Mitochondrial ADP/ATP Carrier: Preventing Conformational Changes by Point Mutations Inactivates Nucleotide Transport Activity

Abstract

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family (MCF); its members allow metabolic fluxes between mitochondria and the cytosol. The members of the MCF share numerous structural and functional characteristics. Ancp is very specifically inhibited by two classes of compounds, which stabilize the carrier in two different conformations involved in nucleotide transport. Resolution of the atomic structure of the bovine Ancp, in complex with one of its specific inhibitors, is that of the carrier open toward the intermembrane space. To gain insights into the interconversion from one conformation to the other, we introduced point mutations in the yeast carrier at positions Cys73 in the first matrix loop and Tyr97 and Gly298 in transmembrane helices 2 and 6. We demonstrate in this paper that they impair stabilization of the carrier in one conformation or the other, resulting in an almost complete inactivation of nucleotide transport in both cases. The results are discussed on the basis of the atomic structure of the conformation open to the cytosol. These mutant proteins could afford convenient tools for undertaking structural studies of both conformations of the yeast carrier.