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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

Fission yeast ts-537 and ts-88 are mutants of PPCS/Ppc1, auxotrophic to pantothenate, and lack functional acetyl-CoA. (a) The substitution mutation sites (T48I and M209T) determined for ts-537 and ts-88 strains, respectively, reside within the coding region of PPCS/Ppc1 (top and bottom aligned sequences are S. pombe and human, respectively). Identical residues are in red. (b) The location of mutation sites in the three-dimensional structure of human PPCS [40]. T48I mutation site in ppc1-537 locates near the catalytic centre, while the site for ppc1-88 locates at the periphery of PPCS. (c) Pantothenate is needed for ppc1-537 but not for the WT. Spot tests were done for the WT and ppc1-537 in the presence or the absence of pantothenate. The exponentially growing cells were spotted by a serial cell concentration on the EMM2 synthetic medium plates either containing or lacking 1 mg l−1 pantothenate, followed by incubation at the indicated temperatures for 4 days. (d) Cell extracts of growing WT and ppc1-537 mutant cells were made from the complete culture at 26°C or 36°C. Histone acetyltransferase (HAT) activities of these extracts were measured using the kit containing histone H4 peptide in the addition (+) or the non-addition (−) of human acetyltransferase PCAF recombinant protein and acetyl-CoA. The degree of acetylation that occurred at histone H4 peptide was assayed by the optical density using anti-acetyllysine first antibody and HRP conjugated second antibody. The WT extracts did not need the addition of acetyl-CoA, but the mutant extracts made at 36°C required acetyl-CoA for the HAT activity.
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RSOB120117F3: Fission yeast ts-537 and ts-88 are mutants of PPCS/Ppc1, auxotrophic to pantothenate, and lack functional acetyl-CoA. (a) The substitution mutation sites (T48I and M209T) determined for ts-537 and ts-88 strains, respectively, reside within the coding region of PPCS/Ppc1 (top and bottom aligned sequences are S. pombe and human, respectively). Identical residues are in red. (b) The location of mutation sites in the three-dimensional structure of human PPCS [40]. T48I mutation site in ppc1-537 locates near the catalytic centre, while the site for ppc1-88 locates at the periphery of PPCS. (c) Pantothenate is needed for ppc1-537 but not for the WT. Spot tests were done for the WT and ppc1-537 in the presence or the absence of pantothenate. The exponentially growing cells were spotted by a serial cell concentration on the EMM2 synthetic medium plates either containing or lacking 1 mg l−1 pantothenate, followed by incubation at the indicated temperatures for 4 days. (d) Cell extracts of growing WT and ppc1-537 mutant cells were made from the complete culture at 26°C or 36°C. Histone acetyltransferase (HAT) activities of these extracts were measured using the kit containing histone H4 peptide in the addition (+) or the non-addition (−) of human acetyltransferase PCAF recombinant protein and acetyl-CoA. The degree of acetylation that occurred at histone H4 peptide was assayed by the optical density using anti-acetyllysine first antibody and HRP conjugated second antibody. The WT extracts did not need the addition of acetyl-CoA, but the mutant extracts made at 36°C required acetyl-CoA for the HAT activity.

Mentions: Plasmids that fully rescued the ts phenotype in ts-537 were isolated using S. pombe genomic DNA sequence-containing plasmid library as described previously [27]. The subcloned DNA sequences capable of promoting ts-537 colony formation at 36°C contained the single gene SPCC4B3.18 that was designated ppc1+ as it encodes PPCS, an intermediate enzyme in the biosynthetic pathway to produce CoA from pantothenate. Genetic analysis by tetrad dissection confirmed that ts-537 was linked (6.2 cM) to the sti1+ locus that is situated 120 kb apart from the Ppc1/SPCC4B3.18 locus. We then isolated the ppc1+ gene from the genomic DNA of the ts-537 mutant and its DNA sequence was determined. Only a single nucleotide change was found in the mutant ppc1 gene that corresponded to the amino acid substitution T48I in the amino-terminal region. This site is highly conserved among human, fly and budding yeast (figure 3a(i)). We hence concluded that ts-537 is a mutant of the PPCS/ppc1+/SPCC4B3.18 gene and designated it ppc1-537.Figure 3.


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)

Fission yeast ts-537 and ts-88 are mutants of PPCS/Ppc1, auxotrophic to pantothenate, and lack functional acetyl-CoA. (a) The substitution mutation sites (T48I and M209T) determined for ts-537 and ts-88 strains, respectively, reside within the coding region of PPCS/Ppc1 (top and bottom aligned sequences are S. pombe and human, respectively). Identical residues are in red. (b) The location of mutation sites in the three-dimensional structure of human PPCS [40]. T48I mutation site in ppc1-537 locates near the catalytic centre, while the site for ppc1-88 locates at the periphery of PPCS. (c) Pantothenate is needed for ppc1-537 but not for the WT. Spot tests were done for the WT and ppc1-537 in the presence or the absence of pantothenate. The exponentially growing cells were spotted by a serial cell concentration on the EMM2 synthetic medium plates either containing or lacking 1 mg l−1 pantothenate, followed by incubation at the indicated temperatures for 4 days. (d) Cell extracts of growing WT and ppc1-537 mutant cells were made from the complete culture at 26°C or 36°C. Histone acetyltransferase (HAT) activities of these extracts were measured using the kit containing histone H4 peptide in the addition (+) or the non-addition (−) of human acetyltransferase PCAF recombinant protein and acetyl-CoA. The degree of acetylation that occurred at histone H4 peptide was assayed by the optical density using anti-acetyllysine first antibody and HRP conjugated second antibody. The WT extracts did not need the addition of acetyl-CoA, but the mutant extracts made at 36°C required acetyl-CoA for the HAT activity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120117F3: Fission yeast ts-537 and ts-88 are mutants of PPCS/Ppc1, auxotrophic to pantothenate, and lack functional acetyl-CoA. (a) The substitution mutation sites (T48I and M209T) determined for ts-537 and ts-88 strains, respectively, reside within the coding region of PPCS/Ppc1 (top and bottom aligned sequences are S. pombe and human, respectively). Identical residues are in red. (b) The location of mutation sites in the three-dimensional structure of human PPCS [40]. T48I mutation site in ppc1-537 locates near the catalytic centre, while the site for ppc1-88 locates at the periphery of PPCS. (c) Pantothenate is needed for ppc1-537 but not for the WT. Spot tests were done for the WT and ppc1-537 in the presence or the absence of pantothenate. The exponentially growing cells were spotted by a serial cell concentration on the EMM2 synthetic medium plates either containing or lacking 1 mg l−1 pantothenate, followed by incubation at the indicated temperatures for 4 days. (d) Cell extracts of growing WT and ppc1-537 mutant cells were made from the complete culture at 26°C or 36°C. Histone acetyltransferase (HAT) activities of these extracts were measured using the kit containing histone H4 peptide in the addition (+) or the non-addition (−) of human acetyltransferase PCAF recombinant protein and acetyl-CoA. The degree of acetylation that occurred at histone H4 peptide was assayed by the optical density using anti-acetyllysine first antibody and HRP conjugated second antibody. The WT extracts did not need the addition of acetyl-CoA, but the mutant extracts made at 36°C required acetyl-CoA for the HAT activity.
Mentions: Plasmids that fully rescued the ts phenotype in ts-537 were isolated using S. pombe genomic DNA sequence-containing plasmid library as described previously [27]. The subcloned DNA sequences capable of promoting ts-537 colony formation at 36°C contained the single gene SPCC4B3.18 that was designated ppc1+ as it encodes PPCS, an intermediate enzyme in the biosynthetic pathway to produce CoA from pantothenate. Genetic analysis by tetrad dissection confirmed that ts-537 was linked (6.2 cM) to the sti1+ locus that is situated 120 kb apart from the Ppc1/SPCC4B3.18 locus. We then isolated the ppc1+ gene from the genomic DNA of the ts-537 mutant and its DNA sequence was determined. Only a single nucleotide change was found in the mutant ppc1 gene that corresponded to the amino acid substitution T48I in the amino-terminal region. This site is highly conserved among human, fly and budding yeast (figure 3a(i)). We hence concluded that ts-537 is a mutant of the PPCS/ppc1+/SPCC4B3.18 gene and designated it ppc1-537.Figure 3.

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