Mitochondrial import of cyclophilin-D.

Mammalian mitochondria contain an inner membrane pore which opens following an increase in matrix calcium. Pore opening has been implicated in tissue damage following ischaemic injury. The pore is reversibly blocked by cyclosporin A [l] suggesting a role for cyclophilin in pore control. A mitochondria1 isoform of cyclophilin (CyP-D) [2,3,4,5] has now been identified. Analysis of the N-terminus of the sequence suggests that it could form an amphipathic helix, typical of many presequences [6]. Cleavage of the precursor is believed to occur between Thr29 and Cys30, to yield mature cyclophilin. The primer pair CypDl and CypD2 was used to amplify the entire cyclophilin coding sequence from a cloned CyP-D sequence [S. Virji, J.M. Ward and M. Crompton, unpublished data] introducing an upstream T7 promoter. The product of this amplification was introduced directly into a coupled in vitro transcription/translation reaction (Fig. 1.). Full length precursor CyP-D (pCyP-D) was expressed as a 23kDa radiolabelled protein by the addition of ["Slmethionine to the translation reaction.