A family of related carrier proteins mediates the exchange of metabolites across the mitochondrial inner membrane. The carrier signature Px[D/E]xx[K/R] is a highly conserved sequence motif in all members of this family. To determine its function in the biogenesis of carrier proteins, we used the dicarboxylate carrier (DIC) of yeast as a model protein. We found that the carrier signature was dispensable in binding of the newly synthesized protein to the import receptor Tom70, but that it was specifically required for efficient translocation across the mitochondrial outer membrane. To determine the relevance of individual amino acid residues of the carrier signature in the transport activity of the protein, we exchanged defined residues with alanine and reconstituted the mutant proteins in vitro. Substitution of the carrier signature in helix H1 reduced the transport activity for [(33)P]-phosphate by approximately 90% and an additional substitution of the carrier signature in helix H5 blocked the transport activity completely. We conclude that the carrier signature of the dicarboxylate carrier is involved both in the biogenesis and in the transport activity of the functional protein.