title: Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of heterologous proteins to endoplasmic reticulum and mitochondria.
abstract: Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.
authors.affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6047, USA.
authors.forename: S V
authors.initials: SV
authors.lastname: Bhagwat
authors.forename: G
authors.initials: G
authors.lastname: Biswas
authors.forename: H K
authors.initials: HK
authors.lastname: Anandatheerthavarada
authors.forename: S
authors.initials: S
authors.lastname: Addya
authors.forename: W
authors.initials: W
authors.lastname: Pandak
authors.forename: N G
authors.initials: NG
authors.lastname: Avadhani
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D053493:Cholestanetriol 26-Monooxygenase; D019363:Cytochrome P-450 CYP1A1 / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007150:Immunohistochemistry; D008928:Mitochondria / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D053493:Cholestanetriol 26-Monooxygenase; D013762:Tetrahydrofolate Dehydrogenase