A Novel Nuclear Localization Signal in the Auxiliary Domain of Apobec-1 Complementation Factor Regulates Nucleocytoplasmic Import and Shuttling

• Published in: The Journal of biological chemistry 2003-10, Vol.278 (42), p.41198-41204
• Main Authors: Blanc, Valerie, Kennedy, Susan, Davidson, Nicholas O.
• Format: Article
• Language: English
• Subjects: Active Transport, Cell Nucleus; Amino Acid Motifs; Amino Acid Sequence; Animals; Antibiotics, Antineoplastic - pharmacology; beta-Galactosidase - metabolism; Cell Nucleus - metabolism; COS Cells; Cytoplasm - metabolism; Dactinomycin - pharmacology; Fatty Acids, Unsaturated - pharmacology; HeLa Cells; Humans; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Sequence Data; NIH 3T3 Cells; Nuclear Localization Signals; Protein Structure, Tertiary; RNA - metabolism; RNA, Messenger - metabolism; RNA-Binding Proteins - genetics; Temperature; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M302951200

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-β-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.