Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism

Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible. Synopsis The human mitochondrial oxoglutarate carrier (OGC), citrate carrier (CIC), dicarboxylate carrier (DIC) and aspartate/glutamate carrier (AGC2) of the SLC25 family were all thought to be homodimers, operating with a sequential mechanism in which the two exchanged substrates bind at the same time to the transporter. This study shows that they are monomers, except for the dimeric aspartate/glutamate carrier (AGC2), and that they import one substrate first before the other is exported. The human mitochondrial citrate, dicarboxylate and oxoglutarate carriers are structural monomers. In contrast to these carriers, the mitochondrial aspartate/glutamate carrier is a structural dimer. They all operate with a ping-pong kinetic mechanism, where one substrate is imported first, before the the counter-substrate is exported. These kinetic and structural observations are consistent with an alternating access mechanism for mitochondrial carriers using a single substrate-binding site. The oxoglutarate, citrate and dicarboxylate carriers are monomeric in contrast