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Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Nakamura T, Pluskal T, Nakaseko Y, Yanagida M - Open Biol (2012)

Bottom Line: The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation.Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence.Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Tancha 1919-1, Onna, Okinawa 904-0495, Japan.

ABSTRACT
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

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Related in: MedlinePlus

Detection of CoA, acetyl-CoA and Ppc1/PPCS in Schizosaccharomyces pombe. (a) Metabolomic analysis was performed for the specimens for LC/MS prepared as described [23,24] (§5) using an LTQ Orbitrap mass spectrometer. The amounts of ATP, ADP, AMP, CoA, acetyl-CoA and pantothenate are shown with the m/z values, the integrated peak areas and the retention times (min). (b) WT and ppc1-537 grown at 26°C were transferred to 36°C for 4 h in the EMM2 medium. Metabolites were extracted from WT and mutant cells and analysed. The levels of CoA, acetyl-CoA and 4′-phosphopantothenate are shown, normalized by the amount of internal standard (PIPES) using the MZmine 2 software [43]. (c) The precursor compound 4′-phosphopantothenate, accumulated in ppc1-537, disappeared, and CoA and acetyl-CoA were restored when plasmid expressing the WT ppc1+ gene was introduced into the mutant cells. (d) The level of Ppc1 protein was measured using the chromosomally integrated strain with the Ppc1 tagged with GFP and expressed under the native promoter. Two other integrant strains carrying Ptk1-GFP or Acs1-GFP are shown as the control. (e) Localization of Ppc1-GFP chromosomally integrated and expressed under the native promoter. Cells were cultured at 26°C and the GFP signal was observed with DAPI co-staining. Scale bar, 10 μm.
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RSOB120117F4: Detection of CoA, acetyl-CoA and Ppc1/PPCS in Schizosaccharomyces pombe. (a) Metabolomic analysis was performed for the specimens for LC/MS prepared as described [23,24] (§5) using an LTQ Orbitrap mass spectrometer. The amounts of ATP, ADP, AMP, CoA, acetyl-CoA and pantothenate are shown with the m/z values, the integrated peak areas and the retention times (min). (b) WT and ppc1-537 grown at 26°C were transferred to 36°C for 4 h in the EMM2 medium. Metabolites were extracted from WT and mutant cells and analysed. The levels of CoA, acetyl-CoA and 4′-phosphopantothenate are shown, normalized by the amount of internal standard (PIPES) using the MZmine 2 software [43]. (c) The precursor compound 4′-phosphopantothenate, accumulated in ppc1-537, disappeared, and CoA and acetyl-CoA were restored when plasmid expressing the WT ppc1+ gene was introduced into the mutant cells. (d) The level of Ppc1 protein was measured using the chromosomally integrated strain with the Ppc1 tagged with GFP and expressed under the native promoter. Two other integrant strains carrying Ptk1-GFP or Acs1-GFP are shown as the control. (e) Localization of Ppc1-GFP chromosomally integrated and expressed under the native promoter. Cells were cultured at 26°C and the GFP signal was observed with DAPI co-staining. Scale bar, 10 μm.

Mentions: To characterize mutant phenotypes, the metabolomic analysis recently developed for S. pombe [23–25] was applied to assay the level of CoA and acetyl-CoA present in WT and mutant cell extracts, using an LTQ Orbitrap mass spectrometer. In S. pombe, using our extraction and detection conditions, the levels of CoA and acetyl-CoA were relatively low, nearly 1/100-fold in the peak areas in comparison with other abundant metabolites such as ATP, ADP and AMP (figure 4a). However, we could detect reproducible peaks of ionized CoA, acetyl-CoA and their precursor compounds in repeated experiments [24]. The identity of CoA and acetyl-CoA peaks was verified using purchased standard compounds.Figure 4.


Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Nakamura T, Pluskal T, Nakaseko Y, Yanagida M - Open Biol (2012)

Detection of CoA, acetyl-CoA and Ppc1/PPCS in Schizosaccharomyces pombe. (a) Metabolomic analysis was performed for the specimens for LC/MS prepared as described [23,24] (§5) using an LTQ Orbitrap mass spectrometer. The amounts of ATP, ADP, AMP, CoA, acetyl-CoA and pantothenate are shown with the m/z values, the integrated peak areas and the retention times (min). (b) WT and ppc1-537 grown at 26°C were transferred to 36°C for 4 h in the EMM2 medium. Metabolites were extracted from WT and mutant cells and analysed. The levels of CoA, acetyl-CoA and 4′-phosphopantothenate are shown, normalized by the amount of internal standard (PIPES) using the MZmine 2 software [43]. (c) The precursor compound 4′-phosphopantothenate, accumulated in ppc1-537, disappeared, and CoA and acetyl-CoA were restored when plasmid expressing the WT ppc1+ gene was introduced into the mutant cells. (d) The level of Ppc1 protein was measured using the chromosomally integrated strain with the Ppc1 tagged with GFP and expressed under the native promoter. Two other integrant strains carrying Ptk1-GFP or Acs1-GFP are shown as the control. (e) Localization of Ppc1-GFP chromosomally integrated and expressed under the native promoter. Cells were cultured at 26°C and the GFP signal was observed with DAPI co-staining. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472395&req=5

RSOB120117F4: Detection of CoA, acetyl-CoA and Ppc1/PPCS in Schizosaccharomyces pombe. (a) Metabolomic analysis was performed for the specimens for LC/MS prepared as described [23,24] (§5) using an LTQ Orbitrap mass spectrometer. The amounts of ATP, ADP, AMP, CoA, acetyl-CoA and pantothenate are shown with the m/z values, the integrated peak areas and the retention times (min). (b) WT and ppc1-537 grown at 26°C were transferred to 36°C for 4 h in the EMM2 medium. Metabolites were extracted from WT and mutant cells and analysed. The levels of CoA, acetyl-CoA and 4′-phosphopantothenate are shown, normalized by the amount of internal standard (PIPES) using the MZmine 2 software [43]. (c) The precursor compound 4′-phosphopantothenate, accumulated in ppc1-537, disappeared, and CoA and acetyl-CoA were restored when plasmid expressing the WT ppc1+ gene was introduced into the mutant cells. (d) The level of Ppc1 protein was measured using the chromosomally integrated strain with the Ppc1 tagged with GFP and expressed under the native promoter. Two other integrant strains carrying Ptk1-GFP or Acs1-GFP are shown as the control. (e) Localization of Ppc1-GFP chromosomally integrated and expressed under the native promoter. Cells were cultured at 26°C and the GFP signal was observed with DAPI co-staining. Scale bar, 10 μm.
Mentions: To characterize mutant phenotypes, the metabolomic analysis recently developed for S. pombe [23–25] was applied to assay the level of CoA and acetyl-CoA present in WT and mutant cell extracts, using an LTQ Orbitrap mass spectrometer. In S. pombe, using our extraction and detection conditions, the levels of CoA and acetyl-CoA were relatively low, nearly 1/100-fold in the peak areas in comparison with other abundant metabolites such as ATP, ADP and AMP (figure 4a). However, we could detect reproducible peaks of ionized CoA, acetyl-CoA and their precursor compounds in repeated experiments [24]. The identity of CoA and acetyl-CoA peaks was verified using purchased standard compounds.Figure 4.

Bottom Line: The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation.Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence.Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation.

View Article: PubMed Central - PubMed

Affiliation: Okinawa Institute of Science and Technology Graduate University, Tancha 1919-1, Onna, Okinawa 904-0495, Japan.

ABSTRACT
Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

Show MeSH
Related in: MedlinePlus