A novel nuclear export signal sensitive to oxidative stress in the fission yeast transcription factor Pap1
A novel nuclear export signal sensitive to oxidative stress in the fission yeast transcription factor Pap1
复制标题
DOI：
10.1074/jbc.274.21.15151
复制
发表时间：
1999-05-21
影响因子：
4.8
通讯作者：
Horinouchi, S
中科院分区：
文献类型：
--
作者：
Kudo, N;Taoka, H;Horinouchi, S
Pap 1是一种裂殖酵母AP-1样转录因子，受核输出因子CRM 1/exportin 1负调控。Pap 1通常定位于细胞质中，但当Crm 1被温度敏感突变或用一种特异性输出抑制剂来霉素B处理而失活时，Pap 1在细胞核中积累。C-末端富含半胱氨酸结构域（CRD）的缺失导致Pap 1在细胞核中积累，而谷胱甘肽S-转移酶-绿色荧光蛋白-CRD融合蛋白以Crm 1依赖的方式定位于细胞质中。缺失和突变分析鉴定了CRD中19个氨基酸区域中的几个重要氨基酸作为核输出信号（内斯）。引人注目的是，除了两个亮氨酸和一个异亮氨酸之外，半胱氨酸残基（Cys-532）对于内斯功能是重要的，并且两个半胱氨酸残基中至少一个的存在是必需的。与经典的NES如人类免疫缺陷病毒Rev内斯不同，Pap 1内斯在用氧化剂如马来酸二乙酯处理后失去功能。氧化应激反应是保守的，通过进化，作为绿色荧光蛋白融合蛋白携带Pap 1内斯在哺乳动物细胞中表达的马来酸二乙酯。这些结果表明，Pap 1中含有两个重要半胱氨酸的疏水氨基酸富集区是一个新的内斯，对氧化应激敏感。
Pap1, a fission yeast AP-l-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.