Figure 3

LETM1 forms a Ca²⁺/H⁺ antiporter in vitro.

(A) Gel-filtration chromatography analysis of the purified LETM1. The peak fractions were analyzed via SDS-PAGE. The calculated molecular weight was approximately 404 kDa. (B) The pH-dependent conformational change was analyzed using circular dichroism. LETM1 exhibits an obvious conformational change when the pH shifts from alkaline to acidic. (C) Schematic drawing of the liposome assay system. The liposomes were prepared with 50 μM of the Ca²⁺-sensitive dye, Fura-2, inside and the Ca²⁺ influx was initiated by adding 100 μM of CaCl₂. (D) LETM1 wild-type Ca²⁺/H⁺ antiporter activities. The Ca²⁺ transport ability increased as the pH gradient increased. (E) Three different views of the negative staining EM density map of LETM1 at pH 8.0. The diameter of the central cavity was indicated by dotted black lines and arrows. (F) Views of the negative staining EM map of LETM1 at pH 6.5. The plunger was indicated by the black arrow.