Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells

1981 ◽  
Vol 1 (4) ◽  
pp. 321-329
Author(s):  
C J Doersen ◽  
E J Stanbridge
Keyword(s):  
Protein Synthesis ◽  
Infected Cell ◽  
Cell Lines ◽  
Resistant Strain ◽  
Drug Sensitivity ◽  
Mitochondrial Protein ◽  
Phenotypic Expression ◽  
Mitochondrial Protein Synthesis ◽  
Protein Synthesis Inhibitors

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.

scholarly journals Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

1981 ◽  
Vol 1 (4) ◽  
pp. 321-329 ◽  
Author(s):  
C J Doersen ◽  
E J Stanbridge
Keyword(s):  
Protein Synthesis ◽  
Infected Cell ◽  
Cell Lines ◽  
Resistant Strain ◽  
Drug Sensitivity ◽  
Mitochondrial Protein ◽  
Phenotypic Expression ◽  
Mitochondrial Protein Synthesis ◽  
Protein Synthesis Inhibitors

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.

scholarly journals Mitochondrial nucleoids maintain genetic autonomy but allow for functional complementation

2008 ◽  
Vol 181 (7) ◽  
pp. 1117-1128 ◽  
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.

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

1981 ◽  
Vol 90 (1) ◽  
pp. 108-115 ◽  
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.

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

1972 ◽  
Vol 54 (1) ◽  
pp. 56-74 ◽  
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.

Mitochondrial protein synthesis in vitro is not an artifact

FEBS Letters ◽  
1973 ◽  
Vol 29 (1) ◽  
pp. 73-76 ◽  
Author(s):  
Nader G. Ibrahim ◽  
James P. Burke ◽  
Diana S. Beattie
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Synthesis

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

1991 ◽  
Vol 11 (4) ◽  
pp. 2236-2244 ◽  
Author(s):  
A Chomyn ◽  
G Meola ◽  
N Bresolin ◽  
S T Lai ◽  
G Scarlato ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Protein Synthesis ◽  
Human Cell ◽  
Mitochondrial Myopathy ◽  
Mitochondrial Protein ◽  
Human Cell Lines ◽  
Mitochondrial Protein Synthesis ◽  
Direct Link ◽  
Genetic Transfer

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.

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

1988 ◽  
Vol 8 (8) ◽  
pp. 3311-3315 ◽  
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.

scholarly journals The Molecular Pharmacology of Pateamine A

2021 ◽  
Author(s):  
◽  
James Henry Matthews
Keyword(s):  
Protein Synthesis ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Mode Of Action ◽  
Protein Synthesis Inhibitors ◽  
Primary Target ◽  
Dead Box ◽  
Mammalian Cell Lines ◽  
Pateamine A

<p>Pateamine A is a cytotoxic terpenoid isolated from the marine sponge Mycale hentscheli that induces apoptosis in mammalian cell lines and is growth inhibitory to yeasts and fungi, yet shows no inhibitory action in prokaryotes. The targets of pateamine in mammalian cell lines were isolated and identified using a combination of affinity chromatography and mass spectrometry, putative targets included the DEAD-Box helicase eIF4A family of proteins, β-tubulin and actin. In vitro assessment of tubulin and actin polymerization showed pateamine was able to affect them only at high micromolar concentrations, whereas the effect on eIF4A in vitro was shown by others to occur at nanomolar concentrations. Additionally, pateamine was shown to inhibit cap-dependent protein synthesis in vivo, suggesting eIF4A as a primary target. The generation of a pateamine resistance-conferring mutation in the yeast eIF4A encoding gene TIF1, suggested further that eIF4A is a primary target in both mammalian and yeast cells, and allows the speculation of the position of the binding site for pateamine on the N-terminal lobe of eIF4A and the proposal of potential covalent interaction between this drug and its target. Given the size of the DEAD-Box helicase family, all of which share considerable homology with the eIF4As, FAL1 especially which is essential for rRNA maturation, a chemogenomic screen was performed in an attempt to establish the breadth of functional interactions of pateamine. The results of hierarchical clustering of these screen results suggest that pateamine has a mode-of-action distinct from other compounds screened previously, despite its effect on protein synthesis it failed to cluster with any other protein synthesis inhibitors regardless of their separate mechanisms, though, as a class, protein synthesis inhibitors were not found to form a discrete cluster in any of the variations of cluster analysis performed. Functional analysis, by GO term enrichment, of the genes whose deletions are hypersensitive to pateamine indicates that deletions of genes involved in numerous aspects of RNA metabolism affect pateamine sensitivity, however clear results regarding the involvement of FAL1 or any other non-eIF4A target in pateamine’s mode-of-action were not found.</p>

scholarly journals NOA1 is an essential GTPase required for mitochondrial protein synthesis

2011 ◽  
Vol 22 (1) ◽  
pp. 1-11 ◽  
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.

Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions

1993 ◽  
Vol 264 (2) ◽  
pp. C383-C389 ◽  
Author(s):  
A. M. Cogswell ◽  
R. J. Stevens ◽  
D. A. Hood
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Mitochondrial Fraction ◽  
Biochemical Properties ◽  
Leucine Incorporation ◽  
Mitochondrial Protein Synthesis ◽  
Skeletal Muscle Mitochondria ◽  
Mitochondrial Fractions

Two mitochondrial fractions, termed intermyofibrillar (IMF) and subsarcolemmal (SS), were isolated from skeletal muscle, and their biochemical properties were related to differences in respiration and mitochondrial protein synthesis. State III respiration was 2.3- to 2.8-fold greater in IMF than in SS mitochondria. Site 1 inhibition of respiration with rotenone reduced this difference to 1.4-fold. When sites 1 and 2 were inhibited with antimycin, the 1.4-fold differences remained. The activities of cytochrome-c oxidase (CYTOX) and succinate dehydrogenase (SDH) could account for some of these differences, since CYTOX was 20% greater (P < 0.05) in IMF mitochondria, and SDH was 40% greater (P < 0.05) in SS mitochondria. Cytochromes a, b, c, and c1 contents were similar in the two fractions. Cardiolipin (CL) content was higher (P < 0.05) in SS mitochondria, indicating a less dense mitochondrial fraction with respect to CL. In vitro [3H]leucine incorporation was 1.8-fold higher (P < 0.05) in IMF than in SS mitochondria. Thus compositional differences between IMF and SS fractions exist, perhaps representing mitochondria at different stages of biogenesis. The biochemical and functional differences could not solely be due to differences in mitochondrial protein synthesis but could also be due to nuclear-directed protein synthesis specific to each mitochondrial fraction.

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