Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition

A fusion protein between cyclophilin-D (CyP-D) and glutathione S-transferas e (GST) was shown to bind to purified liver inner mitochondrial membranes ( IMMs) in a cyclosporin A (CsA)-sensitive manner. Binding was enhanced by di amide treatment of the IMMs. Immobilized GST-CyP-D avidly bound a single 30 kDa protein present in Triton X-100-solubilized IMMs; immunoblotting showe d this to be the adenine nucleotide translocase (ANT). Binding was prevente d by pretreatment of the CyP-D with CsA, but not with cyclosporin H. Purifi ed ANT also bound specifically to GST-CyP-D, but porin did not, even in the presence of ANT.