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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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Viability decrease of ts-537 in the G0 phase and in mitosis. (a) Wild-type (WT) and ts-537 cells were brought into the G0 quiescent state at 26°C (i) under the nitrogen source deficiency for 24 h [26]. Resulting G0 quiescent cultures were shifted to (ii) 36°C for 4 days. As the control, portions of the cultures were kept at 26°C. The cell viability percentages at different time points (days) were obtained by plating cells on the complete medium at 26°C, and calculated as the percentage of the number of formed colonies against the number of plated cells. ts-537 mutant cells lost the viability within 2 days at the restrictive temperature. (b) Light micrographs of non-dividing (i) WT and (ii) ts-537 G0 cells. Cells were cultured in the nitrogen-deficient medium (EMM2-N) at 26°C for 24 h and transferred to 36°C for 3 days. Cells were then fixed and stained with DAPI. (c)(i) WT and (ii) ts-537 cells, which had been kept in the G0 quiescent medium (EMM2-N) for 24 h at 26°C, were nitrogen source replenished and cultured at 36°C for 14 h. Cells were collected at the time points indicated, and their DNA contents were measured by the Beckton–Dickinson FACscan. See text. (d)(i) The cell numbers (blue, WT; red, ts-537). (ii) The frequencies of aberrant mitotic cells. Red, ts-537; blue, WT. See text. (e)(i) Cell viability percentages of WT (blue) and ts-537 (red). (ii) Septation index of WT (blue) and ts-537 (red).
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RSOB120117F2: Viability decrease of ts-537 in the G0 phase and in mitosis. (a) Wild-type (WT) and ts-537 cells were brought into the G0 quiescent state at 26°C (i) under the nitrogen source deficiency for 24 h [26]. Resulting G0 quiescent cultures were shifted to (ii) 36°C for 4 days. As the control, portions of the cultures were kept at 26°C. The cell viability percentages at different time points (days) were obtained by plating cells on the complete medium at 26°C, and calculated as the percentage of the number of formed colonies against the number of plated cells. ts-537 mutant cells lost the viability within 2 days at the restrictive temperature. (b) Light micrographs of non-dividing (i) WT and (ii) ts-537 G0 cells. Cells were cultured in the nitrogen-deficient medium (EMM2-N) at 26°C for 24 h and transferred to 36°C for 3 days. Cells were then fixed and stained with DAPI. (c)(i) WT and (ii) ts-537 cells, which had been kept in the G0 quiescent medium (EMM2-N) for 24 h at 26°C, were nitrogen source replenished and cultured at 36°C for 14 h. Cells were collected at the time points indicated, and their DNA contents were measured by the Beckton–Dickinson FACscan. See text. (d)(i) The cell numbers (blue, WT; red, ts-537). (ii) The frequencies of aberrant mitotic cells. Red, ts-537; blue, WT. See text. (e)(i) Cell viability percentages of WT (blue) and ts-537 (red). (ii) Septation index of WT (blue) and ts-537 (red).

Mentions: To determine whether ts-537 could maintain the viability in the quiescent G0 phase, we monitored the time course of cell viability under nitrogen starvation [26]. WT and mutant ts-537 cells first grown in the synthetic Edinburgh minimal medium (EMM2) were transferred to the nitrogen-deficient EMM2-N at 26°C for 24 h, and the resulting quiescent cells, arrested in the pre-replicative G0 phase, were cultivated at either 26°C or 36°C for 4 days. Aliquots of the cultures were taken at intervals, and their cell viability percentage was assayed in the nutrient medium at 26°C (figure 2a). The cell viability of ts-537 was diminished at 36°C after 2 days, whereas the viability of the WT was high even after 4 days, suggesting that ts-537 failed to restore the cell cycle following a period of G0 maintenance. DAPI-stained cells of WT and ts-537 are shown in figure 2b. No significant difference of cell shape between WT and mutant cells was observed. The nucleus, however, was positioned closely to the plasma membrane in approximately 50 per cent of the mutant cells.Figure 2.


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)

Viability decrease of ts-537 in the G0 phase and in mitosis. (a) Wild-type (WT) and ts-537 cells were brought into the G0 quiescent state at 26°C (i) under the nitrogen source deficiency for 24 h [26]. Resulting G0 quiescent cultures were shifted to (ii) 36°C for 4 days. As the control, portions of the cultures were kept at 26°C. The cell viability percentages at different time points (days) were obtained by plating cells on the complete medium at 26°C, and calculated as the percentage of the number of formed colonies against the number of plated cells. ts-537 mutant cells lost the viability within 2 days at the restrictive temperature. (b) Light micrographs of non-dividing (i) WT and (ii) ts-537 G0 cells. Cells were cultured in the nitrogen-deficient medium (EMM2-N) at 26°C for 24 h and transferred to 36°C for 3 days. Cells were then fixed and stained with DAPI. (c)(i) WT and (ii) ts-537 cells, which had been kept in the G0 quiescent medium (EMM2-N) for 24 h at 26°C, were nitrogen source replenished and cultured at 36°C for 14 h. Cells were collected at the time points indicated, and their DNA contents were measured by the Beckton–Dickinson FACscan. See text. (d)(i) The cell numbers (blue, WT; red, ts-537). (ii) The frequencies of aberrant mitotic cells. Red, ts-537; blue, WT. See text. (e)(i) Cell viability percentages of WT (blue) and ts-537 (red). (ii) Septation index of WT (blue) and ts-537 (red).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120117F2: Viability decrease of ts-537 in the G0 phase and in mitosis. (a) Wild-type (WT) and ts-537 cells were brought into the G0 quiescent state at 26°C (i) under the nitrogen source deficiency for 24 h [26]. Resulting G0 quiescent cultures were shifted to (ii) 36°C for 4 days. As the control, portions of the cultures were kept at 26°C. The cell viability percentages at different time points (days) were obtained by plating cells on the complete medium at 26°C, and calculated as the percentage of the number of formed colonies against the number of plated cells. ts-537 mutant cells lost the viability within 2 days at the restrictive temperature. (b) Light micrographs of non-dividing (i) WT and (ii) ts-537 G0 cells. Cells were cultured in the nitrogen-deficient medium (EMM2-N) at 26°C for 24 h and transferred to 36°C for 3 days. Cells were then fixed and stained with DAPI. (c)(i) WT and (ii) ts-537 cells, which had been kept in the G0 quiescent medium (EMM2-N) for 24 h at 26°C, were nitrogen source replenished and cultured at 36°C for 14 h. Cells were collected at the time points indicated, and their DNA contents were measured by the Beckton–Dickinson FACscan. See text. (d)(i) The cell numbers (blue, WT; red, ts-537). (ii) The frequencies of aberrant mitotic cells. Red, ts-537; blue, WT. See text. (e)(i) Cell viability percentages of WT (blue) and ts-537 (red). (ii) Septation index of WT (blue) and ts-537 (red).
Mentions: To determine whether ts-537 could maintain the viability in the quiescent G0 phase, we monitored the time course of cell viability under nitrogen starvation [26]. WT and mutant ts-537 cells first grown in the synthetic Edinburgh minimal medium (EMM2) were transferred to the nitrogen-deficient EMM2-N at 26°C for 24 h, and the resulting quiescent cells, arrested in the pre-replicative G0 phase, were cultivated at either 26°C or 36°C for 4 days. Aliquots of the cultures were taken at intervals, and their cell viability percentage was assayed in the nutrient medium at 26°C (figure 2a). The cell viability of ts-537 was diminished at 36°C after 2 days, whereas the viability of the WT was high even after 4 days, suggesting that ts-537 failed to restore the cell cycle following a period of G0 maintenance. DAPI-stained cells of WT and ts-537 are shown in figure 2b. No significant difference of cell shape between WT and mutant cells was observed. The nucleus, however, was positioned closely to the plasma membrane in approximately 50 per cent of the mutant cells.Figure 2.

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