A genetic approach to identifying mitochondrial proteins
A genetic approach to identifying mitochondrial proteins
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DOI：
10.1038/nbt791
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发表时间：
2003-03-01
影响因子：
46.9
通讯作者：
Umezawa, Y
中科院分区：
文献类型：
--
作者：
Ozawa, T;Sako, Y;Umezawa, Y
真核细胞内复杂网络的控制依赖于蛋白质的差异区室化。我们已经开发了一种方法，允许快速识别新的蛋白质划分在线粒体中筛选大规模的cDNA文库。该原理是基于通过来自集胞藻属PCC 6803的DnaE的蛋白质剪接来重建分裂增强的绿色荧光蛋白（EGFP）。cDNA文库在用逆转录病毒感染后在哺乳动物细胞中表达。如果测试蛋白含有功能性线粒体靶向信号（MTS），则其易位到线粒体基质中，然后通过蛋白质剪接形成EGFP。通过荧光激活的细胞分选快速筛选携带该重建的EGFP的细胞，并从细胞中分离和鉴定cDNA。对258个cDNA的分析揭示了各种MTS，其中我们鉴定了对应于线粒体蛋白的新转录本。这种方法应该提供一种手段，映射蛋白质分布在细胞内细胞器在广泛的不同组织和疾病状态。
The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.