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

Abstract

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.

Major concerns with the integrity of the mitochondrial ADP/ATP carrier in dodecyl-phosphocholine used for solution NMR studies

“…1c). When CATR was added at a molar ratio of one or above, a marked shift in stability of AAC3 to higher temperatures is observed, consistent with earlier observations 14,16 . When the same AAC3 preparation was diluted into 3 mM DPC, a high signal was observed with no transition, showing that AAC3 in DPC is in a non--native state (Fig.…”

Calcium regulation of the human mitochondrial ATP-Mg/Pi carrier SLC25A24 uses a locking pin mechanism

“…Thermostability assays have previously been used to probe conformational changes of mitochondrial carriers by assessing their thermostability in different states252627. Here, they have been applied to determine the differences in thermostability between the calcium-bound and calcium-free state of detergent-solubilised APC1.…”

In vitro reconstitution, functional dissection, and mutational analysis of metal ion transport by mitoferrin-1

“…1, S1). These residues are thought be important for stabilizing the matrix-or the cytosol-facing conformations of the carriers (16). Ablating one, two, or three C-gate salt bridges in TMfrn1 by replacing charged K or D/E with uncharged A results in essentially non-expressing protein (Fig.…”

“…Yet they transport a wide array of small-and medium-size organic molecules and metabolites with a large stereochemical diversity (12). The conserved topology is a tripartite repeat of about 100 amino acids folded into two transmembrane helices connected by an amphipathic helix at the membrane-aqueous interface in the mitochondrial matrix ( 2 [K/R] motifs, one on each side of the membrane, are hypothesized to constitute two salt-bridge networks which alternate in gating the MCs during their transport cycles (13,15,16). The conserved topology, in combination with the diversity of the structures of molecules transported, pose intriguing questions about the mechanism of transport used by the MCs.…”