Biogenesis of eel liver citrate carrier (CIC): negative charges can substitute for positive charges in the presequence

A family of structurally related carrier proteins mediates the flux of metabolites across the mitochondrial inner membrane. Differently from most other mitochondrial proteins, members of the carrier family are synthesized without an amino-terminal targeting sequence. However, in some mammalian and plant species, representatives were identified that carry a positively charged presequence. To obtain data on a carrier protein from lower vertebrates, we determined the primary structure of eel mitochondrial citrate carrier (CIC) and investigated its import pathway into the target organelle. To our knowledge this is the first study on the molecular mechanisms of protein import into fish mitochondria. The N-terminal extension of eel CIC comprises 20 residues, including two positively charged amino acids (K6 and R13). The cleavage site is recognized by a magnesium-dependent peptidase in the intermembrane space. We found that the presequence was neither required for targeting nor for import into mitochondria but significantly increased the solubility of the preprotein. An exchange of both positively charged amino acids against negatively charged residues completely retained the chaperoning effect of the presequence. Strikingly, the authentic sequences of several carrier proteins show both positively and negatively charged residues in the N-termini, suggesting that these sequences may act as non-cleavable intramolecular chaperones similar to the cleavable presequences of vertebrate CIC.