title: A novel nuclear localization signal in the auxiliary domain of apobec-1 complementation factor regulates nucleocytoplasmic import and shuttling.
abstract: C to U editing of the nuclear apolipoprotein B (apoB) transcript is mediated by a core enzyme containing a catalytic deaminase, apobec-1, and an RNA binding subunit, apobec-1 complementation factor (ACF). ACF expression is predominantly nuclear, including mutant proteins with deletions of a putative nuclear localization signal. We have now identified a novel 41-residue motif (ANS) in the auxiliary domain of ACF that functions as an authentic nuclear localization signal. ANS-green fluorescence protein and ANS-beta-galactosidase chimeras were both expressed exclusively in the nucleus, whereas wild-type chimeras or an ACF deletion mutant lacking the ANS were cytoplasmic. Nuclear accumulation of ACF is transcription-dependent, temperature-sensitive, and reversible, features reminiscent of a shuttling protein. ACF relocates to the cytoplasm after actinomycin D treatment, an effect blocked by the CRM1 inhibitor leptomycin B. Heterokaryon assays confirmed directly that ACF shuttles in vivo. ACF binds to the protein carrier, transportin 2 in vivo, and colocalizes to the nucleus as determined by confocal microscopy. Co-immunoprecipitation experiments revealed that transportin 2 binds directly to the ANS motif. These data suggest that directed nuclear localization and compartmentalization of the core complex of the apoB RNA editing enzyme is regulated through a dominant targeting sequence (ANS) contained within ACF.
authors.affiliation: Departments of Internal Medicine and Pharmacology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
authors.forename: Valerie
authors.initials: V
authors.lastname: Blanc
authors.forename: Susan
authors.initials: S
authors.lastname: Kennedy
authors.forename: Nicholas O
authors.initials: NO
authors.lastname: Davidson
mesh_terms: D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics*; D013696:Temperature; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: C411647:A1CF protein, human; D000903:Antibiotics, Antineoplastic; D005231:Fatty Acids, Unsaturated; D019913:Nuclear Localization Signals; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D003609:Dactinomycin; D012313:RNA; D001616:beta-Galactosidase; C038753:leptomycin B