Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier.

Mitochondrial ADP/ATP carriers catalyze the equimolar exchange of ADP and ATP across the mitochondrial inner membrane. Structurally, they consist of three homologous domains with a single substrate binding site. They alternate between a cytoplasmic and matrix state in which the binding site is accessible to these compartments for binding of ADP or ATP. It has been proposed that cycling between states occurs by disruption and formation of a matrix and cytoplasmic salt bridge network in an alternating way, but formation of the latter has not been shown experimentally. Here, we show that state-dependent formation of the cytoplasmic salt bridge network can be demonstrated by measuring the effect of mutations on the thermal stability of detergent-solubilized carriers locked in a specific state. For this purpose, mutations were made to increase or decrease the overall interaction energy of the cytoplasmic network. When locked in the cytoplasmic state by the inhibitor carboxyatractyloside, the thermostabilities of the mutant and wild-type carriers were similar, but when locked in the matrix state by the inhibitor bongkrekic acid, they correlated with the predicted interaction energy of the cytoplasmic network, demonstrating its formation. Changing the interaction energy of the cytoplasmic network also had a profound effect on the kinetics of transport, indicating that formation of the network is a key step in the transport cycle. These results are consistent with a unique alternating access mechanism that involves the simultaneous rotation of the three domains around a central translocation pathway.