INVOLVEMENT OF CYCLOPHILIN-D IN THE ACTIVATION OF A MITOCHONDRIAL PORE BY CA2+ AND OXIDANT STRESS

Heart and liver mitochondria contain a structure that is able to form a large non-selective pore in the inner membrane under conditions of h igh matrix Ca2+ and oxidant stress. The pore is blocked by cyclosporin A (CSA). Ln this study, rat liver mitochondria were covalently labell ed with a photoactive CSA derivative in the presence and absence of th e pore ligands Ca2+ and ADP. Photolabelling of a 21-kDa protein was se lectively depressed by Ca2+ in a manner reversed by ADP. The protein e xhibited peptidyl-prolyl cis-trans isomerase (PPIase) activity and was inhibited by CSA (K-i, 8 nM). The PPIase was associated with the outs ide of sonicated submitochondrial particles but dissociated in 0.5 M N aCl. When mitochondria were treated with increasing, concentrations of digitonin, the 21-kDa PPIase fractionated with the matrix marker enzy me, malate; dehydrogenase. A second PPIase of 18 kDa fractionated with the intermembrane-space marker, adenylate kinase. Photolabelling of t he 18-kDa PPIase was unaffected by Ca2+ or ADP. The 21-kDa PPIase was digested with endoproteinase Asp-N and 11 of the peptides were N-termi nally sequenced. The sequences were most similar to those of human cyc lophilin-D, and it is concluded that this protein is probably the CSA receptor during pore blockade by CSA. The implications of these findin gs are discussed.