Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1) - Implications for interspecies differences in mitochondrial toxicity of fialuridine
Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1) - Implications for interspecies differences in mitochondrial toxicity of fialuridine
复制标题
DOI：
10.1074/jbc.m513825200
复制
发表时间：
2006-06-16
影响因子：
4.8
通讯作者：
Unadkat, Jashvant D.
中科院分区：
文献类型：
--
作者：
Lee, Eun-Woo;Lai, Yurong;Unadkat, Jashvant D.
我们先前已经表明，人平衡型核苷转运蛋白1（hENT 1）在线粒体膜中表达并起作用，并且这种表达增强了核苷药物氟替尼定（FIAU）的线粒体毒性（Lai，Y.，谢氏角M.，和Unadkat，J. D. (004)J. Biol. Chem. 279，4490 4497）。在这里，我们报告的鉴定线粒体靶向序列的hENT 1。使用共聚焦显微镜和不同的截断和点突变的hENT 1-YFP（黄色荧光蛋白）在Madin-Darby犬肾细胞中表达，我们确定了氨基酸残基Pro（71），Glu（72），和Asn（74）（PESN基序）的hENT 1作为重要的线粒体靶向hENT 1。这种线粒体靶向序列的鉴定为FIAU在人类和啮齿动物之间线粒体毒性的显著差异提供了可能的解释。虽然在Madin-Darby犬肾细胞中表达的小鼠ENT 1（mENT 1）可以转运FIAU，但共聚焦显微镜显示mENT 1-GFP（绿色荧光蛋白）不定位于线粒体。与此观察结果一致，从小鼠肝脏分离的线粒体不运输FIAU。hENT 1、mENT 1和大鼠ENT 1（rENT 1）的序列比对显示，在mENT 1和rENT 1中，hENT 1的PEXN基序被PAXS基序取代。用PEXN取代mENT 1中的PAXS（以产生mENT 1-PEXN-GFP）和用PAXS取代hENT 1中的PEXN（以产生hENT 1-PAXS-YFP）导致mENT 1-PEXN-GFP的部分线粒体定位和hENT 1-PAXS-YFP的线粒体定位丧失。这是首次发现hENT 1的线粒体靶向信号。我们的数据表明，FIAU在小鼠中缺乏线粒体毒性是由于缺乏mENT 1靶向线粒体和在线粒体中表达。
We have previously shown that the human equilibrative nucleoside transporter 1 (hENT1) is expressed and functional in the mitochondrial membrane and that this expression enhances the mitochondrial toxicity of the nucleoside drug, fialuridine (FIAU) (Lai, Y., Tse, C. M., and Unadkat, J. D. (004) J. Biol. Chem. 279, 4490 4497). Here we report on identification of the mitochondrial targeting sequence of hENT1. Using confocal microscopy and different truncated and point mutants of hENT1-YFP (yellow fluorescent protein) expressed in Madin-Darby canine kidney cells, we identified amino acid residues Pro(71), Glu(72), and Asn(74) (the PEXN motif) of hENT1 as important in mitochondrial targeting of hENT1. Identification of this mitochondrial targeting sequence provides a possible explanation for the dramatic difference in mitochondrial toxicity of FIAU between humans and rodents. Although the mouse ENT1 (mENT1), expressed in Madin-Darby canine kidney cells, can transport FIAU, confocal microscopy showed that mENT1-GFP (green fluorescent protein) was not localized to the mitochondria. Consistent with this observation, mitochondria isolated from mouse livers did not transport FIAU. Sequence alignment of hENT1, mENT1, and rat ENT1 (rENT1) showed that the PEXN motif of hENT1 was substituted with a PAXS motif in both mENT1 and rENT1. Substitution of PAXS in mENT1 with PEXN (to create mENT1-PEXN-GFP) and of PEXN in hENT1 with PAXS (to create hENT1-PAXS-YFP) resulted in partial mitochondrial localization of mENT1-PEXN-GFP and loss of mitochondrial localization of hENT1-PAXS-YFP. This is the first time that the mitochondrial targeting signal of hENT1 has been identified. Our data suggest that the lack of mitochondrial toxicity of FIAU in mice is due to the lack of mENT1 targeting to and expression in the mitochondria.