Dominant-negative VDAC1 mutants reveal oligomeric VDAC1 to be the active unit in mitochondria-mediated apoptosis

Mitochondria play a central role in the intrinsic pathway of apoptosis. Oligomerization of the mitochondrial protein VDAC1 (voltage-dependent anion channel 1) has been proposed to play a role in apoptosis in various studies. In the present study, we have generated dimeric fusion proteins consisting of tandem-linked wild-type and RuR (Ruthenium Red)-insensitive mutant VDAC1 monomers and studied the capacity of RuR to protect against apoptosis, as induced by various means. Fusion proteins composed of wild-type and/or E72Q-VDAC1 were successfully expressed in T-REx-293 cells. Bilayer-reconstituted dimeric rVDAC1 (rat VDAC1) functions as a channel-forming protein, showing typical voltage-dependence conductance, but with a unitary conductance higher than that of monomeric VDAC. As with wild-type VDAC1, overexpression of either the wild-type or mutated VDAC1 dimeric fusion protein induced apoptotic cell death. In addition, as shown previously, the anti-apoptotic effect of RuR was not observed in cells expressing E72Q-VDAC1, despite endogenous VDAC1 being present in these cells. Similar RuR insensitivity governed the VDAC1 fusion proteins comprising the E72Q mutation in either the first, second or both VDAC1 monomers of the same dimer. RuR-mediated protection against apoptosis in T-REx-293 cells, as induced by staurosporine, was observed in cells expressing VDAC1 or dimeric wild-type VDAC1. However, RuR offered no protection against staurosporine-induced apoptosis in cells expressing E72Q-VDAC1 or E72Q-containing dimeric VDAC1. These results suggest that E72Q-VDAC1 has a dominant-negative effect and implies that VDAC1 homo-oligomerization, involving intermolecular interactions, might be involved in the apoptotic process. Abbreviations: AIF, apoptosis-inducing factor; DMEM, Dulbecco's modified Eagle's medium; EGS, ethylene glycol bis(succinimidylsuccinate); GFP, green fluorescent protein; HEK-293T cells, HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40); HK, hexokinase; HRP, horseradish peroxidase; LDAO, N,N-dimethyldodecylamine-N-oxide; OMM, outer mitochondrial membrane; PLB, planar lipid bilayer; RuR, Ruthenium Red; siRNA, small interfering RNA; STS, staurosporine; TEV, tobacco etch virus; T-REx-293, HEK-293 cells that express the tetracycline repressor; VDAC, voltage-dependent anion channel; hVDAC1, human VDAC1; mVDAC1, murine VDAC1; rVDAC1, rat VDAC1