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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.
Highlights
The LETM1 gene was first reported in 1999 and is missing in most patients who suffer from Wolf-Hirschhorn Syndrome (WHS), which is a syndrome that includes severe mental and development retardation, muscle weakness and seizures[21]
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)
HEK293T cells were transfected with full length mouse LETM1 containing a haemagglutinin (HA) tag that was inserted between Glu[115] and Asp[116]
Summary
The LETM1 gene was first reported in 1999 and is missing in most patients who suffer from Wolf-Hirschhorn Syndrome (WHS), which is a syndrome that includes severe mental and development retardation, muscle weakness and seizures[21]. LETM1 affects cell growth and survival in C. elegans and in mice[26,27]. LETM1 contains an N-terminal domain (NTD) and a C-terminal domain (CTD) that are connected by one transmembrane helix. It has been argued that LETM1 cannot act as a transporter that mediates ion flux because it only has one transmembrane helix[28]. The reconstitution of the purified protein LETM1 into the liposome showed that it can transport Ca2+ ions that are dependent on the H+ concentration[30]. For the first time, we observed that LETM1 has two conformational states and functions as a Ca2+/H+ antiporter using biochemical and cell biology methods, an in vitro liposome assay and negative stain electron microscopy
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