MITOCHONDRIAL TARGETING MECHANISMS OF PROTOPORPHYRINOGEN OXIDASE: IDENTIFICATION AND CHARACTERIZATION OF INTERNAL TARGETING SEQUENCES

von und zu Fraunberg M, Kauppinen R

Department of Medicine, Division of Endocrinology, University Central Hospital of Helsinki, Biomedicum Helsinki, Finland

Recent studies have revealed that a relatively short 28-amino acid segment in the amino terminus of protoporphyrinogen oxidase (PPOX) contains information, which is sufficient to direct the protein into mitochondria. Furthermore, our experiments with amino-terminally truncated green fluorescent protein (GFP)-fusion polypeptides showed that amino acids 25-477 of PPOX contained an additional mitochondrial targeting signal(s), which could direct the reporter protein into mitochondria even in the absence of the amino-terminal signal. We have investigated the internal signal sequences for mitochondrial transport of PPOX by performing a secondary structure analysis of the polypeptide and studying the intracellular localization of mutated PPOX-GFP-fusion proteins in COS-1 cells. Since the model for the interaction between the amino terminus of PPOX and the putative mitochondrial receptor protein Tom20 suggested that leucine and isoleucine residues forming an alpha-helical hydrophobic motif LXXXIXXL were crucial for the recognition of the targeting signal, we searched for helical, leucine-rich segments with net positive charge and chose four most promising candidate segments. Three of the segments could direct PPOX into mitochondria when replacing the authentic amino-terminal targeting signal. Substituting polar glutamine for non-polar leucine residues of these segments could impair the mitochondrial transport in the absence of the amino-terminal targeting signal indicating that these regions contribute to internal mitochondrial targeting signaling of PPOX. The importance of leucine and isoleucine residues for the mitochondrial targeting suggests that hydrophobic interactions are involved also in the recognition of internal targeting signals, possibly through interaction with Tom20 or other mitochondrial receptors