title: Mitochondrial targeting of normal and mutant protoporphyrinogen oxidase.
abstract: We have investigated the signal sequence for mitochondrial transport of mutants (I12T, 78insC, IVS2-2a-->c, 338G-->C, R152C, 470A-->C, and L401F) and the wild type protoporphyrinogen oxidase (PPOX), which is the penultimate enzyme in the heme biosynthesis. We constructed the corresponding green fluorescent protein fusion proteins and studied their intracellular localization in COS-1 cells. We showed that 28 amino acids in the amino terminus of PPOX contain an independently functioning signal for mitochondrial targeting. The experiments with amino-terminally truncated green fluorescent protein fusion proteins revealed that amino acids 25-477 of PPOX contained an additional mitochondrial targeting signal(s). We constructed a structural model for the interaction between the amino-terminal end of PPOX and the putative mitochondrial receptor protein Tom20. The model suggests that leucine and isoleucine residues Leu-8, Ile-12, and Leu-15 forming an alpha-helical hydrophobic motif, LXXXIXXL, were crucial for the recognition of the targeting signal. The validity of the model was tested using mutants L8Q, I12T, and L15Q disrupting the hydrophobic surface of the LXXXIXXL helix. The results from in vitro expression studies and molecular modeling were in accordance supporting the hypothesis that the recognition of the mitochondrial targeting signal is dependent on hydrophobic interactions between the targeting signal and the mitochondrial receptor.
authors.affiliation: Department of Medicine, Division of Endocrinology, University of Helsinki, Biomedicum Helsinki, 00029 HUS, Helsinki, Finland. mikael.fraunberg@hus.fi
authors.forename: Mikael
authors.initials: M
authors.lastname: von und zu Fraunberg
authors.forename: Tommi
authors.initials: T
authors.lastname: Nyröen
authors.forename: Raili
authors.initials: R
authors.lastname: Kauppinen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017931:DNA Primers; D005420:Flavoproteins; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D050768:Protoporphyrinogen Oxidase; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D017931:DNA Primers; D005420:Flavoproteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C493978:PPOX protein, human; D050768:Protoporphyrinogen Oxidase