Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore

A cyclophilin-D affinity matrix was employed to isolate components of the m itochondrial permeability transition pore. A cDNA encoding cyclophilin-D wa s cloned from a rat liver library and ligated into pGEX to allow expression of a glutathione S-transferase/cyclophilin-D fusion protein in Escherichia coli XL1 cells. The cyclophilin-D in the fusion was functionally normal as judged by its peptidylprolyl cis-trans-isomerase activity and its inhibiti on by cyclosporin A. The fusion protein was bound to glutathione-agarose to form the cyclophilin-D affinity matrix. The matrix selectively bound 32-kD a proteins of mitochondrial membrane extracts, but no H2O-soluble proteins were bound. The 32-kDa band on SDS/PAGE resolved into a doublet and reacted with antibodies against the voltage-dependent anion channel (porin) and th e adenine nucleotide translocase. These two proteins were also selectively retained by the affinity matrix in the presence of cyclosporin A. The thus- purified voltage-dependent anion channel, adenine nucleotide translocase an d the fusion protein were incorporated into phosphatidylcholine liposomes c ontaining fluorescein sulphonate. The proteoliposomes were permeabilized by Ca2+ plus phosphate, and this was blocked completely by cyclosporin A. The se properties are identical to those of the permeability transition pore in mitochondria. It is concluded that the basic permeability transition pore structure comprises the voltage-dependent anion channel (outer membrane), a denine nucleotide translocase (inner membrane) and cyclophilin-D, and forms at contact sites between the two membranes.