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. 2015 Jan 2;34(1):67-80.
doi: 10.15252/embj.201488806. Epub 2014 Nov 5.

Differential retrotranslocation of mitochondrial Bax and Bak

Franziska Todt  1 , Zeynep Cakir  2 , Frank Reichenbach  1 , Frederic Emschermann  3 , Joachim Lauterwasser  2 , Andrea Kaiser  4 , Gabriel Ichim  5 , Stephen W G Tait  5 , Stephan Frank  6 , Harald F Langer  3 , Frank Edlich  7
Affiliations

Differential retrotranslocation of mitochondrial Bax and Bak

Franziska Todt et al. EMBO J. .

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.

Keywords: Bcl‐2 proteins; apoptosis; membrane association; tail anchor.

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Figures

Figure 1
Figure 1. Bak is present in the cytosol

  1. Flow chart of human tissue homogenization and subsequent fractionation into cytosolic (C) and mitochondria-containing heavy membrane (HM) fraction. With authorization by the local ethics committee, human tissue samples were obtained from the Institute of Pathology, University Hospitals of Basel.

  2. Analysis of anti-Bax and anti-Bak antibody specificity using whole-cell lysates from HCT116 wild-type, Bax KO, Bak KO and Bax/Bak DKO. After detection of Bax the membrane was redecorated with anti-Bak antibodies (Bax/Bak). Actin is used as a loading control.

  3. Western blot analysis of Bak in the heavy membrane (HM) fraction of human heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue. The cytosol fraction obtained from CNS cortex (C), LDH, and Tom20 serve as fractionation and loading controls. n = 3.

  4. Presence of Bak in the cytosol of heart, cerebral cortex (CNS GM), kidney, brain white matter (CNS WM), lung, liver, and skeletal muscle tissue analyzed by Western blot in parallel to (C). The heavy membrane heart fraction (HM), LDH and Tom20 serve as fractionation and loading controls. n = 3.

  5. Carbonate extraction of the heavy membrane protein fraction of human brain white matter (CNS WM) and lung tissue analyzed for the presence of Bak by Western blot. Membrane-associated protein supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the mitochondrial intermembrane space (IMS) during carbonate extraction and CoxIV remains in the pellet. n = 2.

Source data are available online for this figure.
Figure 2
Figure 2. Bcl-xL retrotranslocates Bak

  1. Confocal images of HCT116 Bax/Bak DKO cells transfected with GFP-Bak in the absence (left) or the presence (right) of Bcl-xL overexpression. Scale bar, 15 μm. n ≥ 10.

  2. FLIP (Fluorescence Loss in Photobleaching) of mitochondrial GFP-Bak in the absence (•, straight line) or the presence (○, broken line) of overexpressed Bcl-xL. Fluorescence of the neighboring cell (C) is shown as a control (▾, dotted line). Data represent averages ± SEM from 16 (-Bcl-xL) and 40 (+Bcl-xL) ROI measurements.

  3. FLIP of GFP-Bak in the absence (top) or the presence (bottom) of overexpressed Bcl-xL diminishes GFP-Bak fluorescence in the cytosol of targeted cells (circled) completely after 90 s and GFP fluorescence is detected only on the mitochondria (arrows). During FLIP measurements mitochondrial GFP-Bak fluorescence is monitored, while the cytosol is bleached repeatedly. Time points in seconds are displayed above the images.

Figure 3
Figure 3. Bak is shifted into the cytosol in parallel to Bax by retrotranslocation

  1. Western blot analysis of GFP-Bak localization in cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells with or without overexpressed wild-type Bcl-xL or Bcl-xL G138A. GAPDH and Tom20 serve as fractionation controls. n = 10. Bak shuttling by Bcl-xL is independent of the GFP fusion (Supplementary Fig S2A).

  2. Quantification of predominant mitochondrial localization (light grey bars) and mixed cytosolic and mitochondrial localization (dark grey bars) of Bak in HCT116 Bax/Bak DKO cells expressing GFP-Bak with or without Bcl-xL overexpression. Data represent averages ± SD from three independent experiments with n ≥ 100.

  3. Retrotranslocation rates measured for wild-type Bak or Bak D83R in the presence or the absence of Bcl-2, Mcl-1, wild-type Bcl-xL or Bcl-xL G138A. Data represent averages ± SD.

  4. Analysis of endogenous Bak localization in cytosol (C) and heavy membrane fraction (HM) of HeLa cells expressing degradation-prone DD-FLAG-Bcl-xL. In the absence of Shield-1 DD-FLAG-Bcl-xL is readily degraded, resulting in unaltered Bcl-xL levels. Addition of 0.5 μM Shield-1 instantly stabilizes DD-FLAG-Bcl-xL resulting in elevated Bcl-xL levels that increase cytosolic levels of endogenous Bak (Supplementary Fig S3C and D). Akt1 and Tom20 serve as fractionation controls. n = 3.

Source data are available online for this figure.
Figure 4
Figure 4. The C-terminal membrane anchor determines the localization of Bax and Bak

  1. The influence of the C-terminal membrane anchor (MA) on the differential localization and function of Bax and Bak has been analyzed using the wild-type proteins of Bax (blue) and Bak (green) and MA substitutions of both proteins resulting in the chimeras BaxTBak and BakTBax.

  2. Quantification of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak with the expressed protein being present largely cytosolic (white bars), in a mixed distribution between cytosol and mitochondria (grey bars) or largely mitochondrial (black bars). Data represent averages ± SEM from seven independent experiments with n ≥ 100 cells.

  3. Confocal images of HCT116 Bax/Bak DKO cells expressing Bax, BaxTBak, BakTBax or Bak. The GFP/YFP fluorescence of the expressed protein variants is depicted in the top panels and in green in the merged image on the bottom. The mitochondria were stained by MitoTracker far red depicted in red in the merged images (bottom row). Scale bar, 10 μm. n ≥ 5.

  4. Western blot analysis of Bax, BaxTBak, Bak and BakTBax localization expressed in HCT116 Bax/Bak DKO cells. Cytosol (C) and heavy membrane fraction (HM) of HCT116 Bax/Bak DKO cells are displayed. GAPDH and VDAC serve as fractionation controls. n = 3.

Source data are available online for this figure.
Figure 5
Figure 5. Mitochondrial Bax is activated apoptosis stimulus-independently

  1. Caspase-3/7 activity measured in HCT116 Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression in the absence of apoptosis stimuli. Caspase activity is displayed in relative fluorescence units (RFU). pcDNA3.1-transfected cells served as a control. Data represent averages ± SEM. n ≥ 3. P-values according to one-way ANOVA are displayed.

  2. Staurosporine (STS, 1 μM)-induced caspase-3/7 activity of Bax/Bak DKO cells overexpressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression displayed in relative fluorescence units (RFU). Data represent averages ± SEM. n ≥ 3. P-values according to one-way ANOVA. BaxTBak activities with and without Bcl-xL expression revealed no significant difference (n.s.), while in the presence of Bcl-xL overexpression BaxTBak activity is significantly higher than Bax, Bak or BakTBax activities (P < 0.001).

  3. Analysis of the active Bax conformation in HCT116 Bax/Bak DKO cells expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL overexpression (light grey bars) by the monoclonal antibody 6A7 (Sigma) detecting the active Bax protein fold by fluorescence imaging. Cells were analyzed prior to or after treatment with 1 μM STS in the presence of the pan-caspase inhibitor qVD. Data are represented as % of the expressing cell population ± SEM. n = 4.

  4. HCT116 Bax/Bak DKO cells ectopically expressing wild-type Bax or BaxTBak with (dark grey bars) or without Bcl-xL (light grey bars) were analyzed in the presence or the absence of 1 μM STS and qVD for retained mitochondrial cyt c. Data are represented as % of the expressing cell population ± SEM. n = 4.

  5. Flow cytometry analysis of Annexin V staining of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in the absence (red line) or the presence of Bcl-xL overexpression (black line), following STS treatment. The percentage of gated cells is displayed in the color of the corresponding graph. Data represent averages ± SD. n = 4.

  6. Colony formation of Bax/Bak DKO cells transfected with pcDNA, Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression. STS (1 μM) was added for 24 h before cells were replated and colonies were stained with methylene blue typically 14 days after treatment.

  7. Quantification of colony formation (F) of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax with or without Bcl-xL overexpression after STS treatment. Data represent averages ± SEM. n = 4. P-values according to one-way ANOVA. BaxTBak-expressing cells with or without Bcl-xL expression showed no significant difference (n.s.).

  8. Quantification of the colony formation of Bax/Bak DKO cells expressing Bax, BaxTBak, Bak or BakTBax in presence of Bcl-xL overexpression without apoptosis stimulation. Data represent averages ± SEM. n = 5. P-values according to one-way ANOVA.

Figure 6
Figure 6. Bax and Bak are regulated by the same mechanism

  1. FLIP of BaxTBakSS without (○, broken line) or with (•, straight line) overexpressed Bcl-xL. Fluorescence of a neighboring cell is shown as control (▾, dotted line). Data represent averages ± SEM from 20 (-Bcl-xL) and 16 (+Bcl-xL) ROI measurements.

  2. Western blot analysis of PARP cleavage in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. The experiment was carried out in the absence of apoptotic stimuli. Cells transfected with pcDNA3.1 vector serve as a control for PARP cleavage. Similar sample loading is controlled by actin. n = 4.

  3. Colony formation of Bax/Bak DKO cells transfected with pcDNA, BaxTBak or BaxTBakSS with Bcl-xL overexpression in the absence of apoptotic stimuli. n = 3.

  4. Caspase-3/7 activity was measured in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BaxTBak or BaxTBak V197/198S. Caspase activity is displayed in relative fluorescence units (RFU). The experiment was carried out in the absence of apoptotic stimuli and pcDNA3.1-transfected cells served as a control. Data represent averages ± SEM. n = 4. P-values according to one-way ANOVA using the Holm–Sidak method are displayed.

  5. Comparison of Bax and Bak localization and activity dependent on the MA (Bax MA, blue rectangle) using wild-type Bax (i, blue), Bax S184V (ii), BakTBax (iv) and BakTBax S184V (iii). Direct comparison of Bax S184V (ii) and BakTBax S184V (iii) reveals MA-independent influences of Bax and Bak on protein localization and activity (I). The impact of the S184V substitution (*) on translocation, retrotranslocation and activity is shown by differences between BakTBax and BakTBax S184V (II). The additional effect of Bax MA binding to the hydrophobic groove of Bax (III) is indicated by the comparison of both Bax variants (i + ii) versus both Bak variants (iii + iv).

  6. STS-induced caspase-3/7 activity of HCT116 Bax/Bak DKO cells expressing Bax, Bax S184V, BakTBax or BakTBax S184V is displayed normalized to Bax activity. Data represent averages ± SEM. n ≥ 3.

  7. Western blot analysis of PARP cleavage after STS treatment in HCT116 Bax/Bak DKO cells overexpressing Bax, Bax S184V, BakTBax or BakTBax S184V. Empty vector-transfected cells serve as a control for PARP cleavage and actin is the loading control. n = 3.

  8. Plot of Bax (•) and Bak (○) retrotranslocation rates versus hydrophobicity of the MA of the constructs. R2 = 0.95.

Source data are available online for this figure.
Figure 7
Figure 7. Bax activation does not per se depend on mitochondrial Bax levels

  1. Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing wild-type Bcl-xL in the presence or the absence of Bak or Bax analyzed by Western blot. Carbonate-extractable supernatant (S) and membrane-integral protein pellet (P) are displayed. Smac is released from the IMS during carbonate extraction and VDAC remains in the pellet. n = 4.

  2. Western blot analysis of Bcl-xLTBax carbonate extraction from the heavy membrane fraction from Bax/Bak DKO cells expressing the Bcl-xL variant with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.

  3. Carbonate extraction of the HM protein fraction of Bax/Bak DKO cells expressing Bcl-xL G138A with or without Bak or Bax. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.

  4. HM protein fraction and carbonate extraction of Bax/Bak DKO cells expressing Bax, BaxTBak or Bax S184V. Smac and VDAC serve as controls for supernatant (S) and pellet (P). n = 4.

  5. Smac release from the heavy membrane fraction (HM) into the cytosol (C) in HCT116 Bax/Bak DKO cells overexpressing Bax and BaxTBak without apoptotic stimuli analyzed by Western blot. Bax and BaxTBak were expressed differentially to produce similar levels of mitochondrial Bax. GAPDH and Tom20 serve as fractionation controls. n = 3.

  6. PARP cleavage in HCT116 Bax/Bak DKO cells expressing similar levels of mitochondrial Bax or BaxTBak analyzed by Western blot without apoptotic stimuli. Actin is used as a loading control. n = 3.

  7. Bax (blue, left) and Bak (green, right) are retrotranslocated similarly by pro-survival Bcl-2 proteins. The shuttling shifts also OMM-integral Bax and Bak into the cytosol dependent on the interactions between hydrophobic Bcl-xL groove and BH3 motif of either Bax or Bak. While Mcl-1 shuttles Bax and Bak like Bcl-xL, Bcl-2 only accelerates Bax retrotranslocation. Fast shuttling from the mitochondria into the cytosol at the level of Bax retrotranslocation in proliferating cells shifts Bax and Bak into the cytosol and inhibits their pro-apoptotic activities in parallel (top). Wild-type Bak is usually shuttled at low retrotranslocation rates and thus accumulates on the mitochondria (bottom, right). Shuttling at similar speed also causes mitochondrial Bax accumulation but results in stimulus-independent Bax activation (bottom, left). Therefore cells are required to shuttle Bax at high rates to prevent commitment to apoptosis.

Source data are available online for this figure.
See this image and copyright information in PMC

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