Article

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

ABSTRACT

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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Available from: Soojay Banerjee
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    • "Because of different rates for the retrotranslocation from the mitochondria to the cytosol, Bak resides predominantly in the mitochondrial outer membrane, whereas the majority of the Bax molecules are in the cytosol (Edlich et al, 2011;Schellenberg et al, 2013;Todt et al, 2015). Upon apoptosis, active Bax is not retrotranslocated, as Bax activation blocks shuttling into the cytosol and consequently Bax accumulates on the mitochondria (Wolter et al, 1997;Edlich et al, 2011). In the course of the translocation, Bax changes its conformation , oligomerizes, and inserts into the outer membrane (Hsu et al, 1997;Gross et al, 1998;Eskes et al, 2000). "
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    ABSTRACT: The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells.
    Full-text · Article · Jan 2016 · The EMBO Journal
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    • "For cell fractionation , cells were cultured in 15-cm dishes and transfected with 10 lg DNA and 5 ll Lipofectamine 2000 (HeLa) or 10 lg DNA and 12 ll Turbofect (HCT116). The construct GFP-Bax was a generous gift from Dr. Nathan Brady (DKFZ, Heidelberg); GFP-Bax 1-2/L-6 was kindly given by Dr. Frank Edlich (Institute for Biochemistry and Molecular Biology, University of Freiburg) (Edlich et al, 2011). For mitochondrial staining, cells were incubated with 100 nM Mito- Tracker Red or TMRE (Life Technologies, Germany) for 30 min. "
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    ABSTRACT: Bax is a key regulator of apoptosis that, under cell stress, accumulates at mitochondria, where it oligomerizes to mediate the permeabilization of the mitochondrial outer membrane leading to cytochrome c release and cell death. However, the underlying mechanism behind Bax function remains poorly understood. Here, we studied the spatial organization of Bax in apoptotic cells using dual-color single-molecule localization-based super-resolution microscopy. We show that active Bax clustered into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies that localized in discrete foci along mitochondria. Remarkably, both rings and arcs assemblies of Bax perforated the membrane, as revealed by atomic force microscopy in lipid bilayers. Our data identify the supramolecular organization of Bax during apoptosis and support a molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.
    Full-text · Article · Jan 2016 · The EMBO Journal
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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.
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