The mitochondrial tricarboxylate transport protein. cDNA cloning, primary structure, and comparison with other mitochondrial transport proteins.

The amino acid sequence of the rat liver mitochondrial tricarboxylate transport protein has been deduced from its corresponding cDNA. Using the polymerase chain reaction, with primers derived from amino acid sequence information that we obtained by direct sequencing of the purified transporter and its internal peptides, a cDNA fragment was amplified that encodes approximately two-thirds of the tricarboxylate transport protein. This cDNA fragment was used to screen a rat liver lambda gt11 cDNA library which permitted the isolation and sequencing of a cDNA clone that encodes the entire tricarboxylate transporter. The clone is 1927 base pairs in length with 5'- and 3'-untranslated regions of 419 and 572 base pairs, respectively. The open reading frame encodes a mature transport protein of 298 amino acids preceded by a presequence of 13 residues. Analysis of the tricarboxylate transporter sequence indicates that it contains three related sequence domains, each of approximately 100 amino acid residues in length. Dot plot comparisons and sequence alignment indicate that these domains are related to each other, as well as to domains of similar length that are present in other mitochondrial transporters. Hydrophobicity analysis predicts that the tricarboxylate carrier contains six membrane-spanning alpha-helices (two per 100-amino acid sequence domain) and has permitted the construction of an initial model for the topography of this transporter within the mitochondrial inner membrane. Finally, Southern blot analysis of both rat and human genomic DNA demonstrates the presence of multiple sequences related to the tricarboxylate transporter in both genomes. These studies provide the first information on the primary structure of the mitochondrial tricarboxylate transport protein. We are now able, on the basis of both structural and functional considerations, to assign this metabolically important transporter to the mitochondrial carrier family, the members of which are likely to have evolved from a common genetic origin.