Dissecting structure and function of the monovalent cation/H⁺ antiporters Mdm38 and Ylh47 in Saccharomyces cerevisiae

Saccharomyces cerevisiae Mdm38 and Ylh47 are homologs of the Ca²⁺/H⁺ antiporter Letm1, a candidate gene for seizures associated with Wolf-Hirschhorn syndrome in humans. Mdm38 is important for K⁺/H⁺ exchange across the inner mitochondrial membrane and contributes to membrane potential formation and mitochondrial protein translation. Ylh47 also localizes to the inner mitochondrial membrane. However, knowledge of the structures and detailed transport activities of Mdm38 and Ylh47 is limited. In this study, we conducted characterization of the ion transport activities and related structural properties of Mdm38 and Ylh47. Growth tests using Na⁺/H⁺ antiporter-deficient Escherichia coli strain TO114 showed that Mdm38 and Ylh47 had Na⁺ efflux activity. Measurement of transport activity across E. coli-inverted membranes showed that Mdm38 and Ylh47 had K⁺/H⁺, Na⁺/H⁺, and Li⁺/H⁺ antiport activity, but unlike Letm1, they lacked Ca²⁺/H⁺ antiport activity. Deletion of the ribosome-binding domain resulted in decreased Na⁺ efflux activity in Mdm38. Structural models of Mdm38 and Ylh47 identified a highly conserved glutamic acid in the pore-forming membrane-spanning region. Replacement of this glutamic acid with alanine, a non-polar amino acid, significantly impaired the ability of Mdm38 and Ylh47 to complement the salt sensitivity of E. coli TO114. These findings not only provide important insights into the structure and function of the Letm1-Mdm38-Ylh47 antiporter family but by revealing their distinctive properties also shed light on the physiological roles of these transporters in yeast and animals.

Importance: The inner membrane of mitochondria contains numerous ion transporters, including those facilitating H⁺ transport by the electron transport chain and ATP synthase to maintain membrane potential. Letm1 in the inner membrane of mitochondria in animals functions as a Ca²⁺/H⁺ antiporter. However, this study reveals that homologous antiporters in mitochondria of yeast, Mdm38 and Ylh47, do not transport Ca²⁺ but instead are selective for K⁺ and Na⁺. Additionally, the identification of conserved amino acids crucial for antiporter activity further expanded our understanding of the structure and function of the Letm1-Mdm38-Ylh47 antiporter family.