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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 is responsible for mitochondrial Ca2+ output.(A) Putative domain organization of LETM1. (B) Protease K digestion of LETM1 in mitochondria. The mitochondria were isolated from cells that overexpressed LETM1 and were incubated with increasing concentrations of digitonin in the presence of Protease K. The samples were analyzed using western blotting. An inner membrane protein TIM23, a mitochondrial matrix protein PRX3, and HA-LETM1 were tested. The HA tag position in LETM1 is indicated. (C) Schematic representation of the LETM1 localization on the inner mitochondrial membrane. The N-terminus of LETM1 is located on the mitochondrial intermembrane space and the C-terminus extends into the matrix. The orange sphere represents Ca2+. (D) Responses of the mitochondrial Ca2+ level in LETM1 knockdown HeLa cells after stimulation of 100 μM of histamine. (E) Western blot analysis of LETM1 protein levels in HeLa cells. Full gel and blots were shown in Figure S4. (F) Quantify human LETM1 protein levels only in HeLa cells by integrated density.
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f1: LETM1 is responsible for mitochondrial Ca2+ output.(A) Putative domain organization of LETM1. (B) Protease K digestion of LETM1 in mitochondria. The mitochondria were isolated from cells that overexpressed LETM1 and were incubated with increasing concentrations of digitonin in the presence of Protease K. The samples were analyzed using western blotting. An inner membrane protein TIM23, a mitochondrial matrix protein PRX3, and HA-LETM1 were tested. The HA tag position in LETM1 is indicated. (C) Schematic representation of the LETM1 localization on the inner mitochondrial membrane. The N-terminus of LETM1 is located on the mitochondrial intermembrane space and the C-terminus extends into the matrix. The orange sphere represents Ca2+. (D) Responses of the mitochondrial Ca2+ level in LETM1 knockdown HeLa cells after stimulation of 100 μM of histamine. (E) Western blot analysis of LETM1 protein levels in HeLa cells. Full gel and blots were shown in Figure S4. (F) Quantify human LETM1 protein levels only in HeLa cells by integrated density.

Mentions: LETM1 has been predicted to be a single transmembrane helix membrane protein that is located in the mitochondrial inner membrane (Fig. 1A). To verify the LETM1 localization in the mitochondria, we transfected the full length mouse LETM1-GFP into HeLa cells. The result shows that mouse LETM1-GFP were co-localized with Mito Tracker Red nicely, indicating that LETM1 is localized in the mitochondria (Figure S1). To further investigate the orientation of LETM1, a protease K digestion assay was performed. HEK293T cells were transfected with full length mouse LETM1 containing a haemagglutinin (HA) tag that was inserted between Glu115 and Asp116. The mitochondria were isolated from those HEK293T cells and then incubated with different concentrations of digitonin. PRX3, a mitochondrial matrix protein and Tim23, a mitochondria inner membrane protein, were used as the controls. Our data shows that as the mitochondria inner membrane remained intact, the N-terminus of LETM1 was partially digested, indicating that the N-terminus of LETM1 was first accessed by the protease K (Fig. 1B). When PRX3 was partially degraded by the protease K, the N-terminus of HA tagged LETM1 was digested completely. Thus, it is likely that the N-terminus of LETM1 is located in the intermembrane space, and the C-terminus extends to the mitochondrial matrix (Fig. 1C). Our results are consistent with several previous reports2231.


Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) forms a Ca 2+ /H + antiporter
LETM1 is responsible for mitochondrial Ca2+ output.(A) Putative domain organization of LETM1. (B) Protease K digestion of LETM1 in mitochondria. The mitochondria were isolated from cells that overexpressed LETM1 and were incubated with increasing concentrations of digitonin in the presence of Protease K. The samples were analyzed using western blotting. An inner membrane protein TIM23, a mitochondrial matrix protein PRX3, and HA-LETM1 were tested. The HA tag position in LETM1 is indicated. (C) Schematic representation of the LETM1 localization on the inner mitochondrial membrane. The N-terminus of LETM1 is located on the mitochondrial intermembrane space and the C-terminus extends into the matrix. The orange sphere represents Ca2+. (D) Responses of the mitochondrial Ca2+ level in LETM1 knockdown HeLa cells after stimulation of 100 μM of histamine. (E) Western blot analysis of LETM1 protein levels in HeLa cells. Full gel and blots were shown in Figure S4. (F) Quantify human LETM1 protein levels only in HeLa cells by integrated density.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037442&req=5

f1: LETM1 is responsible for mitochondrial Ca2+ output.(A) Putative domain organization of LETM1. (B) Protease K digestion of LETM1 in mitochondria. The mitochondria were isolated from cells that overexpressed LETM1 and were incubated with increasing concentrations of digitonin in the presence of Protease K. The samples were analyzed using western blotting. An inner membrane protein TIM23, a mitochondrial matrix protein PRX3, and HA-LETM1 were tested. The HA tag position in LETM1 is indicated. (C) Schematic representation of the LETM1 localization on the inner mitochondrial membrane. The N-terminus of LETM1 is located on the mitochondrial intermembrane space and the C-terminus extends into the matrix. The orange sphere represents Ca2+. (D) Responses of the mitochondrial Ca2+ level in LETM1 knockdown HeLa cells after stimulation of 100 μM of histamine. (E) Western blot analysis of LETM1 protein levels in HeLa cells. Full gel and blots were shown in Figure S4. (F) Quantify human LETM1 protein levels only in HeLa cells by integrated density.
Mentions: LETM1 has been predicted to be a single transmembrane helix membrane protein that is located in the mitochondrial inner membrane (Fig. 1A). To verify the LETM1 localization in the mitochondria, we transfected the full length mouse LETM1-GFP into HeLa cells. The result shows that mouse LETM1-GFP were co-localized with Mito Tracker Red nicely, indicating that LETM1 is localized in the mitochondria (Figure S1). To further investigate the orientation of LETM1, a protease K digestion assay was performed. HEK293T cells were transfected with full length mouse LETM1 containing a haemagglutinin (HA) tag that was inserted between Glu115 and Asp116. The mitochondria were isolated from those HEK293T cells and then incubated with different concentrations of digitonin. PRX3, a mitochondrial matrix protein and Tim23, a mitochondria inner membrane protein, were used as the controls. Our data shows that as the mitochondria inner membrane remained intact, the N-terminus of LETM1 was partially digested, indicating that the N-terminus of LETM1 was first accessed by the protease K (Fig. 1B). When PRX3 was partially degraded by the protease K, the N-terminus of HA tagged LETM1 was digested completely. Thus, it is likely that the N-terminus of LETM1 is located in the intermembrane space, and the C-terminus extends to the mitochondrial matrix (Fig. 1C). Our results are consistent with several previous reports2231.

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.