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

Abstract

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.

Keywords: S. cerevisiae; cation/H+ antiporter; mitochondria; yeast.

Fig 1

Growth of Na⁺ efflux or K⁺ uptake-deficient E. coli strains containing Mdm38 or Ylh47. (A and B) Complementation assay with Na⁺/H⁺ antiporter-deficient E. coli strain TO114 transformed with plasmids encoding EcNhaA, Mdm38, Ylh47, or the empty vector pTrcHis2B. Tenfold serial dilutions of the cells were spotted on KLB with 0 mM or 25 mM NaCl and 10 µM (A) or 100 µM (B) IPTG. A representative example of three biological replicates is shown. (C) Growth of K⁺ uptake-deficient E. coli strain ∆dghu containing plasmids encoding AtKEA3, Mdm38, Ylh47, or the empty vector pTrcHis2B. Tenfold serial dilutions of the cells were spotted on KLB medium or K⁺ minimal medium supplemented with 10, 20, or 30 mM KCl. A representative example of three biological replicates is shown.

Fig 2

Cation/H⁺ antiport activities of Mdm38 and Ylh47. (A) Determination of K⁺/H⁺ antiporter activities in inverted membranes by dequenching of acridine orange fluorescence. The closed triangle and open triangle indicate the time when L-lactate and KCl were added, respectively. Finally, CCCP was added (gray triangle) to collapse the ∆pH across the inverted membranes, and the filled arrow indicates when that was achieved. Solid lines represent the results with Mdm38 and Ylh47, and dashed lines indicate the data obtained with membranes isolated from TO114 containing the empty vector (EV). Traces shown are representative examples of three biological replicates. (B) Determination of K⁺, Na⁺, Li⁺, Rb⁺, and Ca²⁺ transport activity of Mdm38 and YLH47. Assays were performed as for (A) but instead of KCl, NaCl, LiCl, RbCl, or CaCl₂ was added to the inverted membranes. Error bars represent SD (n = 3). The significance of differences between the control of E. coli TO114 with the EV and TO114 expressing Mdm38 and Ylh47 was analyzed by Students’ t-test (**P < 0.01, *P < 0.05, n = 3).

Fig 3

Michaelis-Menten kinetics of K⁺/H⁺ antiport and Na⁺/H⁺ antiport activities of Mdm38 and Ylh47 K⁺/H⁺ antiport activity and Na⁺/H⁺ antiport activity of Mdm38 (A) and Ylh47 (B) with varying concentrations of KCl or NaCl. The experiments were conducted by measuring dequenching of acridine orange fluorescence in inverted membranes as in Fig. 2. The error bars represent SD (n = 3) at different concentrations of KCl and NaCl.

Fig 4

The C-terminal motif in Mdm38 is required for ion transport activity. (A) Model showing the predicted domains in Letm1, Mdm38, and Ylh47. EF, EF-hand domain. The black arrows indicate coiled-coil regions. (B) Complementation of salt sensitivity of E. coli TO114 by EcNhaA, Mdm38, Mdm38 ∆RBD, and Mdm38 ∆cc. Tenfold serial dilutions of the cells were spotted on KLB containing 10 µM IPTG plus 0 mM or 25 mM NaCl. A representative example of three biological replicates is shown.

Fig 5

Structural model of Letm1, Mdm38, and Ylh47 showing the conserved glutamic acid in the transmembrane region. The amino acid sequence alignment of the transmembrane regions of Letm1, Mdm38, and Ylh47 is shown at the top, and the conserved glutamic acid is shown in red. The model at the bottom shows the structure for members of the Letm1-Mdm38-Ylh47 family as a hexamer. The side view is shown on the left, and the bottom view focused on the TM region (i.e., pore) on the right. The dashed six-pointed star approximates the shape of the RBDs (although the actual size would be much larger). The conserved glutamic acids are represented by red and gray spheres, with a bound ion at the center (manually placed).

Fig 6

Ion transport activities of Mdm38 E152A and Ylh47 E150A. Growth assay of E. coli TO114 containing EcNhaA, Mdm38, Mdm38 E152A, Ylh47, or Ylh47 E150A on media with 0 or 25 mM NaCl. Tenfold serial dilutions of the cells were spotted on KLB containing 10 µM IPTG (A) or 100 µM IPTG (B).The representative data of three biological replicates are shown. A representative example of three biological replicates is shown.

Fig 7

Comparison of cation/H⁺ antiport activities and structures of Mdm38, Ylh47, and Letm1. LETM1, which has an EF-hand motif in its C-terminal region, has Ca²⁺/H⁺ antiport activity and also contributes to K⁺/H⁺ antiport activity. Mdm38 and Ylh47, which contain no EF-hand motif, showed K⁺/H⁺ and Na⁺/H⁺ antiport activity. The conserved glutamic acid residues in the transmembrane region of Mdm38 (E152), Ylh47 (E150), and Letm1 (E222) are labeled.