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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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ts-537 strain is defective in the accurate chromosome segregation. (a) The biosynthetic pathway of CoA from pantothenate. (b) Light micrographs of WT and ts mutant ts-537, which were cultured at 36°C for 4 h. Cells were fixed and stained with DAPI. Scale bar, 10 μm. The arrows and arrowheads indicate cells with the defective phenotypes of unequal segregation and displaced nucleus, respectively. (c) The WT and mutant ts-537 cells exponentially grown at 26°C in the complete YPD medium were transferred to 36°C for 0–8 h. The cell number and cell viability percentage were scored at 1-h intervals under microscope and by plating. Aliquots of cells were fixed and stained with DAPI, and the frequency of aberrant mitosis was scored under the microscope. See text. (d) WT and ts-537 strains chromosomally integrated with GFP-tagged Lac repressor that was bound to the peri-centromeric region of chromosome I (§5) were cultured at 36°C for 4 h, then fixed with methanol. The peri-centromeric GFP signals are shown together with DAPI staining. Scale bar, 10 μm. (e) WT and ts-537 strains carrying an artificial minichromosome Ch10-CN2 were cultured in YPD medium at 26°C for 10 generations and plated on the complete medium. The colonies with high frequencies of minichromosome loss showed red–orange colour. The frequencies of minichromosome loss rates were scored as the frequency of Ade− red colonies. (f) Additive defects were observed for the double mutants between ts-537 and mis6, mis12 or mis16 (see text).
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RSOB120117F1: ts-537 strain is defective in the accurate chromosome segregation. (a) The biosynthetic pathway of CoA from pantothenate. (b) Light micrographs of WT and ts mutant ts-537, which were cultured at 36°C for 4 h. Cells were fixed and stained with DAPI. Scale bar, 10 μm. The arrows and arrowheads indicate cells with the defective phenotypes of unequal segregation and displaced nucleus, respectively. (c) The WT and mutant ts-537 cells exponentially grown at 26°C in the complete YPD medium were transferred to 36°C for 0–8 h. The cell number and cell viability percentage were scored at 1-h intervals under microscope and by plating. Aliquots of cells were fixed and stained with DAPI, and the frequency of aberrant mitosis was scored under the microscope. See text. (d) WT and ts-537 strains chromosomally integrated with GFP-tagged Lac repressor that was bound to the peri-centromeric region of chromosome I (§5) were cultured at 36°C for 4 h, then fixed with methanol. The peri-centromeric GFP signals are shown together with DAPI staining. Scale bar, 10 μm. (e) WT and ts-537 strains carrying an artificial minichromosome Ch10-CN2 were cultured in YPD medium at 26°C for 10 generations and plated on the complete medium. The colonies with high frequencies of minichromosome loss showed red–orange colour. The frequencies of minichromosome loss rates were scored as the frequency of Ade− red colonies. (f) Additive defects were observed for the double mutants between ts-537 and mis6, mis12 or mis16 (see text).

Mentions: The CoA synthetic pathway, present in prokaryotes, fungi, plants and animals, consists of five steps, requiring four molecules of nucleotide triphosphate (ATP or CTP) [7–10] (figure 1a). Pantothenate is a specific precursor for CoA that is phosphorylated by pantothenate kinase (PANK) to 4′-phosphopantothenate as the initial step. In the next step, phosphopantothenoylcysteine synthetase (PPCS) catalyses the condensation reaction of 4′-phosphopantothenate and cysteine [11]. The third reaction involves decarboxylation reaction to 4′-phosphopantetheine [12]. Finally, the AMP moiety is added to form dephospho-CoA, which is subsequently phosphorylated on the 3′-OH of the ribose to yield CoA [13].Figure 1.


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)

ts-537 strain is defective in the accurate chromosome segregation. (a) The biosynthetic pathway of CoA from pantothenate. (b) Light micrographs of WT and ts mutant ts-537, which were cultured at 36°C for 4 h. Cells were fixed and stained with DAPI. Scale bar, 10 μm. The arrows and arrowheads indicate cells with the defective phenotypes of unequal segregation and displaced nucleus, respectively. (c) The WT and mutant ts-537 cells exponentially grown at 26°C in the complete YPD medium were transferred to 36°C for 0–8 h. The cell number and cell viability percentage were scored at 1-h intervals under microscope and by plating. Aliquots of cells were fixed and stained with DAPI, and the frequency of aberrant mitosis was scored under the microscope. See text. (d) WT and ts-537 strains chromosomally integrated with GFP-tagged Lac repressor that was bound to the peri-centromeric region of chromosome I (§5) were cultured at 36°C for 4 h, then fixed with methanol. The peri-centromeric GFP signals are shown together with DAPI staining. Scale bar, 10 μm. (e) WT and ts-537 strains carrying an artificial minichromosome Ch10-CN2 were cultured in YPD medium at 26°C for 10 generations and plated on the complete medium. The colonies with high frequencies of minichromosome loss showed red–orange colour. The frequencies of minichromosome loss rates were scored as the frequency of Ade− red colonies. (f) Additive defects were observed for the double mutants between ts-537 and mis6, mis12 or mis16 (see text).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120117F1: ts-537 strain is defective in the accurate chromosome segregation. (a) The biosynthetic pathway of CoA from pantothenate. (b) Light micrographs of WT and ts mutant ts-537, which were cultured at 36°C for 4 h. Cells were fixed and stained with DAPI. Scale bar, 10 μm. The arrows and arrowheads indicate cells with the defective phenotypes of unequal segregation and displaced nucleus, respectively. (c) The WT and mutant ts-537 cells exponentially grown at 26°C in the complete YPD medium were transferred to 36°C for 0–8 h. The cell number and cell viability percentage were scored at 1-h intervals under microscope and by plating. Aliquots of cells were fixed and stained with DAPI, and the frequency of aberrant mitosis was scored under the microscope. See text. (d) WT and ts-537 strains chromosomally integrated with GFP-tagged Lac repressor that was bound to the peri-centromeric region of chromosome I (§5) were cultured at 36°C for 4 h, then fixed with methanol. The peri-centromeric GFP signals are shown together with DAPI staining. Scale bar, 10 μm. (e) WT and ts-537 strains carrying an artificial minichromosome Ch10-CN2 were cultured in YPD medium at 26°C for 10 generations and plated on the complete medium. The colonies with high frequencies of minichromosome loss showed red–orange colour. The frequencies of minichromosome loss rates were scored as the frequency of Ade− red colonies. (f) Additive defects were observed for the double mutants between ts-537 and mis6, mis12 or mis16 (see text).
Mentions: The CoA synthetic pathway, present in prokaryotes, fungi, plants and animals, consists of five steps, requiring four molecules of nucleotide triphosphate (ATP or CTP) [7–10] (figure 1a). Pantothenate is a specific precursor for CoA that is phosphorylated by pantothenate kinase (PANK) to 4′-phosphopantothenate as the initial step. In the next step, phosphopantothenoylcysteine synthetase (PPCS) catalyses the condensation reaction of 4′-phosphopantothenate and cysteine [11]. The third reaction involves decarboxylation reaction to 4′-phosphopantetheine [12]. Finally, the AMP moiety is added to form dephospho-CoA, which is subsequently phosphorylated on the 3′-OH of the ribose to yield CoA [13].Figure 1.

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