A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functionalin vivo

The adenine nucleotide carrier, or Ancp, is an integral protein of the inne r mitochondrial membrane. It is established that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, but different contradictory models were proposed over the past years to describe the organization of t he active Ancp. In order to decide in favor of a single model, it is necess ary to establish the orientations of the N- and C-termini and thus the pari ty of the Ancp transmembrane segments (TMS, According to this, we have cons tructed a gene encoding a covalent tandem dimer of the Saccharomyces cerevi siae Anc2p and we demonstrate that it is stable and active in vivo as well as in vitro. The properties of the isolated dimer are strongly similar to t hose of the native Ancp, as seen from nucleotide exchange and inhibitor bin ding experiments. We can therefore conclude that the native Anc2p has an ev en number of TMS and that the N- and C-terminal regions are exposed to the same cellular compartment. Furthermore, our results support the idea of a m inimal dimeric functional organization of the Ancp in the mitochondrial mem brane and we can suggest that TMS 1 of one monomer and TMS 6 of the other m onomer in the native dimer are very close to each other, (C) 2000 Elsevier Science B.V, All rights reserved.