Molecular cloning of aralar, a new member of the mitochondrial carrier superfamily that binds calcium and is present in human muscle and brain

Abstract

We have identified a new calcium-dependent subfamily of mitochondrial carrier proteins with members in Saccharomyces cerevisiae, Caenorhabditis elegans, and various mammalian species. The members of this subfamily have a bipartite structure: a carboxyl-terminal half with the characteristic features of the mitochondrial solute carrier superfamily and an amine- terminal extension harboring various EF-hand domains. A member of this subfamily (that we have termed Aralar) was cloned from a human heart cDNA library. The corresponding cDNA comprises an open reading frame of 2037 base pairs encoding a polypeptide of 678 amine acids. The carboxyl-terminal half of Aralar (amine acids 321-678) has high similarity with the oxoglutarate, citrate, and adenine nucleotide carriers (28-29% identity), whereas the amine-terminal half (amine acids 1-320) contains three canonical EF-hands. Aralar amine-terminal half was shown to bind calcium by 45Ca2+ overlay and calcium-dependent mobility shift assays. The subcellular localization of the protein in COS cells transfected with Aralar was exclusively mitochondrial. Antibodies against Aralar amine-terminal fusion protein recognized a 70-kDa protein in brain mitochondrial fractions. Northern blot analysis showed that the protein was expressed in heart, brain, and skeletal muscle. The domain structure, mitochondrial localization, and presence in excitable tissues suggests a possible function of Aralar as calcium- dependent mitochondrial solute carrier.