Publications (2)8.01 Total impact
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ABSTRACT: Lipid droplets (LDs) are intracellular structures that regulate neutral lipid homeostasis. In mammals, LD synthesis is inhibited by rapamycin, a known inhibitor of the mTORC1 pathway. In S. cerevisiae, LD dynamics is modulated by the growth phase, however the regulatory pathways involved are unknown. Therefore, we decided to study the role of the TORC1 pathway on LD metabolism in S. cerevisiae. Interestingly, rapamycin treatment resulted in a fast LDs replenishment and growth inhibition. The discovery that osmotic stress (1M sorbitol) also induced LD synthesis but not growth inhibition, suggested that the induction of LDs in yeast is not a secondary response to reduced growth. Induction of LDs by rapamycin was due to increased triacylglycerol but not sterol esters synthesis. Induction was dependent on the TOR downstream effectors, the PP2A-related phosphatase Sit4p and the regulatory protein Tap42p. The TORC1-controlled transcriptional activators GLN3, GAT1, RTG1 and RTG3 but not MSN2 and MSN4 were required for full induction of LDs by rapamycin. Furthermore, we show that deletion of GLN3 and GAT1 transcription factors, which are activated in response to nitrogen availability, led to abnormal LD dynamics. These results reveal that the TORC1 pathway is involved in neutral lipid homeostasis in yeast. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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ABSTRACT: In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.
Federal University of Rio de Janeiro
Rio de Janeiro, Rio de Janeiro, Brazil
- Programa Biologia Molecular e Biotecnologia