Identification of nuclear export signals in antizyme-1.
Antizyme-1 (AZ1) is a protein that negatively regulates polyamine synthesis by inhibiting the key synthetic enzyme ornithine decarboxylase and targeting it for degradation by the 26 S proteasome. Recent work shows that antizyme protein translocates to the nucleus during mouse development (Gritli-Linde, A., Nilssom, J., Bohlooly, Y. M., Heby, O., and Linde, A. (2001) Dev. Dyn. 220, 259-275). However, the significance and mechanism of this phenomenon remain unclear. In this study, we expressed AZ1 fused with enhanced green fluorescent protein (EGFP) to study its localization in a living cell. We found that EGFP-AZ1 was predominantly localized in the cytoplasm and that treatment with leptomycin B, a specific inhibitor of chromosomal region maintenance 1 (CRM1) induced nuclear accumulation of EGFP-AZ1 in Chinese hamster ovary and NIH3T3 cells. Two independent nuclear export signal (NES) sequences, each containing essential hydrophobic residues, were identified in the 50 N-terminal amino acid residues and in the central part of AZ1. The activity of the second NES was inhibited by an N-terminal adjacent region and was only revealed in N-terminal truncated constructs. Both NESs were active when fused to an artificial nuclear protein SV40-NLS-EGFP-EGFP. The ability of AZ1 to shuttle between the nucleus and the cytoplasm suggests that it has a novel function in the nucleus.
Murai
Noriyuki
N
Second Department of Biochemistry, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan. nmurai@jikei.ac.jp
Murakami
Yasuko
Y
Matsufuji
Senya
S
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004330:Drosophila; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D065108:Ornithine Decarboxylase Inhibitors; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D015398:Signal Transduction; D014162:Transfection
D005231:Fatty Acids, Unsaturated; D008164:Luminescent Proteins; D065108:Ornithine Decarboxylase Inhibitors; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; C031017:ornithine decarboxylase antizyme; D049452:Green Fluorescent Proteins; C038753:leptomycin B