate cells deficient for all eight proapoptotic BH3-only proteins (OctaKO) and those that lack the entire Bcl-2 family (Bcl-2 allKO). Although the OctaKO cells were resistant to most apoptotic stimuli tested, they underwent Bax/Bak-dependent and p53/Rb-independent apoptosis efficiently when both Bcl-xL and Mcl-1, two anti-apoptotic Bcl-2 proteins, were inactivated or eliminated. Strikingly, when expressed in the Bcl-2 allKO cells, both Bax and Bak spontaneously associated with the outer mitochondrial membrane (OMM) through their respective helix 9, and this association triggered their homo-oligomerization/activation. Together, these results strongly suggest that the OMM, not BH3-only proteins or p53/Rb, is the long-sought-after direct activator of Bax/Bak following BH3-only-mediated neutralization of anti-apoptotic Bcl-2 proteins.

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“…Nevertheless, inducible systems based around tamoxifen/ER type control methods or degronbased approaches should facilitate this approach 112,113 . In a complementary approach, experimentally engaging different levels of tumour cell apoptosis in vivo would directly allow one to test the effects of tumour cell apoptosis on tumour progression.…”

Section: Modeling Oncogenic Effects Of Apoptosismentioning

confidence: 99%