The molecular mechanism of substrate binding and transport of the mitochondrial ADP/ATP carrier

Abstract

Mitochondrial ADP/ATP carriers provide key transport steps in eukaryotic oxidative phosphorylation by importing ADP into the mitochondrial matrix and by exporting syn- thesized ATP to fuel cellular processes. Structures of the in- hibited cytoplasmic- and matrix-open states have confirmed an alternating access mechanism, but the molecular nature of substrate binding is unresolved. Here, we investigate all solvent-exposed residues in the translocation pathway in both conformational states of the mitochondrial ADP/ATP carrier. By combining binding studies with functional assays using single alanine replacement variants, we identify a single substrate binding site in the center of the cavity. It consists of three positively charged residues, which could bind the phosphate groups, and a cluster of three aliphatic and one aromatic residue, which could bind the adenosine moiety of M the nucleotides. In addition, two arginine/asparagine pairs are identified, which are unique to ADP/ATP carriers but play a role in binding. Importantly, the same residues are O involved in binding of both ADP and ATP, implying that the import and export steps occur consecutively and in a N reversible way. The features of the identified binding site explain the electrogenic nature of transport