The mitochondrial inner membrane is host to integral membrane proteins that play indispensable roles in the metabolic pathways of the mitochondria. Excluding select oxidative phosphorylation components, inner membrane proteins are encoded in the nucleus and imported, undergoing a sequence of chaperoned transfers en route. Dedicated mitochondrial machinery mediates the linked processes of translocation, membrane insertion and assembly (reviewed in 1).

This study focuses on understanding the molecular basis of import of inner membrane carrier proteins, of which the ATP/ADP carrier protein (AAC), also known as the Adenine Nucleotide Translocase (ANT) is a well-known representative. Members of this class of proteins are characterized by the presence of six membrane-spanning helices that correspond to three internal two-helix repeats connected by short hydrophilic loops. Although their general import pathway has been elucidated, the targeting signals of the inner membrane proteins, their molecular interactions with translocase components, and the sequence of events, remain to be unraveled.

In this study, we seek to identify and characterize the interactions of human ANT isoform 1 precursor with translocase components viz., Tom70, the outer surface receptor and TIM9.10, the intermembrane space chaperone complex. In preparation for structural studies, a combination of purified recombinant components and shorter transmembrane polypeptides have been produced in house. We are developing in vitro fluorescence based assays to determine the binding affinities and co-crystallisation trials have been initiated with the aim of obtaining molecular snapshots of precursor-translocase complexes. These are important steps toward elucidating the mechanistic basis of targeting and transfer and could offer valuable insights into the pathway as a whole.