Nakamura Yasuhiko, Suzuki Hiroyuki, Sakaguchi Masao, Mihara Katsuyoshi
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-0054, Japan.
J Biol Chem. 2004 May 14;279(20):21223-32. doi: 10.1074/jbc.M314156200. Epub 2004 Feb 25.
Tom22 is a preprotein receptor and organizer of the mitochondrial outer membrane translocase complex (TOM complex). Rat Tom22 (rTOM22) is a 142-residue protein, embedded in the outer membrane through the internal transmembrane domain (TMD) with 82 N-terminal residues in the cytosol and 41 C-terminal residues in the intermembrane space. We analyzed the signals that target rTOM22 to the mitochondrial outer membrane and assembly into the TOM complex in cultured mammalian cells. Deletions or mutations were systematically introduced into the molecule, and the intracellular localization of the mutant constructs in HeLa cells was examined by confocal microscopy and cell fractionation. Their assembly into the TOM complex was also examined using blue native gel electrophoresis. These experiments revealed three separate structural elements: a cytoplasmic 10-residue segment with an acidic alpha-helical structure located 30 residues upstream of the TMD (the import sequence), TMD with an appropriate hydrophobicity, and a 20-residue C-terminal segment located 22 residues downstream of the TMD (C-tail signal). The import sequence and TMD were both essential for targeting and integration into the TOM complex, whereas the C-tail signal affected the import efficiency. The import sequence combined with foreign TMD functioned as a mitochondrial targeting and anchor signal but failed to integrate the construct into the TOM complex. Thus, the mitochondrial-targeting and TOM integration signal could be discriminated. A yeast two-hybrid assay revealed that the import sequence interacted with two intramolecular elements, the TMD and C-tail signal, and that it also interacted with the import receptor Tom20.
Tom22是一种前体蛋白受体，也是线粒体外膜转位酶复合物（TOM复合物）的组织者。大鼠Tom22（rTOM22）是一种由142个氨基酸残基组成的蛋白质，通过内部跨膜结构域（TMD）嵌入外膜，其82个N端残基位于胞质溶胶中，41个C端残基位于膜间隙中。我们分析了在培养的哺乳动物细胞中将rTOM22靶向线粒体外膜并组装到TOM复合物中的信号。对该分子进行了系统的缺失或突变，并通过共聚焦显微镜和细胞分级分离法检测了HeLa细胞中突变构建体的细胞内定位。还使用蓝色天然凝胶电泳检测了它们组装到TOM复合物中的情况。这些实验揭示了三个独立的结构元件：一个位于TMD上游30个残基处具有酸性α螺旋结构的10个残基的胞质段（导入序列）、具有适当疏水性的TMD以及位于TMD下游22个残基处的20个残基的C端段（C尾信号）。导入序列和TMD对于靶向和整合到TOM复合物中都是必不可少的，而C尾信号影响导入效率。与外源TMD结合的导入序列起到了线粒体靶向和锚定信号的作用，但未能将构建体整合到TOM复合物中。因此，可以区分线粒体靶向信号和TOM整合信号。酵母双杂交试验表明，导入序列与两个分子内元件TMD和C尾信号相互作用，并且它还与导入受体Tom20相互作用。
