Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2-independent permeability transition pore opening and apoptosis

Mitochondrial membrane permeabilization is a critical event in the process leading to physiological or chemotherapy-induced apoptosis. This permeabili zation event is at least in part under the control of the permeability tran sition pore complex (PTPC), which interacts with oncoproteins from the Bcl- 2 family as well as with tumor suppressor proteins from the Bar family, whi ch inhibit or facilitate membrane permeabilization, respectively. Here we s how that thiol crosslinking agents including diazenedicarboxylic acid bis S N,N-dimethylamide (diamide), dithiodipyridine (DTDP), or bis-maleimido-hexa ne (BMH) can act on the adenine nucleotide translocator (ANT), one of the p roteins within the PTPC. ANT alone reconstituted into artificial lipid bila yers suffices to confer a membrane permeabilization response to thiol cross linking agents. Diamide, DTDP, and BMH but not tert-butylhydroperoxide or a rsenite cause the oxidation of a critical cysteine residue (Cys 56) of ANT. Thiol modification within ANT is observed in intact cells, isolated mitoch ondria, and purified ANT. Recombinant Bcl-2 fails to prevent thiol modifica tion of ANT. Concomitantly, a series of different thiol crosslinking agents (diamide, DTDP, and BMH, phenylarsine oxide) but not tert-butylhydroperoxi de or arsenite induce mitochondrial membrane permeabilization and cell deat h irrespective of the expression level of Bcl-2. These data indicate that t hiol crosslinkers cause a covalent modification of ANT which, beyond any co ntrol by Bcl-2, leads to mitochondrial membrane permeabilization and cell d eath.