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Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) forms a Ca 2+ /H + antiporter

View Article: PubMed Central - PubMed

ABSTRACT

Leucine zipper-EF-hand-containing transmembrane protein1 (LETM1) is located in the mitochondrial inner membrane and is defective in Wolf-Hirschhorn syndrome. LETM1 contains only one transmembrane helix, but it behaves as a putative transporter. Our data shows that LETM1 knockdown or overexpression robustly increases or decreases mitochondrial Ca2+ level in HeLa cells, respectively. Also the residue Glu221 of mouse LETM1 is identified to be necessary for Ca2+ flux. The mutation of Glu221 to glutamine abolishes the Ca2+-transport activity of LETM1 in cells. Furthermore, the purified LETM1 exhibits Ca2+/H+ anti-transport activity, and the activity is enhanced as the proton gradient is increased. More importantly, electron microscopy studies reveal a hexameric LETM1 with a central cavity, and also, observe two different conformational states under alkaline and acidic conditions, respectively. Our results indicate that LETM1 is a Ca2+/H+ antiporter and most likely responsible for mitochondrial Ca2+ output.

No MeSH data available.


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.
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f3: 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.

Mentions: To explore the possibility whether LETM1 forms a functional transporter even though it only has one transmembrane helix (Fig. 1A), we looked for additional evidence in vitro. We first expressed and purified the recombinant mouse LETM1 (residues 116–698) with the mitochondrial signal peptide deleted, referred as LETM1-delta hereafter. The oligomerization analysis of LETM1-delta via gel-filtration show that the purified LETM1-delta is a hexamer with a molecular weight of approximately 404 kDa (the theoretical LETM1-delta monomer is 66 kDa) (Fig. 3A). We further verified the secondary structure via circular dichroism, which surprisingly shows that LETM1 possibly exists as two conformational states under different pH conditions (Fig. 3B).


Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) forms a Ca 2+ /H + antiporter
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.
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Related In: Results  -  Collection

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f3: 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.
Mentions: To explore the possibility whether LETM1 forms a functional transporter even though it only has one transmembrane helix (Fig. 1A), we looked for additional evidence in vitro. We first expressed and purified the recombinant mouse LETM1 (residues 116–698) with the mitochondrial signal peptide deleted, referred as LETM1-delta hereafter. The oligomerization analysis of LETM1-delta via gel-filtration show that the purified LETM1-delta is a hexamer with a molecular weight of approximately 404 kDa (the theoretical LETM1-delta monomer is 66 kDa) (Fig. 3A). We further verified the secondary structure via circular dichroism, which surprisingly shows that LETM1 possibly exists as two conformational states under different pH conditions (Fig. 3B).

View Article: PubMed Central - PubMed

ABSTRACT

Leucine zipper-EF-hand-containing transmembrane protein1 (LETM1) is located in the mitochondrial inner membrane and is defective in Wolf-Hirschhorn syndrome. LETM1 contains only one transmembrane helix, but it behaves as a putative transporter. Our data shows that LETM1 knockdown or overexpression robustly increases or decreases mitochondrial Ca2+ level in HeLa cells, respectively. Also the residue Glu221 of mouse LETM1 is identified to be necessary for Ca2+ flux. The mutation of Glu221 to glutamine abolishes the Ca2+-transport activity of LETM1 in cells. Furthermore, the purified LETM1 exhibits Ca2+/H+ anti-transport activity, and the activity is enhanced as the proton gradient is increased. More importantly, electron microscopy studies reveal a hexameric LETM1 with a central cavity, and also, observe two different conformational states under alkaline and acidic conditions, respectively. Our results indicate that LETM1 is a Ca2+/H+ antiporter and most likely responsible for mitochondrial Ca2+ output.

No MeSH data available.