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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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Both TG synthases Dga1 and Lro1 function at the large ER-associated lipid droplets in cds1-9 S. pombe cells. (A) Dga1-GFP localizes to the periphery of LDs in cds1-9 cells at the restrictive temperature of 36°C. (B) Lro1-GFP localizes to the ER, including the ER membranes associated with large LDs in cds1-9 cells, at the restrictive temperature of 36°C. Insets, magnified views. (C) TLC shows that deletion of dga1 massively reduces cellular TG content in cds1-9 genetic background at the restrictive temperature of 36°C. cds1-9 lro1Δ cells also show reduced total TG. (D) BODIPY 493/503–stained LDs in cds1-9 cells lacking the TG synthases Lro1 or Dga1 at the restrictive temperature of 36°C. (A–D) Cells grown in the chemically defined minimal medium. Scale bar, 5 μm.
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Figure 7: Both TG synthases Dga1 and Lro1 function at the large ER-associated lipid droplets in cds1-9 S. pombe cells. (A) Dga1-GFP localizes to the periphery of LDs in cds1-9 cells at the restrictive temperature of 36°C. (B) Lro1-GFP localizes to the ER, including the ER membranes associated with large LDs in cds1-9 cells, at the restrictive temperature of 36°C. Insets, magnified views. (C) TLC shows that deletion of dga1 massively reduces cellular TG content in cds1-9 genetic background at the restrictive temperature of 36°C. cds1-9 lro1Δ cells also show reduced total TG. (D) BODIPY 493/503–stained LDs in cds1-9 cells lacking the TG synthases Lro1 or Dga1 at the restrictive temperature of 36°C. (A–D) Cells grown in the chemically defined minimal medium. Scale bar, 5 μm.

Mentions: Fission yeast genomes encode orthologues of the two budding yeast TG synthases, Dga1 and Lro1. Both proteins contributed to TG synthesis in exponentially growing S. pombe (Supplemental Figure S7A; see also Zhang et al., 2003). These enzymes exhibited distinct patterns of subcellular localization. In wild type, the GFP-tagged Dga1, the DG acyltransferase that uses acyl-CoA as an acyl donor (Oelkers et al., 2002), was enriched at LDs (Figure 7A). In cds1-9 cells grown at the restrictive temperature of 36°C, Dga1-GFP also localized predominantly to the periphery of large LDs (Figure 7A). On the other hand, the acyl-CoA–independent diacylglycerol acyltransferase Lro1, which is believed to use PE or PC (Oelkers et al., 2000) as acyl donor, exhibited less-defined intracellular localization, with some enrichment at the peripheral ER (Figure 7B). Of interest, we observed enhanced Lro1-GFP signal at the ER membranes surrounding the abnormal LDs and around the NE in cds1-9 cells (Figure 7B). Thus the core machinery for TG biosynthesis appears to be concentrated in the vicinity of large, ER-associated LDs in cells lacking Cds1 activity.


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)

Both TG synthases Dga1 and Lro1 function at the large ER-associated lipid droplets in cds1-9 S. pombe cells. (A) Dga1-GFP localizes to the periphery of LDs in cds1-9 cells at the restrictive temperature of 36°C. (B) Lro1-GFP localizes to the ER, including the ER membranes associated with large LDs in cds1-9 cells, at the restrictive temperature of 36°C. Insets, magnified views. (C) TLC shows that deletion of dga1 massively reduces cellular TG content in cds1-9 genetic background at the restrictive temperature of 36°C. cds1-9 lro1Δ cells also show reduced total TG. (D) BODIPY 493/503–stained LDs in cds1-9 cells lacking the TG synthases Lro1 or Dga1 at the restrictive temperature of 36°C. (A–D) Cells grown in the chemically defined minimal medium. Scale bar, 5 μm.
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Figure 7: Both TG synthases Dga1 and Lro1 function at the large ER-associated lipid droplets in cds1-9 S. pombe cells. (A) Dga1-GFP localizes to the periphery of LDs in cds1-9 cells at the restrictive temperature of 36°C. (B) Lro1-GFP localizes to the ER, including the ER membranes associated with large LDs in cds1-9 cells, at the restrictive temperature of 36°C. Insets, magnified views. (C) TLC shows that deletion of dga1 massively reduces cellular TG content in cds1-9 genetic background at the restrictive temperature of 36°C. cds1-9 lro1Δ cells also show reduced total TG. (D) BODIPY 493/503–stained LDs in cds1-9 cells lacking the TG synthases Lro1 or Dga1 at the restrictive temperature of 36°C. (A–D) Cells grown in the chemically defined minimal medium. Scale bar, 5 μm.
Mentions: Fission yeast genomes encode orthologues of the two budding yeast TG synthases, Dga1 and Lro1. Both proteins contributed to TG synthesis in exponentially growing S. pombe (Supplemental Figure S7A; see also Zhang et al., 2003). These enzymes exhibited distinct patterns of subcellular localization. In wild type, the GFP-tagged Dga1, the DG acyltransferase that uses acyl-CoA as an acyl donor (Oelkers et al., 2002), was enriched at LDs (Figure 7A). In cds1-9 cells grown at the restrictive temperature of 36°C, Dga1-GFP also localized predominantly to the periphery of large LDs (Figure 7A). On the other hand, the acyl-CoA–independent diacylglycerol acyltransferase Lro1, which is believed to use PE or PC (Oelkers et al., 2000) as acyl donor, exhibited less-defined intracellular localization, with some enrichment at the peripheral ER (Figure 7B). Of interest, we observed enhanced Lro1-GFP signal at the ER membranes surrounding the abnormal LDs and around the NE in cds1-9 cells (Figure 7B). Thus the core machinery for TG biosynthesis appears to be concentrated in the vicinity of large, ER-associated LDs in cells lacking Cds1 activity.

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