The great migration of Bax and Bak

Abstract

The Bcl-2 proteins Bcl-2-associated X protein (Bax) and Bcl-2 antagonist killer 1 (Bak) can commit cells to apoptosis. Retrotranslocation of Bax from the mitochondria into the cytosol is essential for cell survival. Recently, we reported that Bak is also present in the cytosol of healthy cells and that Bax and Bak are regulated by the same retrotranslocation process.

Mitochondrial apoptosis signaling converges at activation of the B-cell lymphoma 2 (Bcl-2) proteins Bcl-2-associated X protein (Bax) and Bcl-2 antagonist killer 1 (Bak), which permeabilize the outer mitochondrial membrane (OMM) resulting in mitochondrial dysfunction and efficient dismantling of the cell by caspases. Activation of the proapoptotic Bcl-2 proteins Bax and Bak is regulated by prosurvival Bcl-2 proteins containing 4 Bcl-2 homology domains (BH1-4), such as Bcl-2, B-cell lymphoma-extra large (Bcl-xL), or myeloid cell leukemia 1 (Mcl-1), and a diverse group of proteins sharing only the BH3 domain with Bcl-2 proteins (Bcl-2 homology domain 3-only proteins, or BH3-only proteins). Bax and Bak are inhibited by prosurvival Bcl-2 proteins, either via direct interactions or by sequestering ‘activator’ BH3-only proteins and thereby preventing their interaction with Bax and Bak.^1,2 Upon apoptosis induction, Bax and Bak oligomerize and at least partially insert into the OMM.³ In healthy cells, Bax is primarily cytosolic,⁴ contrasting with the predominantly mitochondrial Bak.⁵ Bax constantly migrates between the cytosol and the OMM. Bax retrotranslocation from the mitochondria requires recognition of its exposed BH3 motif by the hydrophobic groove of Bcl-xL and interaction between the C-terminal Bcl-xL helix and Bax.^6,7 Bax shuttling is also mediated by Bcl-2 and Mcl-1,⁶ but could additionally involve Bcl-2 protein-independent mechanisms.⁸ Compromised Bax shuttling results in mitochondrial Bax accumulation, but further stimulation is required to activate Bax depending on the size of the OMM-localized Bax pool prior to apoptosis stimulation.⁷ The importance of Bax retrotranslocation provoked the question of why cells tolerate Bak on the OMM, but not Bax.

As a starting point, we wanted to analyze whether Bak localizes exclusively to the OMM or, like Bax, could be detected in the cytosol. Fractionation experiments of human tissues revealed the presence of cytosolic Bak in several tissues, among them heart and lung, in addition to a predominant mitochondrial Bak pool.⁹ This finding encouraged us to test whether Bak localization is regulated by an equilibrium similar to Bax shuttling between the cytosol and mitochondria.⁶ Fluorescence loss in photobleaching (FLIP) measurements showed that Bak shuttles from the mitochondria into the cytosol. Strikingly, increased levels of Bcl-xL accelerate Bak retrotranslocation into the cytosol and increase the cytosolic pools of overexpressed and endogenous Bak.⁹ Like Bax retrotranslocation, shuttling of mitochondrial Bak depends on interaction between the BH3 motif of the proapoptotic protein and the hydrophobic groove of the prosurvival Bcl-2 family member. However, Bcl-2 accelerates the shuttling of only Bax, and not that of Bak. The retrotranslocation of these proapoptotic Bcl-2 proteins also differs in the shuttling rate; Bak is retrotranslocated significantly more slowly than Bax, suggesting a plausible reason for the differential localization of Bax and Bak.

We investigated the differential shuttling of Bax and Bak further by generating chimeras of Bax and Bak each harboring the corresponding C-terminal membrane anchor (MA) segment of the other protein (BaxTBak and BakTBax), based on previous reports of the importance of this segment for Bax localization.¹⁰ Indeed, exchange of the MA region reversed shuttling rates and localization of Bax and Bak, showing the important influence of the MA on retrotranslocation and thus localization. Bax and Bak constructs with the same MA showed similar localization, shuttling, and activity following apoptosis stimulation. Strikingly, BaxTBak gains full activity in the absence of apoptotic stimuli that is not completely inhibited by Bcl-xL overexpression, contrasting with the regulation of both wild-type proteins and BakTBax.⁹ Bcl-xL fails to protect cells from BaxTBak activity in the presence and absence of apoptotic stimuli. On one hand, our results establish the link between increased retrotranslocation rates and reduced proapoptotic activity of Bax and Bak. On the other hand, our experiments show that slow Bax retrotranslocation commits cells to apoptosis. Bax activation seems to be determined by the length of time that Bax molecules remain on the OMM, as high mitochondrial Bax levels do not per se commit cells to apoptosis.^7,9 Shuttling rates, localization, and activity of Bax and Bak are determined by the hydrophobicity, but not the sequence, of the MA.⁹

A common retrotranslocation process regulates the localization and activity of Bax and Bak in healthy cells (Fig. 1). Notably, retrotranslocation shuttles OMM-integral forms of Bax, Bak, and Bcl-xL depending on interactions between the BH3 motif of proapoptotic Bcl-2 proteins with the hydrophobic groove of Bcl-xL. The driving force for differential localization and thus retrotranslocation of Bax and Bak is most likely the activation of slowly shuttling Bax. Only Bax is activated at slow retrotranslocation rates in the absence of apoptotic stimuli; therefore, accelerated shuttling of Bax, but not Bak, is essential for cellular survival. Increased Bax retrotranslocation rates and cytosolic localization result from decreased hydrophobicity of the Bax MA segment. Retrotranslocation determines the size of the mitochondrial Bax and Bak pools ⁷ and thus dictates whether or not a cell will commit to apoptosis following severe and persistent stress.

Figure 1.

The great migration of Bax and Bak. The Serengeti silhouette has been adapted from www.balloonsafaris.com. In the Serengeti of B-cell lymphoma (Bcl-2) proteins, shuttling occurs between the cytosol and mitochondria. Unlike the migration of wildebeests and zebras in enormous herds, a fraction of proapoptotic Bax (Bcl-2-associated X protein, blue) constantly migrates to the mitochondria in the absence of apoptosis signaling. At the same time, a mitochondrial Bax pool is retrotranslocated from the mitochondria dependent on the activities of prosurvival Bcl-2 proteins, e.g., Bcl-xL (B-cell lymphoma-extra large, red). The same processes translocate cytosolic Bak (Bcl-2 antagonist killer 1, green) to the mitochondria and retrotranslocate mitochondrial Bak into the cytoplasm. In contrast to the predominantly cytosolic Bax localization, Bak resides primarily on the mitochondria. This differential localization arises from their different shuttling speeds. Prosurvival Bcl-2 proteins co-retrotranslocate with their proapoptotic counterparts from the mitochondria, perhaps as lions would follow wildebeests to the next feeding ground. Since similar amounts of Bax are shuttled in both directions a dynamic equilibrium between cytosolic and mitochondrial Bax pools is established. This equilibrium is disturbed by inhibitors of prosurvival Bcl-2 proteins like BH3-only proteins (Bcl-2 homology domain 3-only proteins, yellow), perhaps comparable to lions getting killed by hunters. BH3-only proteins could mediate the signal of apoptotic stress, e.g., DNA damage. When retrotranslocation is blocked Bax and Bak would accumulate on the mitochondria and induce permeabilization of the outer mitochondrial membrane, thus committing the cell to apoptosis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

I thank Dr. SWG Tait for discussing the manuscript.

Funding

FE is supported by the Emmy Noether program of the German Research Council (Deutsche Forschungsgemeinschaft, DFG), the Else Kröner Fresenius Foundation, the Spemann Graduate School of Biology and Medicine (SGBM, GSC-4), and the Centre for Biological Signalling Studies (BIOSS, EXC-294) funded by the.