Bak/bax-dependent Apoptotic Signaling In Vdac2-/- Cells

Abstract

Bid, a pro-apoptotic Bcl-2 family protein, upon activation forms truncated Bid (tBid) that binds to the outer mitochondrial membrane (OMM) and engages Bak/Bax-dependent release of cytochrome c (cyto c) and other intermembrane space proteins from mitochondria to the cytosol to induce apoptosis. The voltage-dependent anion channel (VDAC) is the major permeability pathway for metabolites and ions in the OMM but its role in the tBid-induced OMM permeabilization remains controversial. Previously we reported that among the VDAC isoform-specific knockout mouse embryonic fibroblasts (MEFs), only VDAC2-/- MEFs lack tBid induced complete cyto c release and loss of ΔΨm. Here we show by single cell fluorescence imaging that permeabilized VDAC2-/- MEFs expressing cyto c-GFP were resistant to tBid (37nM)-induced cyto c-GFP release. Furthermore, by rescuing VDAC2-/- MEFs with VDAC2 the tBid-induced cyto c-GFP release was restored. In addition, tBid adenovirus infection caused less cell death in intact VDAC2-/- MEFs than in wildtype (WT) MEFs. It has been reported that VDAC2 is required for proper targeting of Bak in OMM. Indeed, Bak did not appear in the membrane fraction of VDAC2-/- MEFs. Along this line we show that permeabilized Bak-/- cells were more resistant to tBid induced cyto c release and loss of ΔΨm than WT and Bax-/- MEFs. Strikingly, washout of the cytosol further desensitized the Bak-/- MEFs to tBid. Unlike VDAC2-/- MEFs, the Bak-/- MEFs constitutively overexpressed Bax that was primarily localized in the cytosol. However, recombinant Bax (200nM) could induce cyto c release and depolarization in VDAC2-/- MEFs and also supported the tBid-induced cyto c release. Thus, in VDAC2-/- cells Bak does not localize to the mitochondria and fails to interact with tBid and does not allow a compensatory increase in Bax. The combination of these two mechanisms greatly attenuates tBid-induced OMM permeabilization and apoptosis.