title: Role of protein kinase C-mediated protein phosphorylation in mitochondrial translocation of mouse CYP1A1, which contains a non-canonical targeting signal.
abstract: A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 --> Leu and Tyr-5 --> Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 --> Asn and Leu-17 --> Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 --> Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.
authors.affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Venkata Ramesh
authors.initials: VR
authors.lastname: Dasari
authors.forename: Hindupur K
authors.initials: HK
authors.lastname: Anandatheerthavarada
authors.forename: Marie-Anne
authors.initials: MA
authors.lastname: Robin
authors.forename: Ettickan
authors.initials: E
authors.lastname: Boopathi
authors.forename: Gopa
authors.initials: G
authors.lastname: Biswas
authors.forename: Ji-Kang
authors.initials: JK
authors.lastname: Fang
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Nebert
authors.forename: Narayan G
authors.initials: NG
authors.lastname: Avadhani
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D019363:Cytochrome P-450 CYP1A1 / Q000096:biosynthesis* / Q000235:genetics*; D005288:Ferredoxins / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D051381:Rats; D017386:Sequence Homology, Amino Acid
chemical_list: D005288:Ferredoxins; D019363:Cytochrome P-450 CYP1A1; D011493:Protein Kinase C