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New insights into the bioenergetics of mitochondrial disorders using intracellular ATP reporters.

ABSTRACT: Mutations in mitochondrial DNA (mtDNA) cause impairment of ATP synthesis. It was hypothesized that high-energy compounds, such as ATP, are compartmentalized within cells and that different cell functions are sustained by different pools of ATP, some deriving from mitochondrial oxidative phosphorylation (OXPHOS) and others from glycolysis. Therefore, an OXPHOS dysfunction may affect different cell compartments to different extents. To address this issue, we have used recombinant forms of the ATP reporter luciferase localized in different cell compartments- the cytosol, the subplasma membrane region, the mitochondrial matrix, and the nucleus- of cells containing either wild-type or mutant mtDNA. We found that with glycolytic substrates, both wild-type and mutant cells were able to maintain adequate ATP supplies in all compartments. Conversely, with the OXPHOS substrate pyruvate ATP levels collapsed in all cell compartments of mutant cells. In wild-type cells normal levels of ATP were maintained with pyruvate in the cytosol and in the subplasma membrane region, but, surprisingly, they were reduced in the mitochondria and, to a greater extent, in the nucleus. The severe decrease in nuclear ATP content under "OXPHOS-only" conditions implies that depletion of nuclear ATP plays an important, and hitherto unappreciated, role in patients with mitochondrial dysfunction.

SUBMITTER: Gajewski CD

PROVIDER: S-EPMC196555 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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New insights into the bioenergetics of mitochondrial disorders using intracellular ATP reporters.

Gajewski Carl D CD Yang Lichuan L Schon Eric A EA Manfredi Giovanni G

Molecular biology of the cell 20030627 9

Mutations in mitochondrial DNA (mtDNA) cause impairment of ATP synthesis. It was hypothesized that high-energy compounds, such as ATP, are compartmentalized within cells and that different cell functions are sustained by different pools of ATP, some deriving from mitochondrial oxidative phosphorylation (OXPHOS) and others from glycolysis. Therefore, an OXPHOS dysfunction may affect different cell compartments to different extents. To address this issue, we have used recombinant forms of the ATP ...[more]

PMID: 12972552

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Project description:Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondrial deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Project description:Mutations in nuclear genes encoding for mitochondrial proteins very long-chain acyl-CoA dehydrogenase (VLCAD) and trifunctional protein (TFP) cause rare autosomal recessive disorders. Studies in fibroblasts derived from patients with mutations in VLCAD and TFP exhibit mitochondrial defects. To gain insights on pathological changes that account for the mitochondria l deficits we performed quantitative proteomic, biochemical, and morphometric analyses in fibroblasts derived from subjects with three different VLCAD and three different TFP mutations. Proteomic data that was corroborated by antibody-based detection, indicated reduced levels of VLCAD and TFP protein in cells with VLCAD and TFP mutations respectively, which in part accounted for the diminished fatty acid oxidation capacity. Decreased mitochondrial respiratory capacity in cells with VLCAD and TFP mutations was quantified after glucose removal and cells with TFP mutations had lower levels of glycogen. Despite these energetic deficiencies, the cells with VLCAD and TFP mutations did not exhibit changes in mitochondria morphology, distribution, fusion and fission, quantified by either confocal or transmission electron microscopy and corroborated by proteomic and antibody-based protein analysis. Fibroblasts with VLCAD and to a lesser extend cells with TFP mutations had increased levels of mitochondrial respiratory chain proteins and proteins that facilitate the assembly of respiratory complexes. With the exception of reduced levels of catalase and glutathione S-transferase theta-1 in cells with TFP mutations, the levels of 45 proteins across all major intracellular antioxidant networks were similar between cells with VLCAD and TFP mutations and non-disease controls. Collectively the data indicate that despite the metabolic deficits, cells with VLCAD and TFP mutations maintain their proteomic integrity to preserve cellular and mitochondria architecture, support energy production and protect against oxidative stress.

Dataset Information

New insights into the bioenergetics of mitochondrial disorders using intracellular ATP reporters.

Publications

New insights into the bioenergetics of mitochondrial disorders using intracellular ATP reporters.

Similar Datasets

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