The leucine zipper, EF hand–containing transmembrane protein 1 ( Letm1 ) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca 2+ and K + homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca 2+ fluorophore and 45 Ca 2+ -based assays, we demonstrate directly that Letm1 is a Ca 2+ transporter, with apparent affinities of cations in the sequence of Ca 2+ ≈ Mn 2+ > Gd 3+ ≈ La 3+ > Sr 2+ >> Ba 2+ , Mg 2+ , K + , Na + . Kinetic analysis yields a Letm1 turnover rate of 2 Ca 2+ /s and a K m of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca 2+ /2 H + antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na + /Ca 2+ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H + -dependent Ca 2+ transport mechanism identified in intact mitochondria.
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