Zhao Jingfang, Onduka Toshimitu, Kinoshita Jun-ya, Honsho Masanori, Kinoshita Toshinori, Shimazaki Ken-ichiro, Ito Akio
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581.
J Biochem. 2003 Jan;133(1):115-21. doi: 10.1093/jb/mvg009.
Subfractionation studies showed that cytochrome b(5) (cyt b5), which has been considered to be a typical ER protein, was localized in both the endoplasmic reticulum membrane (ER) and the outer membrane of mitochondria in cauliflower (Brassica olracea) cells and was a component of antimycin A-insensitive NADH-cytochrome c reductase system in both membranes. When cDNA for cauliflower cyt b5 was introduced into mammalian (COS-7) and yeast cells as well as into onion cells, the expressed cytochrome was localized both in the ER and mitochondria in those cells. On the other hand, rat and yeast cyt b5s were specifically localized in the ER membranes even in the onion cells. Mutation experiments showed that cauliflower cyt b5 carries information that targets it to the ER and mitochondria within the carboxy-terminal 10 amino acids, as in the case of rat and yeast cyt b5s, and that replacement of basic amino acids in this region of cauliflower cyt b5 with neutral or acidic ones resulted in its distribution only in the ER. Together with the established findings of the importance of basic amino acids in mitochondrial targeting signals, these results suggest that charged amino acids in the carboxy-terminal portion of cyt b5 determine its location in the cell, and that the same mechanism of signal recognition and of protein transport to organelles works in mammalian, plant, and yeast cells.
亚组分分析研究表明，细胞色素b(5)（cyt b5），一直被认为是一种典型的内质网蛋白，在花椰菜（甘蓝）细胞的内质网膜（ER）和线粒体外膜中均有定位，并且是这两种膜中抗霉素A不敏感的NADH - 细胞色素c还原酶系统的一个组分。当将花椰菜cyt b5的cDNA导入哺乳动物（COS - 7）细胞、酵母细胞以及洋葱细胞时，所表达的细胞色素在这些细胞的内质网和线粒体中均有定位。另一方面，大鼠和酵母的cyt b5即使在洋葱细胞中也特异性地定位于内质网膜。突变实验表明，与大鼠和酵母的cyt b5情况一样，花椰菜cyt b5在其羧基末端的10个氨基酸内携带将其靶向内质网和线粒体的信息，并且将花椰菜cyt b5该区域的碱性氨基酸替换为中性或酸性氨基酸会导致其仅分布在内质网中。连同已确定的碱性氨基酸在线粒体靶向信号中的重要性这一发现，这些结果表明cyt b5羧基末端部分的带电氨基酸决定了其在细胞中的位置，并且相同的信号识别和蛋白质向细胞器运输的机制在哺乳动物、植物和酵母细胞中均起作用。
