Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane
Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane
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DOI：
10.1091/mbc.01-12-0570
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发表时间：
2002-05-01
影响因子：
3.3
通讯作者：
Mihara, K
中科院分区：
文献类型：
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作者：
Horie, C;Suzuki, H;Mihara, K
我们使用酵母 Tom5 作为模型，以绿色荧光蛋白作为报告基因，分析了将带有 C 端跨膜片段 (TMS) 的外膜蛋白导向哺乳动物线粒体的信号。将缺失或突变系统地引入 TMS 或侧翼区域，并使用共聚焦显微镜和细胞分级检查它们在 COS-7 细胞中的细胞内定位。 1) C端五残基片段（C-片段）内的三个碱性氨基酸残基包含线粒体靶向所需的信息。该片段中净正电荷的减少降低了线粒体特异性，并且突变体分布在整个细胞内膜上。 2)TMS的伸长干扰C段的功能并且突变体被递送至细胞内膜。 3) 连接子插入导致 TMS 和 C 段分离，严重损害线粒体靶向功能，导致细胞质错误定位。 4) C 段侧翼 TMS 区域的突变或小缺失也会损害线粒体靶向。因此，TMS 的中等长度、C 段中的正电荷以及 TMS 和 C 段之间的距离或上下文对于靶向信号至关重要。由此定义的信号的结构特征也得到了哺乳动物 C 尾锚定蛋白 OMP25 的证实。
We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.