Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil.
Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil
mBio. 2019 May 7;10(3):e00348-19. doi: 10.1128/mBio.00348-19.
The mitochondrial Ca uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca uptake. MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated and knockout (-KO) cell lines. Ablation of either or showed a significantly reduced mitochondrial Ca uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca concentrations ([Ca]), as occurs with their mammalian orthologs. -KO and -KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles. is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca sensors and gatekeepers of the MCU, preventing Ca uptake under resting conditions and favoring it at high cytosolic Ca concentrations ([Ca]). Using the CRISPR/Cas9 technique, we generated and knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca] but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.
锥虫，属于真核超组外质体，其线粒体钙摄取具有与动物相似的生化特征，动物与真菌一起属于后口动物超组。然而，锥虫的线粒体钙单向转运蛋白（MCU）复合物的组成非常特殊，表明存在谱系特异性适应。在这项工作中，我们使用来研究线粒体钙摄取 1（MICU1）和 MICU2 的同源物在线粒体钙摄取中的作用。MICU1（TcMICU1）和 TcMICU2 具有线粒体靶向信号、两个典型的 EF 手钙结合结构域，并定位于线粒体。使用 CRISPR/Cas9 系统（即，成簇的规则间隔的短回文重复序列与 Cas9），我们生成了和敲除（-KO）细胞系。在通透性的前鞭毛体中，无论是或的缺失都显示出线粒体钙摄取明显减少，而线粒体膜电位没有耗散，也没有影响 AMP/ATP 比或柠檬酸合酶活性。然而，这些蛋白质在低细胞浆 Ca 浓度 ([Ca]) 下都没有门控功能，就像它们的哺乳动物同源物一样。-KO 和 -KO 前鞭毛体的生长速度较慢，氧化代谢受损，而感染性的锥虫动基体侵入宿主细胞并在其中复制为阿米巴体的能力降低。这项工作是首次研究 MICU1 和 MICU2 在与动物进化上遥远的生物体中的作用，结果表明，尽管这些成分可能存在于最后的真核共同祖先（LECA）中，但它们在不同真核超组的进化过程中发展出了不同的作用。这项工作还为锥虫对其特殊生活方式的适应提供了新的见解。是克氏锥虫病的病原体，属于早期分支的真核超组外质体。其线粒体钙单向转运蛋白（MCU）亚基与动物同源物相似，这对于发现其编码基因很重要。在动物细胞中，MICU1 和 MICU2 蛋白作为 Ca 传感器和 MCU 的门控，在静息条件下阻止 Ca 摄取，并在高细胞浆 Ca 浓度 ([Ca]) 下促进其摄取。使用 CRISPR/Cas9 技术，我们生成了和敲除细胞系，并表明 MICU1 和 -2 不在低 [Ca] 下作为门控，但对于正常生长、宿主细胞入侵和细胞内复制是必不可少的，揭示了谱系特异性适应。
