(29) CYCLOSPORIN-SENSITIVE BINDING OF PURIFIED MITOCHONDRIAL CYCLOPHILIN TO THE ADENINE NUCLEOTIDE TRANSLOCASE: IMPLICATIONS FOR THE MECHANISM OF THE PERMEABILITY TRANSITION

A. P. Halestrap, J.P. Gillespie and K-Y. Woodfield
Department of Biochemistry, University of Bristol, Bristol, BS8 1TD, U.K.

The mitochondrial permeability transition (MPT) is caused by the opening of a CsA-inhibitable non-specific pore in the inner membrane under conditions of high matrix [Ca2+] and oxidative stress. We have proposed a model for the mechanism of the MPT which involves an interaction of matrix cyclophilin (CyP-D) with the adenine nucleotide translocase (ANT). In order to demonstrate this directly, rat CyP-D was cloned and over-expressed as a glutathione-S-transferase (GST) fusion protein. The fusion protein had peptidyl-prolyl cis-trans isomerase activity and bound to purified inner mitochondrial membranes (IMMs) in a CsA-sensitive manner. Binding was enhanced when IMMs were from diamide-treated mitochondria. The GST-CyP-D was immobilised on glutathione sepharose and used to bind partially solubilized (0.5% Triton X-100) rat liver inner mitochondrial membrane protein(s). Specifically bound proteins (those whose binding was prevented by CsA but not by its analogue CsH) were eluted with glutathione and the presence of ANT confirmed by Western blotting with anti-ANT antibodies. Both rat and yeast ANT bound specifically under these conditions, but not porin. The binding of ANT to the CyP-D column was prevented by known ligands of the ANT such as carboxyatractyloside, implying that binding is dependent on the conformation of the ANT rather than a non-specific interaction. Purified rat heart ANT was also shown to bind to the GST-CyP in a CsA-sensitive manner. We are now attempting to stabilise the interaction between the ANT and CyP-D using cross-linking reagents. This stable complex will be used to identify other membrane or matrix proteins that may interact with the MPT pore components.