The ADP/ATP carrier protein (AAC) is a member of the solute carrier protein family. These integral membrane proteins control the movement of small molecules across the inner mitochondrial membrane and are vital for energy metabolism and other mitochondrial functions. Being encoded by nuclear genes and expressed in the cytosol, carrier proteins must be specifically imported into mitochondria and nowhere else. However, they lack a cleavable N-terminal presequence and their import through the mitochondrial translocase of the outer membrane (TOM) complex is thought to be directed by multiple permanent internal targeting sequences located in and around their six hydrophobic transmembrane α-helices. These internal targeting sequences are recognized by the Tom70 receptor subunit of the TOM complex.

In the yeast Saccharomyces cerevisiae, there are two members of this class of receptor, Tom70 and Tom71. Here, we characterised the interactions between Tom71 and AAC as a general model for Tom70-mediated import of substrates. Initially, the affinity and stoichiometry of Tom71 binding was measured using synthetic peptides corresponding to the individual transmembrane regions of AAC. Subsequently, these long peptides were subdivided to reveal the specific locations of Tom71 binding. This data suggested multi-site interactions between substrate and receptor. Site-directed mutagenesis of Tom71 was then employed to interrogate potential binding sites on the receptor. Finally, the structures of Tom71 bound to these peptides were solved using X-ray crystallography. These experiments revealed three distinct substrate-binding sites on Tom71. We have concluded from our studies that the Tom70 family of receptors recognize their substrates by reading the sequence context, length and spacing of strings of hydrophobic residues. In this way multiple weak interactions can be combined to generate high affinity and specificity of substrate recognition at the mitochondrial surface, but allows for effective completion by downstream components that pulls the substrate further into the organelle.