Limits...
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.

Show MeSH
The proline 363-to-serine (P363S) mutation in the ER-resident CDP-DG synthase Cds1 is responsible for bbl1 phenotype. (A) Cds1 is an evolutionarily conserved protein. The P363S mutation identified in S. japonicus bbl1 cells is highlighted in red. (B) The bbl1 phenotype is rescued by reintroduction of the WT cds1 copy to the native chromosomal locus, whereas replacement of the WT version with the reconstructed PP363S mutated version induces mutant phenotype de novo. (C) Second-copy introduction of the WT cds1 gene in bbl1 mutant cells rescues the bbl1 phenotype, whereas a second copy of cds1bbl1 mutant in WT cells does not induce it. (D) Both WT and mutant Cds1 proteins tagged with GFP localize to the ER. (B–D) Cells grown in rich yeast extract–based medium. (E) A time course of a typical enzyme activity assay shows that catalytic activity of mutant Cds1bbl1 (red) is lower than with the WT protein (blue). Error bars, SDs (n = 3). (B–D) Scale bar, 5 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263451&req=5

Figure 3: The proline 363-to-serine (P363S) mutation in the ER-resident CDP-DG synthase Cds1 is responsible for bbl1 phenotype. (A) Cds1 is an evolutionarily conserved protein. The P363S mutation identified in S. japonicus bbl1 cells is highlighted in red. (B) The bbl1 phenotype is rescued by reintroduction of the WT cds1 copy to the native chromosomal locus, whereas replacement of the WT version with the reconstructed PP363S mutated version induces mutant phenotype de novo. (C) Second-copy introduction of the WT cds1 gene in bbl1 mutant cells rescues the bbl1 phenotype, whereas a second copy of cds1bbl1 mutant in WT cells does not induce it. (D) Both WT and mutant Cds1 proteins tagged with GFP localize to the ER. (B–D) Cells grown in rich yeast extract–based medium. (E) A time course of a typical enzyme activity assay shows that catalytic activity of mutant Cds1bbl1 (red) is lower than with the WT protein (blue). Error bars, SDs (n = 3). (B–D) Scale bar, 5 μm.

Mentions: We sought to identify the mutation underlying the bbl1 phenotype using a whole-genome resequencing approach (see Materials and Methods for experimental details). Two adjacent CC nucleotides in the SJAG_00426.4 open reading frame (ORF) were found mutated to TT, which resulted in substitution of the evolutionarily conserved proline 363 residue to serine (P363S; Figure 3A and Supplemental Figure S3A). SJAG_00426.4 encodes a lipid biosynthesis enzyme from the CDP-DG synthase family highly conserved across all domains of life (Figure 3A and Supplemental Figure S3B). The budding yeast S. cerevisiae orthologue of SJAG_00426.4 is known as Cds1 (Shen et al., 1996).


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)

The proline 363-to-serine (P363S) mutation in the ER-resident CDP-DG synthase Cds1 is responsible for bbl1 phenotype. (A) Cds1 is an evolutionarily conserved protein. The P363S mutation identified in S. japonicus bbl1 cells is highlighted in red. (B) The bbl1 phenotype is rescued by reintroduction of the WT cds1 copy to the native chromosomal locus, whereas replacement of the WT version with the reconstructed PP363S mutated version induces mutant phenotype de novo. (C) Second-copy introduction of the WT cds1 gene in bbl1 mutant cells rescues the bbl1 phenotype, whereas a second copy of cds1bbl1 mutant in WT cells does not induce it. (D) Both WT and mutant Cds1 proteins tagged with GFP localize to the ER. (B–D) Cells grown in rich yeast extract–based medium. (E) A time course of a typical enzyme activity assay shows that catalytic activity of mutant Cds1bbl1 (red) is lower than with the WT protein (blue). Error bars, SDs (n = 3). (B–D) Scale bar, 5 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263451&req=5

Figure 3: The proline 363-to-serine (P363S) mutation in the ER-resident CDP-DG synthase Cds1 is responsible for bbl1 phenotype. (A) Cds1 is an evolutionarily conserved protein. The P363S mutation identified in S. japonicus bbl1 cells is highlighted in red. (B) The bbl1 phenotype is rescued by reintroduction of the WT cds1 copy to the native chromosomal locus, whereas replacement of the WT version with the reconstructed PP363S mutated version induces mutant phenotype de novo. (C) Second-copy introduction of the WT cds1 gene in bbl1 mutant cells rescues the bbl1 phenotype, whereas a second copy of cds1bbl1 mutant in WT cells does not induce it. (D) Both WT and mutant Cds1 proteins tagged with GFP localize to the ER. (B–D) Cells grown in rich yeast extract–based medium. (E) A time course of a typical enzyme activity assay shows that catalytic activity of mutant Cds1bbl1 (red) is lower than with the WT protein (blue). Error bars, SDs (n = 3). (B–D) Scale bar, 5 μm.
Mentions: We sought to identify the mutation underlying the bbl1 phenotype using a whole-genome resequencing approach (see Materials and Methods for experimental details). Two adjacent CC nucleotides in the SJAG_00426.4 open reading frame (ORF) were found mutated to TT, which resulted in substitution of the evolutionarily conserved proline 363 residue to serine (P363S; Figure 3A and Supplemental Figure S3A). SJAG_00426.4 encodes a lipid biosynthesis enzyme from the CDP-DG synthase family highly conserved across all domains of life (Figure 3A and Supplemental Figure S3B). The budding yeast S. cerevisiae orthologue of SJAG_00426.4 is known as Cds1 (Shen et al., 1996).

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