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Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae.

Flis VV, Fankl A, Ramprecht C, Zellnig G, Leitner E, Hermetter A, Daum G - PLoS ONE (2015)

Bottom Line: Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid.Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway.In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Graz University of Technology, NAWI Graz, Graz, Austria.

ABSTRACT
In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.

No MeSH data available.


Growth phenotype of wild type, cho2Δ, opi3Δ, cho2Δopi3Δ and cki1Δdpl1Δeki1Δ yeast strains.(A): Drop test on YPD plates (30°C and 37°C); on minimal oleate media containing choline and ethanolamine; and on YPO plates are shown. (B) Growth of liquid cultures on YPO.
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pone.0135084.g002: Growth phenotype of wild type, cho2Δ, opi3Δ, cho2Δopi3Δ and cki1Δdpl1Δeki1Δ yeast strains.(A): Drop test on YPD plates (30°C and 37°C); on minimal oleate media containing choline and ethanolamine; and on YPO plates are shown. (B) Growth of liquid cultures on YPO.

Mentions: To analyze the influence of mutations in PC synthesis on growth of yeast cells on different carbon sources drop tests on YPD and YPO agar plates (Fig 2A) were performed and cells were cultivated in liquid media (Fig 2B). These tests showed that strains bearing defects in the CDP-choline pathway of PC synthesis grew normally on YPD at 30°C. The cho2Δopi3Δ mutant showed only slight growth defects on YPD at 30°C. At a temperature of 37°C on YPD, however, cho2Δ, cho2Δopi3Δ and the cki1Δdpl1Δeki1Δ mutants exhibited a growth defect. As shown in Fig 2A growth of the cho2Δopi3Δ mutant was more affected than growth of cki1Δdpl1Δeki1Δ. On minimal media containing oleate as carbon source and supplemented with 5 mM choline and 5 mM ethanolamine the cho2Δopi3Δ mutant grew markedly worse than wild type (see Fig 2A). Under these conditions only a slight growth defect of cho2Δ was observed, and growth of the cki1Δdpl1Δeki1Δ mutant was practically not affected. Surprisingly, on YPO rich media the growth defect of cho2Δ, cho2Δopi3Δ and the cki1Δdpl1Δeki1Δ mutants was more pronounced than on minimal oleate containing media. This effect may be due to the limiting amount of ethanolamine and choline present in YPO. At 37°C, mutant strains did not grow at all on minimal oleate media supplemented with choline and ethanolamine, although the wild type strain showed normal growth (data not shown).


Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae.

Flis VV, Fankl A, Ramprecht C, Zellnig G, Leitner E, Hermetter A, Daum G - PLoS ONE (2015)

Growth phenotype of wild type, cho2Δ, opi3Δ, cho2Δopi3Δ and cki1Δdpl1Δeki1Δ yeast strains.(A): Drop test on YPD plates (30°C and 37°C); on minimal oleate media containing choline and ethanolamine; and on YPO plates are shown. (B) Growth of liquid cultures on YPO.
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pone.0135084.g002: Growth phenotype of wild type, cho2Δ, opi3Δ, cho2Δopi3Δ and cki1Δdpl1Δeki1Δ yeast strains.(A): Drop test on YPD plates (30°C and 37°C); on minimal oleate media containing choline and ethanolamine; and on YPO plates are shown. (B) Growth of liquid cultures on YPO.
Mentions: To analyze the influence of mutations in PC synthesis on growth of yeast cells on different carbon sources drop tests on YPD and YPO agar plates (Fig 2A) were performed and cells were cultivated in liquid media (Fig 2B). These tests showed that strains bearing defects in the CDP-choline pathway of PC synthesis grew normally on YPD at 30°C. The cho2Δopi3Δ mutant showed only slight growth defects on YPD at 30°C. At a temperature of 37°C on YPD, however, cho2Δ, cho2Δopi3Δ and the cki1Δdpl1Δeki1Δ mutants exhibited a growth defect. As shown in Fig 2A growth of the cho2Δopi3Δ mutant was more affected than growth of cki1Δdpl1Δeki1Δ. On minimal media containing oleate as carbon source and supplemented with 5 mM choline and 5 mM ethanolamine the cho2Δopi3Δ mutant grew markedly worse than wild type (see Fig 2A). Under these conditions only a slight growth defect of cho2Δ was observed, and growth of the cki1Δdpl1Δeki1Δ mutant was practically not affected. Surprisingly, on YPO rich media the growth defect of cho2Δ, cho2Δopi3Δ and the cki1Δdpl1Δeki1Δ mutants was more pronounced than on minimal oleate containing media. This effect may be due to the limiting amount of ethanolamine and choline present in YPO. At 37°C, mutant strains did not grow at all on minimal oleate media supplemented with choline and ethanolamine, although the wild type strain showed normal growth (data not shown).

Bottom Line: Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid.Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway.In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Graz University of Technology, NAWI Graz, Graz, Austria.

ABSTRACT
In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.

No MeSH data available.