Yeast Mitochondrial Carriers: Bacterial Expression, Biochemical Identification and Metabolic Significance

The genome of Saccharomyces cerevisiae encodes 35 members of a family proteins thattransport metabolites and substrates across the inner membranes of mitochondria. They includethree isoforms of the ADP/ATP translocase and the phosphate and citrate carriers. At the startof our work, the functions of the remaining 30 members of the family were unknown. We areattempting to identify these 30 proteins by overexpression of the proteins in specially selectedhost strains of Escherichia coli that allow the carriers to accumulate at high levels in the formof inclusion bodies. The purified proteins are then reconstituted into proteoliposomes wheretheir transport properties are studied. Thus far, we have identified the dicarboxylate,succinate-fumarate and ornithine carriers. Bacterial overexpression and functional identification, togetherwith characterization of yeast knockout strains, has brought insight into the physiologicalsignificance of these transporters. The yeast dicarboxylate carrier sequence has been used toidentify the orthologous protein in Caenorhabditis elegans and, in turn, this latter sequencehas been used to establish the sequence of the human ortholog.