Assembly of mitochondrial ATP synthase in cultured human cells: implications for mitochondrial diseases

Unlike most organisms, the mitochondrial DNA (mtDNA) ofChlamydomonas reinhardtii, a green alga, does not encode subunit 6 of F0F1-ATP synthase. We hypothesized that C. reinhardtii ATPase 6 is nucleus encoded and identified cDNAs and a single-copy nuclear gene specifying this subunit (CrATP6, with eight exons, four of which encode a mitochondrial targeting signal). Although the algal and humanATP6 genes are in different subcellular compartments and the encoded polypeptides are highly diverged, their secondary structures are remarkably similar. When CrATP6 was expressed in human cells, a significant amount of the precursor polypeptide was targeted to mitochondria, the mitochondrial targeting signal was cleaved within the organelle, and the mature polypeptide was assembled into human ATP synthase. In spite of the evolutionary distance between algae and mammals, C. reinhardtii ATPase 6 functioned in human cells, because deficiencies in both cell viability and ATP synthesis in transmitochondrial cell lines harboring a pathogenic mutation in the human mtDNA-encoded ATP6 gene were overcome by expression of CrATP6. The ability to express a nucleus-encoded version of a mammalian mtDNA-encoded protein may provide a way to import other highly hydrophobic proteins into mitochondria and could serve as the basis for a gene therapy approach to treat human mitochondrial diseases.

Download Full-text

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

mBio ◽

10.1128/mbio.00268-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 4

Author(s):

Guozhong Huang ◽

Roberto Docampo

Keyword(s):

Trypanosoma Brucei ◽

Atp Synthase ◽

Specific Interaction ◽

Human Cells ◽

Mitochondrial Calcium ◽

Subunit C ◽

Content Type ◽

Mitochondrial Calcium Uniporter ◽

Calcium Uniporter ◽

Mitochondrial Atp Synthase

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.

Download Full-text

ATP5D is differentially expressed following infection by the coronavirus family.

10.31219/osf.io/7jbxz ◽

2020 ◽

Author(s):

Shahan Mamoor

Keyword(s):

Differential Expression ◽

Atp Synthase ◽

Transcriptional Repression ◽

Human Cells ◽

Differentially Expressed ◽

Gene Encoding ◽

Significant Differential Expression ◽

Mitochondrial Atp Synthase ◽

Microarray Datasets

We mined published and public microarray datasets (1-6) to discover conserved host cell machinery transcriptionally modulated by the coronavirus family. We found significant differential expression and transcriptional repression of the gene encoding the mitochondrial F1 ATP synthase subunit 𝛅, ATP5D, following infection of human cells with the Middle East Respiratory Syndrome (MERS) coronavirus in vitro. ATP5D was differentially expressed, to a lesser extent, in the lungs of BALB/c mice infected with the Severe Acute Respiratory Syndrome (SARS) coronavirus 1 (SARS-CoV-1) but not in human cells infected with the human coronavirus HCoV-229E. These data document unique differential expression of a mitochondrial ATP synthase subunit by viruses of the coronavirus family.

Download Full-text

Alpha-Tocopherol Protects Cultured Human Cells from the Acute Lethal Cytotoxicity of Dioxin

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831.72.3.147 ◽

2002 ◽

Vol 72 (3) ◽

pp. 147-153 ◽

Cited By ~ 8

Author(s):

Kei-Ichi Hirai ◽

Jie-Hong Pan ◽

Ying-Bo Shui ◽

Eriko Simamura ◽

Hiroki Shimada ◽

...

Keyword(s):

Cell Death ◽

Cell Damage ◽

Smooth Endoplasmic Reticulum ◽

Dehydroascorbic Acid ◽

Human Cells ◽

Alpha Tocopherol ◽

Cervical Cancer Cells ◽

Cultured Human Cells ◽

Nuclear Damage ◽

Conjunctival Epithelial Cells

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30–40 ng/mL TCDD induced maximal intracellular production of H2O2 at 30 minutes and led to severe cell death (0–31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78–97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.

Download Full-text