In vitro genetic transfer of protein synthesis and respiration defects to mitochondrial DNA-less cells with myopathy-patient mitochondria

A severe mitochondrial protein synthesis defect in myoblasts from a patient with mitochondrial myopathy was transferred with myoblast mitochondria into two genetically unrelated mitochondrial DNA (mtDNA)-less human cell lines, pointing to an mtDNA alteration as being responsible and sufficient for causing the disease. The transfer of the defect correlated with marked deficiencies in respiration and cytochrome c oxidase activity of the transformants and the presence in their mitochondria of mtDNA carrying a tRNA(Lys) mutation. Furthermore, apparently complete segregation of the defective genotype and phenotype was observed in the transformants derived from the heterogeneous proband myoblast population, suggesting that the mtDNA heteroplasmy in this population was to a large extent intercellular. The present work thus establishes a direct link between mtDNA alteration and a biochemical defect.

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Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines

Methods in Molecular Biology - Mitochondrial Gene Expression ◽

10.1007/978-1-0716-0834-0_13 ◽

2020 ◽

pp. 159-181

Author(s):

Matthew Zorkau ◽

Yasmin Proctor-Kent ◽

Rolando Berlinguer-Palmini ◽

Andrew Hamilton ◽

Zofia M. Chrzanowska-Lightowlers ◽

...

Keyword(s):

Protein Synthesis ◽

Cell Lines ◽

Ribosomal Rna ◽

Human Cell ◽

Mitochondrial Protein ◽

Human Cell Lines ◽

Mitochondrial Protein Synthesis

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Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation

The Journal of Cell Biology ◽

10.1083/jcb.200712101 ◽

2008 ◽

Vol 181 (7) ◽

pp. 1117-1128 ◽

Cited By ~ 111

Author(s):

Robert W. Gilkerson ◽

Eric A. Schon ◽

Evelyn Hernandez ◽

Mercy M. Davidson

Keyword(s):

Mitochondrial Dna ◽

Protein Synthesis ◽

Molecular Mechanism ◽

Cell Lines ◽

Mitochondrial Protein ◽

Functional Complementation ◽

Mitochondrial Protein Synthesis ◽

Protein Assemblies ◽

Mtdna Mutations ◽

Mitochondrial Nucleoids

Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.

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Recombination of yeast mitochondrial DNA does not require mitochondrial protein synthesis

Current Genetics ◽

10.1007/bf00413304 ◽

1979 ◽

Vol 1 (1) ◽

pp. 21-31 ◽

Cited By ~ 3

Author(s):

Susan L. Strausberg ◽

C. William Birky

Keyword(s):

Mitochondrial Dna ◽

Protein Synthesis ◽

Mitochondrial Protein ◽

Mitochondrial Protein Synthesis ◽

Yeast Mitochondrial Dna

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Integrity of mitochondria in a mammalian cell mutant defective in mitochondrial protein synthesis.

The Journal of Cell Biology ◽

10.1083/jcb.90.1.108 ◽

1981 ◽

Vol 90 (1) ◽

pp. 108-115 ◽

Cited By ~ 21

Author(s):

K G Burnett ◽

I E Scheffler

Keyword(s):

Protein Synthesis ◽

Mitochondrial Protein ◽

Chinese Hamster ◽

Two Dimensional ◽

Mitochondrial Protein Synthesis ◽

Two Dimensional Gel Electrophoresis ◽

Translational Machinery ◽

Sedimentation Behavior ◽

Rrna Species

A defect in mitochondrial protein synthesis has previously been identified in the respiration-deficient Chinese hamster lung fibroblast mutant V79-G7. The present work extends the characterization of this mutant. A more sensitive analysis has shown that mutant mitochondria synthesize all mitochondrially encoded peptides, but in significantly reduced amounts. This difference is also seen when isolated mitochondria are tested for in vitro protein synthesis. To distinguish between a defect in the translational machinery and a defect in the transcription of mitochondrial DNA, we investigated the synthesis of the 16S and 12S mitochondrial rRNA species and found them to be made in normal amounts in G7 mitochondria. These rRNA species appear to be assembled into subunits whose sedimentation behavior is virtually indistinguishable from that of the wild-type subunits. We also examined the consequences of the defect in mitochondrial protein synthesis on mutant cells and their mitochondria-utilizing techniques of electron microscopy, two-dimensional gel electrophoresis and immunochemical analysis. G7 mitochondria have a characteristic ultrastructure distinguished by predominantly tubular cristae, but the overall biochemical composition of mitochondrial membrane and matrix fractions appears essentially unaltered except for the absence of a few characteristic peptides. Specifically, we identify the absence of two mitochondrially encoded subunits of cytochrome c oxidase on two-dimensional gels and demonstrate a drastic reduction of both cytoplasmically and mitochondrially synthesized subunits of enzyme in immunoprecipitates of G7 mitochondria.

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THE INTRACELLULAR SITE OF SYNTHESIS OF MITOCHONDRIAL RIBOSOMAL PROTEINS IN NEUROSPORA CRASSA

The Journal of Cell Biology ◽

10.1083/jcb.54.1.56 ◽

1972 ◽

Vol 54 (1) ◽

pp. 56-74 ◽

Cited By ~ 39

Author(s):

Paul M. Lizardi ◽

David J. L. Luck

Keyword(s):

Protein Synthesis ◽

Isoelectric Focusing ◽

Gel Electrophoresis ◽

Ribosomal Proteins ◽

Mitochondrial Protein ◽

Selective Inhibitor ◽

Mitochondrial Protein Synthesis ◽

Ribosomal Subunits

The intracellular site of synthesis of mitochondrial ribosomal proteins (MRP) in Neurospora crassa has been investigated using three complementary approaches. (a) Mitochondrial protein synthesis in vitro: Tritium-labeled proteins made by isolated mitochondria were compared to 14C-labeled marker MRP by cofractionation in a two-step procedure involving isoelectric focusing and polyacrylamide gel electrophoresis. Examination of the electrophoretic profiles showed that essentially none of the peaks of in vitro product corresponded exactly to any of the MRP marker peaks. (b) Sensitivity of in vivo MRP synthesis to chloramphenicol: Cells were labeled with leucine-3H in the presence of chloramphenicol, mitochondrial ribosomal subunits were subsequently isolated, and their proteins fractionated by isoelectric focusing followed by gel electrophoresis. The labeling of every single MRP was found to be insensitive to chloramphenicol, a selective inhibitor of mitochondrial protein synthesis. (c) Sensitivity of in vivo MRP synthesis to anisomycin: We have found this antibiotic to be a good selective inhibitor of cytoplasmic protein synthesis in Neurospora. In the presence of anisomycin the labeling of virtually all MRP is inhibited to the same extent as the labeling of cytoplasmic ribosomal proteins. On the basis of these three types of studies we conclude that most if not all 53 structural proteins of mitochondrial ribosomal subunits in Neurospora are synthesized by cytoplasmic ribosomes.

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Mitochondrial protein synthesis in vitro is not an artifact

FEBS Letters ◽

10.1016/0014-5793(73)80019-5 ◽

1973 ◽

Vol 29 (1) ◽

pp. 73-76 ◽

Cited By ~ 33

Author(s):

Nader G. Ibrahim ◽

James P. Burke ◽

Diana S. Beattie

Keyword(s):

Protein Synthesis ◽

Mitochondrial Protein ◽

Mitochondrial Protein Synthesis

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Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.8.3311 ◽

1988 ◽

Vol 8 (8) ◽

pp. 3311-3315 ◽

Cited By ~ 28

Author(s):

T Kaneko ◽

T Watanabe ◽

M Oishi

Keyword(s):

Protein Synthesis ◽

Early Stage ◽

Mitochondrial Protein ◽

Specific Inhibitor ◽

Erythroid Differentiation ◽

Mitochondrial Protein Synthesis ◽

Cell Extracts ◽

Factor Ii ◽

Mouse Erythroleukemia

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.

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Mitochondrial DNA Medicine

Bioscience Reports ◽

10.1007/s10540-007-9032-5 ◽

2007 ◽

Vol 27 (1-3) ◽

pp. 5-9 ◽

Cited By ~ 24

Author(s):

Salvatore DiMauro

Keyword(s):

Mitochondrial Dna ◽

Protein Synthesis ◽

Mitochondrial Protein ◽

Point Mutations ◽

Specific Protein ◽

Mitochondrial Protein Synthesis ◽

Mitochondrial Genetics ◽

Protein Coding ◽

Protein Coding Genes ◽

Mitochondrial Medicine

The small, maternally inherited mitochondrial DNA (mtDNA) has turned out to be a hotbed of pathogenic mutations: 15 years into the era of ‘mitochondrial medicine’, over 150 pathogenic point mutations and countless rearrangements have been associated with a variety of multisystemic or tissue-specific human diseases. MtDNA-related disorders can be divided into two major groups: those due to mutations in genes affecting mitochondrial protein synthesis in toto and those due to mutations in specific protein-coding genes. Here we review the mitochondrial genetics and the clinical features of the mtDNA-related diseases.

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NOA1 is an essential GTPase required for mitochondrial protein synthesis

Molecular Biology of the Cell ◽

10.1091/mbc.e10-07-0643 ◽

2011 ◽

Vol 22 (1) ◽

pp. 1-11 ◽

Cited By ~ 37

Author(s):

Mateusz Kolanczyk ◽

Markus Pech ◽

Tomasz Zemojtel ◽

Hiroshi Yamamoto ◽

Ivan Mikula ◽

...

Keyword(s):

Protein Synthesis ◽

Mammalian Cells ◽

Gradient Centrifugation ◽

Mitochondrial Protein ◽

In Vitro Assays ◽

Developmental Defect ◽

Mitochondrial Protein Synthesis ◽

Mitochondrial Functions ◽

Mitochondrial Ribosome

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1–/– cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1–/– cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis.

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