Membrane maintenance: Regulation of lipid synthesis in S. cerevisiae

Research Areas:

Gene regulation, Lipid metabolism, S. cerevisiae

Imaging Needs:

Visualizing organelle membrane structure

Imaging System:
  • Zeiss Axioplan epifluorescence microscope
  • Zeiss 100× plan-apochromatic 1.4NA objective lens
  • Hamamatsu ORCA-R2 CCD camera
  • Hamamatsu Simple PCI6 software
Measuring protein interactions in cells, whole organisms

Imaging intracellular membranes in living cells takes high resolution. What if you want to pan out and measure interactions within an entire living organism? Find out how Takeaki Ozawa and colleagues screen for GPCR ligands in mice in Illuminating Activity—in vitro and in vivo. Read now.

THE QUESTION

What factors regulate lipid metabolism and membrane dynamics in Saccharomyces cerevisiae?

This ER-associated, CTP-dependent enzyme diacylglycerol (DAG) kinase plays an indirect but essential role in Saccharomyces cerevisiae lipid metabolism. By catalyzing the phosphorylation of DAG to form phosphatidate, it helps to regulate the availability of both enzymes for phospholipid synthesis, membrane growth, and lipid droplet formation.

In mammals, DAG kinases influence numerous cellular processes important to diseases such as cancer, type II diabetes, autoimmunity, and nervous system disorders. Understanding its transcriptional regulation could lead to progress in treating multiple diseases.

THE BARRIERS

S. cerevisiae has a single DAG kinase, encoded by the gene DGK1. Its activity in the cell—phosphorylizing DAG to produce phosphatidate—is countered by the PAH1-encoded enzyme phosphatidate phosphatase, which reverses that reaction. Together, DAG1 and PAH1 regulate levels of lipid synthesis, and deletion of PAH1 has been shown to result in aberrant growth of the nuclear and ER membranes.

THE SOLUTION

Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae
Yixuan Qiu, Stylianos Fakas, Gil-Soo Han, Antonio Daniel Barbosa, Symeon Siniossoglou, and George M. Carman
J Biol Chem. 2013 October. 288(40): 29124–29133. PMCID: PMC3790011.

Using genetic techniques, Qiu, et al,1 showed that DGK1 gene levels are regulated by the transcription factor Reb1p (RNA polymerase I enhancer-binding protein). They then used a Hamamatsu ORCA-R2 CCD camera to image the nuclear and ER membranes in S. cerevisiae expressing SEC63-GFP. Cells lacking both DGK1 and PAH1 did not show the aberrant ER and nuclear membrane growth typical of PAH1-knockout cells, implicating DGK1 and its product DAG kinase in the aberrant membrane growth.

THE POSSIBILITIES

Qiu, et al,1 used fluorescence microscopy and the high-resolution Hamamatsu ORCA-R2 camera to explore lipid metabolism of S. cerevisiae. Similar whole-cell and even whole-organism imaging are possible using luminescence in place of fluorescence. Find out how Takeaki Ozawa and colleagues use Hamamatsu’s ImagEM camera to detect GPCR binding—read Illuminating Activity—in vitro and in vivo.

References

  1. Qiu, et al. Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae. J Biol Chem. 2013 October. 288(40): 29124–29133. PMCID: PMC3790011.
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