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The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans.

ABSTRACT: Mitochondrial Ca2+ 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 Trypanosoma brucei 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. In situ tagging, followed by immunoprecipitation and immunofluorescence microscopy, revealed that T. brucei 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 TbMCUC 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 Trypanosoma cruzi and human cells. Specific interaction between human MCU (HsMCU) and human ATPc (HsATPc) was confirmed in vitro by mutagenesis and MYTH assays and in vivo 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 Pi transport with ATP synthesis, a process that is stimulated by Ca2+ in trypanosomes and human cells.IMPORTANCE The mitochondrial calcium uniporter (MCU) is essential for the regulation of oxidative phosphorylation in mammalian cells, and we have shown that in Trypanosoma brucei, the etiologic agent of sleeping sickness, this channel is essential for its survival and infectivity. Here we reveal that that Trypanosoma brucei MCU subunits interact with subunit c of the mitochondrial ATP synthase (ATPc). Interestingly, the direct physical MCU-ATPc interaction is conserved in T. cruzi and human cells.

SUBMITTER: Huang G

PROVIDER: S-EPMC7078472 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans.

Huang Guozhong G Docampo Roberto R

mBio 20200317 2

Mitochondrial Ca<sup>2+</sup> 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 <i>Trypanosoma brucei</i> 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 ...[more]

PMID: 32184243

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Project description:New findingsWhat is the central question of this study? Humans with obesity have lower ATP synthesis in muscle along with lower content of the β-subunit of the ATP synthase (β-F1-ATPase), the catalytic component of the ATP synthase. Does lower synthesis rate of β-F1-ATPase in muscle contribute to these responses in humans with obesity? What is the main finding and its importance? Humans with obesity have a lower synthesis rate of β-F1 -ATPase and ATP synthase specific activity in muscle. These findings indicate that reduced production of subunits forming the ATP synthase in muscle may contribute to impaired generation of ATP in obesity.AbstractThe content of the β-subunit of the ATP synthase (β-F1 -ATPase), which forms the catalytic site of the enzyme ATP synthase, is reduced in muscle of obese humans, along with a reduced capacity for ATP synthesis. We studied 18 young (37 ± 8 years) subjects of which nine were lean (BMI = 23 ± 2 kg m-2 ) and nine were obese (BMI = 34 ± 3 kg m-2 ) to determine the fractional synthesis rate (FSR) and gene expression of β-F1 -ATPase, as well as the specific activity of the ATP synthase. FSR of β-F1 -ATPase was determined using a combination of isotope tracer infusion and muscle biopsies. Gene expression of β-F1 -ATPase and specific activity of the ATP synthase were determined in the muscle biopsies. When compared to lean, obese subjects had lower muscle β-F1 -ATPase FSR (0.10 ± 0.05 vs. 0.06 ± 0.03% h-1 ; P < 0.05) and protein expression (P < 0.05), but not mRNA expression (P > 0.05). Across subjects, abundance of β-F1 -ATPase correlated with the FSR of β-F1 -ATPase (P < 0.05). The specific activity of muscle ATP synthase was lower in obese compared to lean subjects (0.035 ± 0.004 vs. 0.042 ± 0.007 arbitrary units; P < 0.05), but this difference was not significant after the activity of the ATP synthase was adjusted to the β-F1 -ATPase content (P > 0.05). Obesity impairs the synthesis of β-F1 -ATPase in muscle at the translational level, reducing the content of β-F1 -ATPase in parallel with reduced capacity for ATP generation via the ATP synthase complex.

Dataset Information

The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans.

Publications

The Mitochondrial Calcium Uniporter Interacts with Subunit c of the ATP Synthase of Trypanosomes and Humans.

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