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. 2009 Dec;10(12):1341-7.
doi: 10.1038/embor.2009.219. Epub 2009 Oct 9.

VDAC2 is required for truncated BID-induced mitochondrial apoptosis by recruiting BAK to the mitochondria

Soumya Sinha Roy  1 , Amy M Ehrlich, William J Craigen, György Hajnóczky
Affiliations

VDAC2 is required for truncated BID-induced mitochondrial apoptosis by recruiting BAK to the mitochondria

Soumya Sinha Roy et al. EMBO Rep. 2009 Dec.

Abstract

Truncated BID (tBID), a proapoptotic BCL2 family protein, induces BAK/BAX-dependent release of cytochrome c and other mitochondrial intermembrane proteins to the cytosol to induce apoptosis. The voltage-dependent anion channels (VDACs) are the primary gates for solutes across the outer mitochondrial membrane (OMM); however, their role in apoptotic OMM permeabilization remains controversial. Here, we report that VDAC2(-/-) (V2(-/-)) mouse embryonic fibroblasts (MEFs) are virtually insensitive to tBID-induced OMM permeabilization and apoptosis, whereas VDAC1(-/-), VDAC3(-/-) and VDAC1(-/-)/VDAC3(-/-) MEFs respond normally to tBID. V2(-/-) MEFs regain tBID sensitivity after VDAC2 expression. Furthermore, V2(-/-) MEFs are deficient in mitochondrial BAK despite normal tBID-mitochondrial binding and BAX/BAK expression. tBID sensitivity of BAK(-/-) MEFs is also reduced, although not to the same extent as V2(-/-) MEFs, which might result from their strong overexpression of BAX. Indeed, addition of recombinant BAX also sensitized V2(-/-) MEFs to tBID. Thus, VDAC2 acts as a crucial component in mitochondrial apoptosis by allowing the mitochondrial recruitment of BAK, thereby controlling tBID-induced OMM permeabilization and cell death.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
tBID-induced ΔΨm loss and cytochrome c release in permeabilized VDAC-knockout MEFs and in isolated mitochondria. ΔΨm was monitored in permeabilized wt and V1−/− MEFs (A), in wt, V3−/− and V1/3−/− MEFs (B) and in wt and V2−/− MEFs (C) treated with 37 nM tBID. Mock-treated samples are shown as time control (tc). FCCP (5 μM) was added at the end of each run to depolarize the entire mitochondrial population. (D) Cumulative data showing tBID-induced depolarization of wt, V1−/−, V3−/−, V1/3−/− and V2−/− MEFs. (E) Release of cytochrome c visualized by immunoblotting of rapidly separated pellets (membrane) and supernatants (cytosol). (F) Expression of VDAC2 as visualized by immunoblotting of membrane fractions of MEFs. (G) Isolated mitochondria from wt and V2−/− MEFs were treated with 37 nM tBID. Release of cytochrome c visualized by immunoblotting of rapidly separated mitochondrial supernatants (Sn) and pellets. FCCP, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; MEF, mouse embryonic fibroblast; tBID, truncated BID; V1−/−, VDAC1−/−; V2−/−, VDAC2−/−; V3−/−, VDAC3−/−; VDAC, voltage-dependent anion channel; wt, wild type.
Figure 2
Figure 2
VDAC2 dependence of tBID-induced cyto c-GFP release and cell death in permeabilized and intact MEFs. (A) Fluorescence time-lapse imaging of cyto c-GFP-expressing (green) and mtDsRed-expressing (red), permeabilized wt and V2−/− MEFs. tBID (37 nM) caused release of cyto c-GFP (green to red shift in the overlaid images) only in wt MEFs. At the end of the experiment, 600 μg/ml digitonin was added to discharge the entire mitochondrial pool. Graphs showing the means of tBID-induced cyto c-GFP release kinetics of the entire cell population in the imaging field (15–20 cells; 4–5 measurements). (B) tBID adenovirus (16 h of infection) induced cell death. Means±s.e. (n=3). (C) Immunoblot of cell lysates confirming the rescue of VDAC2 expression in VDAC2-transfected V2−/− MEFs. (D) Fluorescence time-lapse imaging of control and rescued V2−/− MEFs transfected with cyto c-GFP. Addition of 37 nM tBID caused progressive release of cyto c-GFP (shown in greyscale). Graphs showing the mean response of the entire cell population in the imaging field (15–20 cells; 4–5 measurements). The tBID-induced fluorescence change was normalized to the effect of digitonin. Ad-tBID, tBID adenovirus; cyto c, cytochrome c; GFP, green fluorescent protein; MEF, mouse embryonic fibroblast; tBID, truncated BID; V2−/−, VDAC2−/−; VDAC, voltage-dependent anion channel; wt, wild type.
Figure 3
Figure 3
BAX and BAK dependence of tBID-induced ΔΨm loss and cytochrome c release. (A) Immunoblots of 30 μg proteins showing the binding of tBID in the membrane fractions of tBID-treated permeabilized MEFs. (B) The level of BAX and BAK in the membrane and the cytosolic fractions of permeabilized wt, V2−/− and BAK−/− MEFs. (C) The level of BAK in the membrane fractions of permeabilized MEFs and (D) the level of BAK in the whole cell lysates of wt, V2−/− and VDAC2-rescued V2−/− MEFs. ΔΨm was monitored for permeabilized wt, BAK−/− and BAX−/− MEFs treated with either 37 nM (E) or 3.7 nM (F) recombinant tBID for 5 min. (G) Immunoblot of rapidly separated pellets (membrane) and supernatants (cytosol) showing the release of cytochrome c on tBID treatment. FCCP, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; MEF, mouse embryonic fibroblast; tBID, truncated BID; V1−/−, VDAC1−/−; V1/3−/−, VDAC1−/− and VDAC3−/−; V2−/−, VDAC2−/−; V3−/−, VDAC3−/−; VDAC, voltage-dependent anion channel; wt, wild type.
Figure 4
Figure 4
BAX-dependent tBID-induced ΔΨm loss and cyto c release in permeabilized BAK−/− and V2−/− MEFs. (A) Immunoblot showing the presence of BAX in membrane and cytosol (upper left panels) and BAK in membrane (upper right panel) of permeabilized wt, BAK−/− and BAX−/− MEFs. Membranes from the permeabilized, washed cells were immunoblotted for BAX and BAK (lower two panels). (B) ΔΨm was monitored for permeabilized and washed wt, BAK−/− and BAX−/− MEFs treated with 37 nM recombinant tBID. Immunoblot of rapidly separated supernatants (cytosol) showed the release of cyto c. (C) ΔΨm was monitored for permeabilized V2−/− MEFs treated either with 50 nM oligomeric BAX only (light grey trace) or with 50 nM BAX+37 nM tBID (dark grey trace). The supernatants (cytosol) were rapidly separated at different time points (indicated by arrows) and immunoblotted for cyto c. (D) Cumulative data showing BAX and BAX+tBID induced depolarization of V2−/− MEFs. Cyto c, cytochrome c; FCCP, carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone; MEF, mouse embryonic fibroblast; tBID, truncated BID; V2−/−, VDAC2−/−; wt, wild type.
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Comment in

  • Apoptosis: it's BAK to VDAC.
    Shore GC. Shore GC. EMBO Rep. 2009 Dec;10(12):1311-3. doi: 10.1038/embor.2009.249. Epub 2009 Nov 13. EMBO Rep. 2009. PMID: 19949413 Free PMC article. Review. No abstract available.

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