Rada Petr, Makki Abhijith Radhakrishna, Zimorski Verena, Garg Sriram, Hampl Vladimír, Hrdý Ivan, Gould Sven B, Tachezy Jan
Department of Parasitology, Charles University in Prague, Faculty of Science, Prague, Czech Republic.
Institute for Molecular Evolution, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany.
Eukaryot Cell. 2015 Dec;14(12):1264-75. doi: 10.1128/EC.00104-15. Epub 2015 Oct 16.
Mitochondrial evolution entailed the origin of protein import machinery that allows nuclear-encoded proteins to be targeted to the organelle, as well as the origin of cleavable N-terminal targeting sequences (NTS) that allow efficient sorting and import of matrix proteins. In hydrogenosomes and mitosomes, reduced forms of mitochondria with reduced proteomes, NTS-independent targeting of matrix proteins is known. Here, we studied the cellular localization of two glycolytic enzymes in the anaerobic pathogen Trichomonas vaginalis: PPi-dependent phosphofructokinase (TvPPi-PFK), which is the main glycolytic PFK activity of the protist, and ATP-dependent PFK (TvATP-PFK), the function of which is less clear. TvPPi-PFK was detected predominantly in the cytosol, as expected, while all four TvATP-PFK paralogues were imported into T. vaginalis hydrogenosomes, although none of them possesses an NTS. The heterologous expression of TvATP-PFK in Saccharomyces cerevisiae revealed an intrinsic capability of the protein to be recognized and imported into yeast mitochondria, whereas yeast ATP-PFK resides in the cytosol. TvATP-PFK consists of only a catalytic domain, similarly to "short" bacterial enzymes, while ScATP-PFK includes an N-terminal extension, a catalytic domain, and a C-terminal regulatory domain. Expression of the catalytic domain of ScATP-PFK and short Escherichia coli ATP-PFK in T. vaginalis resulted in their partial delivery to hydrogenosomes. These results indicate that TvATP-PFK and the homologous ATP-PFKs possess internal structural targeting information that is recognized by the hydrogenosomal import machinery. From an evolutionary perspective, the predisposition of ancient ATP-PFK to be recognized and imported into hydrogenosomes might be a relict from the early phases of organelle evolution.
线粒体进化涉及蛋白质输入机制的起源，该机制使核编码蛋白质能够靶向该细胞器，以及可裂解的N端靶向序列（NTS）的起源，这些序列允许基质蛋白进行高效分选和输入。在氢化酶体和纺锤剩体中，线粒体的简化形式，蛋白质组减少，已知基质蛋白的NTS非依赖性靶向。在这里，我们研究了厌氧病原体阴道毛滴虫中两种糖酵解酶的细胞定位：焦磷酸依赖性磷酸果糖激酶（TvPPi-PFK），它是原生生物主要的糖酵解PFK活性，以及ATP依赖性PFK（TvATP-PFK），其功能尚不清楚。正如预期的那样，TvPPi-PFK主要在细胞质中被检测到，而所有四个TvATP-PFK旁系同源物都被导入到阴道毛滴虫的氢化酶体中，尽管它们都不具有NTS。TvATP-PFK在酿酒酵母中的异源表达揭示了该蛋白质被识别并导入酵母线粒体的内在能力，而酵母ATP-PFK则位于细胞质中。TvATP-PFK仅由一个催化结构域组成，类似于“短”细菌酶，而ScATP-PFK包括一个N端延伸、一个催化结构域和一个C端调节结构域。ScATP-PFK催化结构域和短大肠杆菌ATP-PFK在阴道毛滴虫中的表达导致它们部分被递送至氢化酶体。这些结果表明，TvATP-PFK和同源ATP-PFK具有内部结构靶向信息，可被氢化酶体输入机制识别。从进化的角度来看，古代ATP-PFK被识别并导入氢化酶体的倾向可能是细胞器进化早期阶段的遗迹。
