Human mitochondrial ADP/ATP carrier SLC25A4 operates with a ping‐pong kinetic mechanism

The mitochondrial ADP/ATP carrier (SLC25A4), also called the adenine nucleotide translocase, imports ADP into the mitochondrial matrix and exports ATP, which are key steps in oxidative phosphorylation. Historically, the carrier was thought to form a homodimer and to operate by a sequential kinetic mechanism, which involves the formation of a ternary complex with the two exchanged substrates bound simultaneously. However, recent structural and functional data have demonstrated that the mitochondrial ADP/ATP carrier works as a monomer and has a single substrate binding site, which cannot be reconciled with a sequential kinetic mechanism. Here, we study the kinetic properties of the human mitochondrial ADP/ATP carrier by using proteoliposomes and transport robotics. We show that the Km/Vmax ratio is constant for all of the measured internal concentrations. Thus, in contrast to earlier claims, we conclude that the carrier operates with a ping‐pong kinetic mechanism in which substrate exchange across the membrane occurs consecutively rather than simultaneously. These data unite the kinetic and structural models, showing that the carrier operates with an alternating access mechanism. Synopsis The human mitochondrial ADP/ATP carrier (SLC25A4) operates with a ping‐pong kinetic mechanism in which one substrate is imported before the other substrate is exported. The kinetics of the mitochondrial ADP/ATP carrier was studied using proteoliposomes and robotic transport assays. Uptake curves were recorded to provide an accurate assessment of the initial transport rates in different substrate concentration gradients. In two‐substrate initial‐velocity studies, the ratio of Km/Vmax is constant for all measured internal concentrations of the substrate. The carrier operates with a ping‐pong kinetic mechanism in which the import and export transport steps occur consecutively. Graphical Abstract The human mitochondrial ADP/ATP carrier (SLC25A4) operates with a ping‐pong kinetic mechanism in which one substrate is imported before the other substrate is exported.