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An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit.

ABSTRACT: Unlike most organisms, the mitochondrial DNA (mtDNA) of Chlamydomonas reinhardtii, a green alga, does not encode subunit 6 of F(0)F(1)-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 human ATP6 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.

SUBMITTER: Ojaimi J

PROVIDER: S-EPMC133596 | biostudies-literature | 2002 Nov

REPOSITORIES: biostudies-literature

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An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit.

Ojaimi Joseline J Pan Junmin J Santra Sumana S Snell William J WJ Schon Eric A EA

Molecular biology of the cell 20021101 11

Unlike most organisms, the mitochondrial DNA (mtDNA) of Chlamydomonas reinhardtii, a green alga, does not encode subunit 6 of F(0)F(1)-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 human ATP6 genes are in different subcellular compartments and the encoded polypeptides are highly diverg ...[more]

PMID: 12429828

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Project description: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.

Dataset Information

An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit.

Publications

An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit.

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