Mitochondrial leader sequences: structural similarities and sequence differences.

While having essentially no amino acid sequence homology, the mitochondrial leader sequences of different pre-proteins carry out the same function of targeting the protein to mitochondria. Among the common attributes that have been noted for leader sequences are a net positive charge and the ability to form amphiphilic alpha-helices. The aim of the research described here was to determine the relative importance of these two attributes in the leader sequence of rat liver mitochondrial aldehyde dehydrogenase. Through site-directed mutagenesis, arginine residues were systematically replaced by glutamine. It was found that individual arginines could be replaced without loss of import competence, so the total charge of the leader sequence was not required for the pre-protein to be imported. However, when two arginines were replaced simultaneously, especially Arg3 and Arg10, the pre-protein lost the ability to be imported. This ability was restored by modifying the leader sequence to increase dramatically its helix-forming potential. This leader sequence did not contain a positive-charged side-chain until Arg11. Therefore, it has been shown that positive charge is not required in the first ten residues provided the sequence could form a relatively more stable alpha-helix. The ability to form an amphiphilic alpha-helix remains the essential factor in determining whether or not a leader sequence can carry out its import function.