Kanaji S, Iwahashi J, Kida Y, Sakaguchi M, Mihara K
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
J Cell Biol. 2000 Oct 16;151(2):277-88. doi: 10.1083/jcb.151.2.277.
Tom20 is a major receptor of the mitochondrial preprotein translocation system and is bound to the outer membrane through the NH(2)-terminal transmembrane domain (TMD) in an Nin-Ccyt orientation. We analyzed the mitochondria-targeting signal of rat Tom20 (rTom20) in COS-7 cells, using green fluorescent protein (GFP) as the reporter by systematically introducing deletions or mutations into the TMD or the flanking regions. Moderate TMD hydrophobicity and a net positive charge within five residues of the COOH-terminal flanking region were both critical for mitochondria targeting. Constructs without net positive charges within the flanking region, as well as those with high TMD hydrophobicity, were targeted to the ER-Golgi compartments. Intracellular localization of rTom20-GFP fusions, determined by fluorescence microscopy, was further verified by cell fractionation. The signal recognition particle (SRP)-induced translation arrest and photo-cross-linking demonstrated that SRP recognized the TMD of rTom20-GFP, but with reduced affinity, while the positive charge at the COOH-terminal flanking segment inhibited the translation arrest. The mitochondria-targeting signal identified in vivo also functioned in the in vitro system. We conclude that NH(2)-terminal TMD with a moderate hydrophobicity and a net positive charge in the COOH-terminal flanking region function as the mitochondria-targeting signal of the outer membrane proteins, evading SRP-dependent ER targeting.
Tom20是线粒体前体蛋白转运系统的主要受体，通过NH(2)-末端跨膜结构域（TMD）以Nin-Ccyt方向与外膜结合。我们在COS-7细胞中分析了大鼠Tom20（rTom20）的线粒体靶向信号，通过系统地在TMD或其侧翼区域引入缺失或突变，使用绿色荧光蛋白（GFP）作为报告基因。适度的TMD疏水性和COOH末端侧翼区域五个残基内的净正电荷对于线粒体靶向均至关重要。侧翼区域内没有净正电荷的构建体以及具有高TMD疏水性的构建体被靶向到内质网-高尔基体区室。通过荧光显微镜确定的rTom20-GFP融合蛋白的细胞内定位通过细胞分级分离进一步得到验证。信号识别颗粒（SRP）诱导的翻译停滞和光交联表明，SRP识别rTom20-GFP的TMD，但亲和力降低，而COOH末端侧翼片段的正电荷抑制了翻译停滞。在体内鉴定的线粒体靶向信号在体外系统中也起作用。我们得出结论，具有适度疏水性的NH(2)-末端TMD和COOH末端侧翼区域的净正电荷作为外膜蛋白的线粒体靶向信号，避免了依赖SRP的内质网靶向。
