Bcl-x(L) Retrotranslocates Bax from the Mitochondria into the Cytosol

Surgical Neurology Branch, NINDS, National Institutes of Health, Bethesda, MD 20892, USA.
Cell (Impact Factor: 32.24). 04/2011; 145(1):104-16. DOI: 10.1016/j.cell.2011.02.034
Source: PubMed


The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-x(L) in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-x(L) inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax.

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    • "Recent data showed that Bcl-xLmediated Bax retrotranslocation may be an important process in regulating Bax mitochondrial content (Schellenberg et al., 2013). This process was first evidenced with GFP-tagged Bax, in mammalian cells where the whole apoptotic network is present (Edlich et al., 2011). It may be noted, however, that a spontaneously mitochondria-localized and inactive mutant of Bax was used in these experiments. "
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    ABSTRACT: Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release. Copyright © 2015. Published by Elsevier Ltd.
    The international journal of biochemistry & cell biology 04/2015; 64. DOI:10.1016/j.biocel.2015.03.020 · 4.05 Impact Factor
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    • "For HCV-induced apoptosis, both host and viral factors have been implicated, however, the molecular mechanisms and the specific factor(s) which perpetuate this process remains largely unknown. The Bcl-2 family members are the major molecular players in programmed cell death (Edlich et al., 2011; Hardwick and Soane, 2013), with some BH3-only proteins reported to play a role in HCV infection (Simonin et al., 2009). The BH3-only pro-apoptotic members, which are the allosteric regulators of the Bcl-2 family members, are known to bind strongly to the pro-survival members through their BH3 domains thereby antagonizing their pro-survival function (Chen et al., 2005). "
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    ABSTRACT: Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3-only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression.
    Virology 11/2014; 474. DOI:10.1016/j.virol.2014.10.027 · 3.32 Impact Factor
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    • "Although only a small fraction of wild-type Bax is present in the OMM-integrated protein pool of proliferating cells, larger pools of Bax variants (e.g. Bax S184V) are membrane integral and shuttle completely into the cytosol (Fig 7D; Supplementary Fig S7C and D; Edlich et al, 2011). Noteworthy, OMM-integral Bax is not in the active conformation (Supplementary Fig S7E). "
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    ABSTRACT: The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death.
    The EMBO Journal 11/2014; 34(1). DOI:10.15252/embj.201488806 · 10.43 Impact Factor
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