Bax interactions with VDAC-ANT mitochondrial intermembrane contact sites during apoptosis: implications for a mechanism of outer mitochondrial membrane permeabilisation.

The Bcl-2 family of proteins regulates the mitochondrial apoptotic pathway and consists of both pro- and anti-apoptotic members. Bax-cc is present in the cytosol of healthy cells. Apoptotic stimuli induce the translocation of Bax-oc to the mitochondria leading to the permeabilisation of the outer mitochondrial membrane (OMM) and the release of downstream pro-apoptotic proteins from the intermembrane space (IMS). The mechanism by which Bax permeabilises the OMM remains unclear. Recent evidence suggests Bax alone might be sufficient to permeabilise the OMM. However, other models indicate the involvement of the mitochondrial permeability transition pore complex or the voltage-dependent anion channel (VDAC) present in the OMM. In this study Bax and C-terminally truncated Bax were expressed as GST-fusion proteins and were immobilised on agarose-GSH. The binding of mitochondrial membrane proteins that might be involved in the Bax mediated release of proteins from the IMS was investigated. The results showed that VDAC and the adenine nucleotide translocase (ANT) were retained by GST-Bax. Exogenous cyclophilin D (Cyp D) was added in the presence of VDAC and ANT and was also retained indicating that Bax interacts with the components of the permeability transition pore complex. The anti-apoptotic Bcl-2 protein Bcl-XL was expressed with a hexahistidine tag at its N-terminus. This was used to investigate its effects on Bax interaction with VDAC and ANT. The ANT ligands atractyloside and bongkrekic acid which promote and inhibit apoptosis respectively were shown to change the relative amounts of VDAC and ANT that bind GST-Bax. Apoptotic cell death has been identified in cardiomyocytes subjected to ischaemia. In this investigation cardiomyocytes transfected with GFP-Bax were treated with cyanide to simulate ischaemia and GFP-Bax translocation was observed using fluorescence microscopy. GFP-Bax co-immunoprecipitated with VDAC and ANT after translocation to mitochondria but not with Cyp D. The implications of these findings are discussed in this thesis