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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.


Western blot analysis of subcellular fractions from S. cerevisiae.Wild type (A) and mutant strains cki1Δdpl1Δeki1Δ (B) and cho2Δopi3Δ (C) were grown on oleic acid as described in the Materials and Methods section. H (homogenate), ER (endoplasmic reticulum), M (mitochondria) and PX (peroxisomes) were isolated according to standard procedures [35]. For electrophoresis 10 μg protein were loaded onto each lane of the gel.
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pone.0135084.g004: Western blot analysis of subcellular fractions from S. cerevisiae.Wild type (A) and mutant strains cki1Δdpl1Δeki1Δ (B) and cho2Δopi3Δ (C) were grown on oleic acid as described in the Materials and Methods section. H (homogenate), ER (endoplasmic reticulum), M (mitochondria) and PX (peroxisomes) were isolated according to standard procedures [35]. For electrophoresis 10 μg protein were loaded onto each lane of the gel.

Mentions: Prerequisite for the analysis of effects on the formation of peroxisomes caused by mutations in the two PC biosynthetic pathways was the isolation of these organelles as described previously by Zinser and Daum [35]. The quality of peroxisome preparations was tested by Western blot analysis to determine the enrichment of peroxisomes and cross contamination with other organelles (Fig 4). Antibodies used for Western blot analysis were directed against a 40 kDa protein (endoplasmic reticulum); Fox1p (multifunctional β-oxidation protein from peroxisomal membranes) and Por1p (outer mitochondrial membrane). As can be seen from Fig 4, Fox1p was highly enriched in peroxisomal fractions from wild type cells. The degree of cross-contamination of peroxisomes with other subcellular fractions was low. Impurities caused by co-isolation of mitochondria were small as can be seen from the patterns of Por1p. Western blot analysis was also routinely performed with subcellular fractions from the mutant strains bearing defects in PC synthesis. Cross contaminations were quantified in Fig 5. Contaminations with mitochondria were found in all fractions most likely due to close contact of mitochondria with other organelles. Endoplasmic reticulum and mitochondria were not contaminated by peroxisomes, and no contamination with endoplasmic reticulum (ER) was found with the other isolated organelles.


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)

Western blot analysis of subcellular fractions from S. cerevisiae.Wild type (A) and mutant strains cki1Δdpl1Δeki1Δ (B) and cho2Δopi3Δ (C) were grown on oleic acid as described in the Materials and Methods section. H (homogenate), ER (endoplasmic reticulum), M (mitochondria) and PX (peroxisomes) were isolated according to standard procedures [35]. For electrophoresis 10 μg protein were loaded onto each lane of the gel.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4524607&req=5

pone.0135084.g004: Western blot analysis of subcellular fractions from S. cerevisiae.Wild type (A) and mutant strains cki1Δdpl1Δeki1Δ (B) and cho2Δopi3Δ (C) were grown on oleic acid as described in the Materials and Methods section. H (homogenate), ER (endoplasmic reticulum), M (mitochondria) and PX (peroxisomes) were isolated according to standard procedures [35]. For electrophoresis 10 μg protein were loaded onto each lane of the gel.
Mentions: Prerequisite for the analysis of effects on the formation of peroxisomes caused by mutations in the two PC biosynthetic pathways was the isolation of these organelles as described previously by Zinser and Daum [35]. The quality of peroxisome preparations was tested by Western blot analysis to determine the enrichment of peroxisomes and cross contamination with other organelles (Fig 4). Antibodies used for Western blot analysis were directed against a 40 kDa protein (endoplasmic reticulum); Fox1p (multifunctional β-oxidation protein from peroxisomal membranes) and Por1p (outer mitochondrial membrane). As can be seen from Fig 4, Fox1p was highly enriched in peroxisomal fractions from wild type cells. The degree of cross-contamination of peroxisomes with other subcellular fractions was low. Impurities caused by co-isolation of mitochondria were small as can be seen from the patterns of Por1p. Western blot analysis was also routinely performed with subcellular fractions from the mutant strains bearing defects in PC synthesis. Cross contaminations were quantified in Fig 5. Contaminations with mitochondria were found in all fractions most likely due to close contact of mitochondria with other organelles. Endoplasmic reticulum and mitochondria were not contaminated by peroxisomes, and no contamination with endoplasmic reticulum (ER) was found with the other isolated organelles.

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.