Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA
mBio. 2020 Mar 17;11(2):e00268-20. doi: 10.1128/mBio.00268-20.
Mitochondrial Ca transport mediated by the uniporter complex (MCUC) plays a key role in the regulation of cell bioenergetics in both trypanosomes and mammals. Here we report that MCU (TbMCU) subunits interact with subunit c of the mitochondrial ATP synthase (ATPc), as determined by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Mutagenesis analysis in combination with MYTH assays suggested that transmembrane helices (TMHs) are determinants of this specific interaction. tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that ATPc (TbATPc) coimmunoprecipitates with TbMCUC subunits and colocalizes with them to the mitochondria. Blue native PAGE and immunodetection analyses indicated that the TbMCUC is present together with the ATP synthase in a large protein complex with a molecular weight of approximately 900 kDa. Ablation of the subunits by RNA interference (RNAi) significantly increased the AMP/ATP ratio, revealing the downregulation of ATP production in the cells. Interestingly, the direct physical MCU-ATPc interaction is conserved in and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed by mutagenesis and MYTH assays and by coimmunoprecipitation. In summary, our study has identified that MCU complex physically interacts with mitochondrial ATP synthase, possibly forming an MCUC-ATP megacomplex that couples ADP and P transport with ATP synthesis, a process that is stimulated by Ca in trypanosomes and human cells. The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in , the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in and human cells.
线粒体钙单向转运体（MCU）复合物（MCUC）介导的钙转运在原生动物和哺乳动物的细胞生物能量调节中起着关键作用。在这里，我们报告说 MCU（TbMCU）亚基与线粒体 ATP 合酶（ATPc）的亚基 c 相互作用，这是通过免疫沉淀和分裂泛素膜酵母双杂交（MYTH）测定确定的。突变分析结合 MYTH 测定表明，跨膜螺旋（TMHs）是这种特定相互作用的决定因素。标记后，通过免疫沉淀和免疫荧光显微镜观察，发现 TbATPc 与 TbMCUC 亚基共沉淀，并与它们一起定位于线粒体。蓝色 native PAGE 和免疫检测分析表明，TbMCUC 与 ATP 合酶一起存在于分子量约为 900 kDa 的大型蛋白质复合物中。通过 RNA 干扰（RNAi）消融亚基显着增加了 AMP/ATP 比，表明细胞中 ATP 产生的下调。有趣的是，在人和细胞中，MCU-ATPc 的直接物理相互作用是保守的。通过突变和 MYTH 测定以及免疫沉淀证实了人 MCU（HsMCU）和人 ATPc（HsATPc）之间的特异性相互作用。总之，我们的研究表明 MCU 复合物与线粒体 ATP 合酶物理相互作用，可能形成一个将 ADP 和 P 转运与 ATP 合成偶联的 MCUC-ATP 超级复合物，该过程在原生动物和人类细胞中受到 Ca 的刺激。线粒体钙单向转运体（MCU）对于哺乳动物细胞氧化磷酸化的调节是必不可少的，我们已经表明，在嗜睡症的病原体中，该通道对于其存活和感染力是必不可少的。在这里，我们揭示了该通道的 TbMCU 亚基与线粒体 ATP 合酶的亚基 c 相互作用。有趣的是，在人和细胞中，MCU-ATPc 的直接物理相互作用是保守的。
