MOLECULAR-CLONING OF A PEROXISOMAL CA2-DEPENDENT MEMBER OF THE MITOCHONDRIAL CARRIER SUPERFAMILY()

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA l ibrary, The full length cDNA clone was 3,298 nt long and coded for a p rotein of 475 amino acids, with four elongation factor-hand motifs loc ated in the N-terminal half of the molecule, The 25-kDa N-terminal pol ypeptide was expressed in Escherichia coli, and it was demonstrated th at it bound Ca2+, undergoing a reversible and specific conformational change as a result, The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (K-d approximate to 0.37 mu M), the apparent Hill coefficient for Ca2+-induced changes being abou t 2.0, The deduced amino acid sequence of the C-terminal half of the m olecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identif ied the protein as a new member of the mitochondrial transporter super family, Northern blot analysis revealed the presence of a single trans cript of about 3,500 bases, and low expression of the transporter coul d be detected in the kidney but none in the liver. The main site of ex pression was the colon with smaller amounts found in the small intesti ne proximal to the ileum, Immunoelectron microscopy localized the tran sporter in the peroxisome, although a minor fraction was found in the mitochondria, The Ca2+ binding N-terminal half of the transporter face s the cytosol.