A novel nuclear export signal sensitive to oxidative stress in the fission yeast transcription factor Pap1.
Pap1, a fission yeast AP-1-like transcription factor, is negatively regulated by CRM1/exportin 1, the nuclear export factor. Pap1 was localized normally in the cytoplasm but was accumulated in the nucleus when Crm1 was inactivated by a temperature-sensitive mutation or by treatment with leptomycin B, a specific export inhibitor. Deletion of the C-terminal cysteine-rich domain (CRD) resulted in nuclear accumulation of Pap1, while a glutathione S-transferase-green fluorescent protein-CRD fusion protein was localized in the cytoplasm in a Crm1-dependent manner. Deletion and mutational analyses identified several important amino acids in a 19-amino acid region in the CRD as a nuclear export signal (NES). Strikingly, a cysteine residue (Cys-532), in addition to two leucines and an isoleucine, was important for the NES function and the presence of at least one of the two cysteine residues was essential. Unlike classical NESs such as the human immunodeficiency virus Rev NES, the Pap1 NES lost the function upon treatment with oxidants such as diethyl maleate. The oxidative stress response is conserved through evolution, as green fluorescent protein-fused proteins bearing the Pap1 NES expressed in mammalian cells responded to diethyl maleate. These results show that the hydrophobic amino acid-rich region containing two important cysteines in Pap1 serves as a novel NES, which is sensitive to oxidative stress.
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000502:physiology*; D000073718:Pancreatitis-Associated Proteins; D029702:Schizosaccharomyces pombe Proteins; D015534:Trans-Activators / Q000378:metabolism*