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. 2018 Sep 18;9(5):e01700-18. doi: 10.1128/mBio.01700-18.
The mitochondrial calcium uniporter complex (MCUC) is a highly selective channel that conducts calcium ions across the organelle inner membrane. We previously characterized 's MCU (TbMCU) as an essential component of the MCUC required for parasite viability and infectivity. In this study, we characterize its paralog MCUb (TbMCUb) and report the identification of two novel components of the complex that we named TbMCUc and TbMCUd. These new MCUC proteins are unique and conserved only in trypanosomatids. tagging and immunofluorescence microscopy revealed that they colocalize with TbMCU and TbMCUb to the mitochondria of Blue Native PAGE and immunodetection analyses indicated that the MCUC proteins exist in a large protein complex with a molecular weight of approximately 380 kDa. RNA interference (RNAi) or overexpression of the TbMCUc and TbMCUd genes significantly reduced or enhanced mitochondrial Ca uptake in , respectively, without affecting the mitochondrial membrane potential, indicating that they are essential components of the MCUC of this parasite. The specific interactions of TbMCU with TbMCUb, TbMCUc, or TbMCUd were confirmed by coimmunoprecipitation and split-ubiquitin membrane-based yeast two-hybrid (MYTH) assays. Furthermore, combining mutagenesis analysis with MYTH assays revealed that transmembrane helices (TMHs) were determinant of the interactions between TbMCUC subunits. In summary, our study has identified two novel essential components of the MCUC of and defined their direct physical interactions with the other subunits that result in a hetero-oligomeric MCUC. causes human African trypanosomiasis and nagana in animals. The finding of a mitochondrial calcium uniporter (MCU) conserved in this parasite was essential for the discovery of the gene encoding the pore subunit. Mitochondrial Ca transport mediated by the MUC complex is critical in for shaping the dynamics of cytosolic Ca increases, for the bioenergetics of the cells, and for viability and infectivity. We found that one component of the complex (MCUb) does not act as a dominant negative effector of the channel as in vertebrate cells and that the TbMCUC possesses two unique subunits (MCUc and MCUd) present only in trypanosomatids and required for Ca transport. The study of the interactions between these four subunits (MCU, MCUb, MCUc, and MCUd) by a variety of techniques that include coimmunoprecipitation, split-ubiquitin membrane-based yeast two-hybrid assays, and site-directed mutagenesis suggests that they interact through their transmembrane helices to form hetero-oligomers.
线粒体钙单向转运体复合物（MCUC）是一种高度选择性的通道，可将钙离子穿过细胞器内膜。我们之前将'的 MCU（TbMCU）鉴定为 MCUC 的必需组成部分，该复合物对于寄生虫的生存力和感染力至关重要。在这项研究中，我们对其同源物 TbMCUb（TbMCUb）进行了表征，并报告了复合物中两个新组件的鉴定，我们将其命名为 TbMCUc 和 TbMCUd。这些新的 MCUC 蛋白是独特的，仅在原生动物中保守。'的标签和免疫荧光显微镜显示，它们与 TbMCU 和 TbMCUb 一起定位于线粒体的蓝色天然 PAGE 和免疫检测分析表明，MCUC 蛋白存在于分子量约为 380 kDa 的大蛋白复合物中。RNA 干扰（RNAi）或 TbMCUc 和 TbMCUd 基因的过表达分别显著降低或增强了 的线粒体 Ca 摄取，但不影响线粒体膜电位，表明它们是寄生虫 MCUC 的必需组成部分。TbMCU 与 TbMCUb、TbMCUc 或 TbMCUd 的特异性相互作用通过共免疫沉淀和分裂泛素膜酵母双杂交（MYTH）测定得到证实。此外，结合突变分析和 MYTH 测定表明，跨膜螺旋（TMHs）是 TbMCUC 亚基相互作用的决定因素。总之，我们的研究鉴定了 TbMCU 的两个新的必需组成部分，并定义了它们与其他亚基的直接物理相互作用，从而形成了异源寡聚 MCUC。导致人类非洲锥虫病和动物中的 nagana。在该寄生虫中发现线粒体钙单向转运体（MCU）对于发现编码孔亚基的基因至关重要。由 MUC 复合物介导的线粒体 Ca 转运对于塑造细胞质 Ca 增加的动力学、细胞的生物能学以及生存力和感染力至关重要。我们发现，复合物的一个组成部分（MCUb）不作为脊椎动物细胞中通道的显性负效应子起作用，并且 TbMCUC 具有两个仅在原生动物中存在且需要 Ca 转运的独特亚基（MCUc 和 MCUd）。通过包括共免疫沉淀、分裂泛素膜酵母双杂交测定和定点突变在内的各种技术研究这四个亚基（MCU、MCUb、MCUc 和 MCUd）之间的相互作用表明，它们通过跨膜螺旋相互作用形成异源寡聚体。
