Kuroda R, Ikenoue T, Honsho M, Tsujimoto S, Mitoma J Y, Ito A
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
J Biol Chem. 1998 Nov 20;273(47):31097-102. doi: 10.1074/jbc.273.47.31097.
Outer mitochondrial membrane cytochrome b5 (OMb), which is an isoform of cytochrome b5 (cyt b5) in the endoplasmic reticulum, is a typical tail-anchored protein of the outer mitochondrial membrane. We cloned cDNA containing the complete amino acid sequence of OMb and found that the protein has no typical structural feature common to the mitochondrial targeting signal at the amino terminus. To identify the region responsible for the mitochondrial targeting of OMb, various mutated proteins were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The deletion of more than 11 amino acid residues from the carboxyl-terminal end of OMb abolished the targeting of the protein to the mitochondria. When the carboxyl-terminal 10 amino acids of OMb were fused to the cyt b5 that was previously deleted in the corresponding 10 residues, the fused protein localized in the mitochondria, thereby indicating that the carboxyl-terminal 10 amino acid residues of OMb have sufficient information to transport OMb to the mitochondria. The replacement of either of the two positively charged residues within the carboxyl-terminal 10 amino acids by alanine resulted in the transport of the mutant proteins to the endoplasmic reticulum. The mutant cyt b5, in which the acidic amino acid in its carboxyl-terminal end was replaced by basic amino acid, could be transported to the mitochondria. It would thus seem that charged amino acids in the carboxyl-terminal portion of these proteins determine their locations in the cell.
线粒体外膜细胞色素b5（OMb）是内质网中细胞色素b5（cyt b5）的一种同工型，是线粒体外膜典型的尾锚定蛋白。我们克隆了包含OMb完整氨基酸序列的cDNA，发现该蛋白在氨基末端没有线粒体靶向信号所共有的典型结构特征。为了确定负责OMb线粒体靶向的区域，在培养的哺乳动物细胞中表达了各种突变蛋白，并分析了所表达蛋白的亚细胞定位。从OMb羧基末端缺失超过11个氨基酸残基会消除该蛋白向线粒体的靶向作用。当将OMb的羧基末端10个氨基酸与先前在相应10个残基中缺失的cyt b5融合时，融合蛋白定位于线粒体，从而表明OMb的羧基末端10个氨基酸残基具有将OMb转运至线粒体的足够信息。将羧基末端10个氨基酸中的两个带正电荷残基中的任何一个替换为丙氨酸会导致突变蛋白转运至内质网。其羧基末端酸性氨基酸被碱性氨基酸取代的突变型cyt b5可以转运至线粒体。因此，这些蛋白羧基末端部分的带电氨基酸似乎决定了它们在细胞中的位置。
