Chimers of Two Fused ADP/ATP Carrier Monomers Indicate a Single Channel for ADP/ATP Transport

The mitochondrial ADP/ATP carrier (AAC) is generally believed to function as a homodimer (Wt · Wt). It remains unknown whether the two monomers possess two independent but fully anticooperative channels or they form a single central channel for nucleotide transport. Here we generated fusion proteins consisting of two tandem covalent-linked AAC monomers and studied the kinetics of ADP/ATP transport in reconstituted proteoliposomes. Functional 64-kDa fusion proteins Wt–Wt and Wt–R294A (wild-type AAC linked to a mutant having low ATP transport activity) were expressed in mitochondria of yeast transformants. Compared to homodimer Wt · Wt, the fusion protein Wt–Wt retained the transport activity and selectivity of ADP versus ATP. The strongly divergent selectivities of Wt and R294A were partially propagated in the Wt–R294A fusion protein, suggesting a limited cooperativity during solute translocation. The rates of ADP or ATP transport were significantly higher than those predicted by the two-channel model. Fusion proteins for Wt–R204L (Wt linked to an inactive mutant) and R204L–Wt were not expressed in aerobically grown yeast cells, which contained plasmid rearrangements that regenerated the fully active 32-kDa homodimer Wt · Wt, suggesting that these fusion proteins are inactive in ADP/ATP transport. These results favor a single binding center gated pore model [Klingenberg, M. (1991) in A Study of Enzymes, Vol. 2: pp. 367–388] in which two AAC subunits cooperate for a coordinated ADP/ATP exchange through a single channel.