Department of Microbiology and Molecular Genetics, Institute of Medical Research Israel-Canada, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
J Mol Biol. 2011 Jun 3;409(2):113-23. doi: 10.1016/j.jmb.2011.03.045. Epub 2011 Apr 6.
The tricarboxylic acid cycle enzyme aconitase in yeast is a single translation product, which is dual targeted and distributed between the mitochondria and the cytosol by a unique mechanism involving reverse translocation. There is limited understanding regarding the precise mechanism of reverse translocation across the mitochondrial membranes. Here, we examined the contribution of the mature part of aconitase to its dual targeting. We created a set of aconitase mutants harboring two kinds of alterations: (1) point mutations or very small deletions in conserved sites and (2) systematic large deletions. These mutants were screened for their localization by a α-complementation assay, which revealed that the aconitase fourth domain that is at the C-terminus (amino acids 517-778) is required for aconitase distribution. Moreover, fusion of this C-terminal domain to mitochondria-targeted passenger proteins such as dihydrofolate reductase and orotidine-5'-phosphate decarboxylase, conferred dual localization on them. These results indicate that the aconitase C-terminal domain is both necessary and sufficient for dual targeting, thereby functioning as an "independent signal". In addition, the same C-terminal domain was shown to be necessary for aconitase efficient posttranslational import into mitochondria.
酵母中三羧酸循环酶顺乌头酸酶是单一翻译产物，通过涉及反向易位的独特机制，双重靶向并分布在线粒体和细胞质之间。对于线粒体膜反向易位的确切机制，人们的了解有限。在这里，我们研究了成熟的顺乌头酸酶部分对其双重靶向的贡献。我们创建了一组顺乌头酸酶突变体，这些突变体具有两种改变：（1）保守位点的点突变或非常小的缺失，以及（2）系统的大片段缺失。通过α互补测定筛选这些突变体的定位，结果表明，位于 C 末端（氨基酸 517-778）的顺乌头酸酶第四结构域对于顺乌头酸酶的分布是必需的。此外，将该 C 末端结构域与靶向线粒体的过客蛋白（如二氢叶酸还原酶和乳清酸 5'-磷酸脱羧酶）融合，赋予它们双重定位。这些结果表明，顺乌头酸酶 C 末端结构域既是必需的又是充分的，从而起到“独立信号”的作用。此外，还表明相同的 C 末端结构域对于顺乌头酸酶有效进行线粒体的翻译后导入是必需的。
