Charged amino acids at the carboxyl-terminal portions determine the intracellular locations of two isoforms of cytochrome b5
Charged amino acids at the carboxyl-terminal portions determine the intracellular locations of two isoforms of cytochrome b5
复制标题
DOI：
10.1074/jbc.273.47.31097
复制
发表时间：
1998-11-20
影响因子：
4.8
通讯作者：
Ito, A
中科院分区：
文献类型：
--
作者：
Kuroda, R;Ikenoue, T;Ito, A
线粒体外膜细胞色素b（5）（OMb）是内质网中细胞色素b（5）（cyt b（5））的同种型，是线粒体外膜的典型尾部锚定蛋白。我们克隆了含有OMb完整氨基酸序列的cDNA，发现该蛋白不具有氨基末端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，并分析了表达蛋白的亚细胞定位。从 OMb 的羧基末端删除超过 2 个氨基酸残基，就消除了该蛋白质对线粒体的靶向作用。当OMb的羧基端10个氨基酸与先前删除的相应10个残基的细胞色素b(5)融合时，融合蛋白定位于线粒体，从而表明OMb的羧基端10个氨基酸残基具有足够的信息将OMb转运至线粒体。用丙氨酸替换羧基末端 10 个氨基酸内的两个带正电荷的残基中的任何一个，导致突变蛋白转运至内质网。突变体细胞色素b(5)的羧基末端的酸性氨基酸被碱性氨基酸取代，可以被转运至线粒体。因此，这些蛋白质的羧基末端部分中的带电氨基酸似乎决定了它们在细胞中的位置。
Outer mitochondrial membrane cytochrome b(5) (OMb), which is an isoform of cytochrome b(5) (cyt b(5)) 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 II 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 b(5) 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 b(5), 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.