Department of Parasitology, Faculty of Science, Charles University, BIOCEV, Prague, Czech Republic.
Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Prague, Czech Republic.
PLoS Biol. 2019 Jan 4;17(1):e3000098. doi: 10.1371/journal.pbio.3000098. eCollection 2019 Jan.
Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.
线粒体起源于变形菌的内共生体，其向细胞器的转变与蛋白质导入途径的建立密切相关。大多数蛋白质的最初导入是由外膜转位酶（TOM）介导的。尽管 TOM 存在于所有形式的线粒体中，但在真核生物谱系之间已经预测到了意想不到的亚基多样性。然而，实验知识仅限于少数几种生物体，到目前为止，TOM 复合物的三聚体孔还是双孔结构仍然没有定论。在这里，我们分析了在阴道毛滴虫中发现的代谢特化的厌氧形式线粒体——氢体中的 TOM 复合物。我们证明高度分化的β桶 T. vaginalis TOM（TvTom）40-2 形成了一个易位通道，以进行氢体蛋白导入。TvTom40-2 存在于高分子量复合物中，对其分析揭示了存在 4 个尾巴锚定（TA）蛋白。其中两个，Tom36 和 Tom46，具有热休克蛋白（Hsp）20 和四肽重复（TPR）结构域，可以结合氢体前蛋白，很可能作为受体发挥作用。第三个亚基，Tom22 样蛋白，具有短的顺式结构域和保守的 Tom22 跨膜段，但缺乏反式结构域。第四个蛋白，氢体外膜蛋白 19（Homp19）没有已知的同源性。此外，我们的数据表明，TvTOM 与分选和装配机器（Sam）50 相关，该机器参与β桶组装。通过电子显微镜观察 TvTOM 的结果表明，它形成了三个孔，并且具有非传统的颅骨状形状。尽管 TvTOM 似乎缺乏 Tom7，但我们的系统发育分析预测了自由生活的挖掘生物中存在 Tom7。总的来说，我们的研究结果表明，由三个保守亚基组成的三聚体孔 TOM 复合物存在于最后共同的真核祖先（LECA）中，而负责底物结合的受体则在不同的真核生物谱系中独立进化。
