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Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function.

He Y, Yam C, Pomraning K, Chin JS, Yew JY, Freitag M, Oliferenko S - Mol. Biol. Cell (2014)

Bottom Line: Understanding what determines the cellular amount of neutral lipids and their packaging into lipid droplets is of fundamental and applied interest.Using two species of fission yeast, we show that cycling cells deficient in the function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol content and assemble large lipid droplets closely associated with the ER membranes.Our results suggest that interfering with the CDP-DG route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, 117604 Singapore Department of Biological Sciences, National University of Singapore, 117543 Singapore.

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A loss-of-function mutation in S. pombe cds1 leads to bbl1-like phenotype. (A) S. pombe cds1-9 mutant cells grown in rich yeast extract–based medium exhibit large BODIPY 493/503–stained lipid droplets (green) at the restrictive temperature of 36°C. Scale bar, 5 μm. (B) Typical time courses of CDP-DG synthesis reactions in crude extracts obtained from wild WT (blue) and cds1-9 mutant cells. (C) Measurements of KmPA and Vmax show that catalytic activity of the mutant enzyme (red) is significantly than that of WT (blue), although the proteins exhibit comparable PA binding. Error bars, SDs (n = 3). (D) Patterns of doubling time of WT (blue) and cds1-9 (red) mutant cells in chemically defined minimal medium with indicated supplements. Cho, choline; Etn, ethanolamine. Error bars, SDs (n = 3). (E) TLC shows that supplementation with choline largely rescues TG accumulation in cds1-9 mutant cells grown in the minimal medium.
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Figure 5: A loss-of-function mutation in S. pombe cds1 leads to bbl1-like phenotype. (A) S. pombe cds1-9 mutant cells grown in rich yeast extract–based medium exhibit large BODIPY 493/503–stained lipid droplets (green) at the restrictive temperature of 36°C. Scale bar, 5 μm. (B) Typical time courses of CDP-DG synthesis reactions in crude extracts obtained from wild WT (blue) and cds1-9 mutant cells. (C) Measurements of KmPA and Vmax show that catalytic activity of the mutant enzyme (red) is significantly than that of WT (blue), although the proteins exhibit comparable PA binding. Error bars, SDs (n = 3). (D) Patterns of doubling time of WT (blue) and cds1-9 (red) mutant cells in chemically defined minimal medium with indicated supplements. Cho, choline; Etn, ethanolamine. Error bars, SDs (n = 3). (E) TLC shows that supplementation with choline largely rescues TG accumulation in cds1-9 mutant cells grown in the minimal medium.

Mentions: The cds1-9 allele was chosen for further characterization because it had a single amino acid substitution, changing cysteine at position 287 to arginine (C287R), and because it exhibited a tight conditional phenotype with respect to abnormal LD formation (Supplemental Figure S5 and Figure 5A). The CDP-DG synthase activity was markedly decreased in cds1-9 cells at 36°C (Figure 5B). The mutant protein appeared to bind PA nearly as well as the wild-type enzyme but exhibited a strongly decreased rate of catalysis (Figure 5C).


Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function.

He Y, Yam C, Pomraning K, Chin JS, Yew JY, Freitag M, Oliferenko S - Mol. Biol. Cell (2014)

A loss-of-function mutation in S. pombe cds1 leads to bbl1-like phenotype. (A) S. pombe cds1-9 mutant cells grown in rich yeast extract–based medium exhibit large BODIPY 493/503–stained lipid droplets (green) at the restrictive temperature of 36°C. Scale bar, 5 μm. (B) Typical time courses of CDP-DG synthesis reactions in crude extracts obtained from wild WT (blue) and cds1-9 mutant cells. (C) Measurements of KmPA and Vmax show that catalytic activity of the mutant enzyme (red) is significantly than that of WT (blue), although the proteins exhibit comparable PA binding. Error bars, SDs (n = 3). (D) Patterns of doubling time of WT (blue) and cds1-9 (red) mutant cells in chemically defined minimal medium with indicated supplements. Cho, choline; Etn, ethanolamine. Error bars, SDs (n = 3). (E) TLC shows that supplementation with choline largely rescues TG accumulation in cds1-9 mutant cells grown in the minimal medium.
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Related In: Results  -  Collection

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Figure 5: A loss-of-function mutation in S. pombe cds1 leads to bbl1-like phenotype. (A) S. pombe cds1-9 mutant cells grown in rich yeast extract–based medium exhibit large BODIPY 493/503–stained lipid droplets (green) at the restrictive temperature of 36°C. Scale bar, 5 μm. (B) Typical time courses of CDP-DG synthesis reactions in crude extracts obtained from wild WT (blue) and cds1-9 mutant cells. (C) Measurements of KmPA and Vmax show that catalytic activity of the mutant enzyme (red) is significantly than that of WT (blue), although the proteins exhibit comparable PA binding. Error bars, SDs (n = 3). (D) Patterns of doubling time of WT (blue) and cds1-9 (red) mutant cells in chemically defined minimal medium with indicated supplements. Cho, choline; Etn, ethanolamine. Error bars, SDs (n = 3). (E) TLC shows that supplementation with choline largely rescues TG accumulation in cds1-9 mutant cells grown in the minimal medium.
Mentions: The cds1-9 allele was chosen for further characterization because it had a single amino acid substitution, changing cysteine at position 287 to arginine (C287R), and because it exhibited a tight conditional phenotype with respect to abnormal LD formation (Supplemental Figure S5 and Figure 5A). The CDP-DG synthase activity was markedly decreased in cds1-9 cells at 36°C (Figure 5B). The mutant protein appeared to bind PA nearly as well as the wild-type enzyme but exhibited a strongly decreased rate of catalysis (Figure 5C).

Bottom Line: Understanding what determines the cellular amount of neutral lipids and their packaging into lipid droplets is of fundamental and applied interest.Using two species of fission yeast, we show that cycling cells deficient in the function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol content and assemble large lipid droplets closely associated with the ER membranes.Our results suggest that interfering with the CDP-DG route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, 117604 Singapore Department of Biological Sciences, National University of Singapore, 117543 Singapore.

Show MeSH