Iwahashi J, Yamazaki S, Komiya T, Nomura N, Nishikawa S, Endo T, Mihara K
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812, Japan.
J Biol Chem. 1997 Jul 18;272(29):18467-72. doi: 10.1074/jbc.272.29.18467.
Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Deltatom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.
Tom20是一种线粒体外膜蛋白，作为细胞质中合成的线粒体前体蛋白的导入受体复合物的一个组成部分发挥作用。它由N端膜锚定片段、四肽重复序列（TPR）基序、膜锚定与TPR基序之间富含带电荷氨基酸的连接片段以及C端酸性氨基酸簇组成。为了评估这些片段在哺乳动物Tom20中的功能意义，我们克隆了大鼠Tom20，并在缺失Tom20的酵母细胞中表达突变型大鼠Tom20蛋白，然后检测它们弥补呼吸驱动生长缺陷和线粒体蛋白导入缺陷的能力。Tom20N69是一种由膜锚定片段和连接片段组成的突变体，它定位于线粒体，并且在弥补生长和导入缺陷方面与野生型Tom20一样有效，而缺失连接片段的突变体则不能。对从互补酵母细胞中分离出的线粒体进行的体外蛋白导入实验表明，靶向酵母Tom70的前体蛋白通过大鼠Tom20N69被有效地导入线粒体。因此，连接片段对大鼠Tom20的功能至关重要，而TPR基序和C端酸性氨基酸则并非如此。
