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.