A novel nuclear localization signal in the auxiliary domain of apobec-1 complementation factor regulates nucleocytoplasmic import and shuttling.
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.
Mesh Terms:
Active Transport, Cell Nucleus, Amino Acid Motifs, Amino Acid Sequence, Animals, Antibiotics, Antineoplastic, COS Cells, Cell Nucleus, Cytoplasm, Dactinomycin, Fatty Acids, Unsaturated, Hela Cells, Humans, Mice, Microscopy, Confocal, Microscopy, Fluorescence, Molecular Sequence Data, NIH 3T3 Cells, Nuclear Localization Signals, Protein Structure, Tertiary, RNA, RNA, Messenger, RNA-Binding Proteins, Temperature, Transfection, beta-Galactosidase
Active Transport, Cell Nucleus, Amino Acid Motifs, Amino Acid Sequence, Animals, Antibiotics, Antineoplastic, COS Cells, Cell Nucleus, Cytoplasm, Dactinomycin, Fatty Acids, Unsaturated, Hela Cells, Humans, Mice, Microscopy, Confocal, Microscopy, Fluorescence, Molecular Sequence Data, NIH 3T3 Cells, Nuclear Localization Signals, Protein Structure, Tertiary, RNA, RNA, Messenger, RNA-Binding Proteins, Temperature, Transfection, beta-Galactosidase
J. Biol. Chem.
Date: Oct. 17, 2003
PubMed ID: 12896982
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