Regulation of triacylglycerol lipases in yeast

In the presence of excessive nutrients eukaryotic cells including yeast form triacylglycerols (TAGs) as depot lipids. These important storage molecules are deposited in special organelles called lipid droplets (LDs) or lipid particles. However, when nutrients become scarce, TAGs are degraded to supply the cell with energy, but also with building blocks for the formation of membrane lipids. The enzymes which catalyze the degradation of TAGs are TAG lipases. These enzymes are localized to the surface of LDs and thus close to their substrate. Thus, the question arises how are TAG lipases regulated to mobilize TAGs only upon requirement, but not during the phase of TAG accumulation? In the proposed project we will address this fundamental question by using the budding yeast Saccharomyces cerevisiae as our experimental system. In yeast, TAGs are degraded by the major TAG lipases Tgl3p, Tgl4p and Tgl5p. In high-throughput studies, these three TAG lipases were identified as phospho-proteins, which are modified by phosphorylation at several sites. Protein phosphorylation/de-phosphorylation is a potent means to modulate the characteristics of a protein. We propose that TAG lipases are regulated via phosphorylation to adjust TAG degradation to the cellular requirements. In our studies we will focus on three particular aspects; (i) which phosphorylation sites of the TAG lipases are involved in regulating their lipolytic activity; (ii) which protein kinases target the respective phosphorylation sites of the lipases; and (iii) do the TAG species and/or the TAG amount affect the regulation of the TAG lipases. These questions will be investigated by using different methods in the fields of molecular biology, cell biology and biochemistry. All required methods are either well established in our laboratory or covered by collaborations with experts in the respective fields. It is expected that our results will provide novel insights into the fundamental process of TAG lipolysis, and - as lipid turnover is evolutionary highly conserved from yeast to humans, - will similarly contribute to a better understanding of this complex process taking place in higher eukaryotes.

Within a cell, triacylglycerols (TAGs) are deposited in special organelles called lipid droplets (LDs). The structure of these organelles is rather simple. TAGs and other hydrophobic molecules form the core of the droplet, which is encompassed by a phospholipid monolayer with a few proteins embedded. Among the proteins forming the LD proteome are also TAG lipases. These enzymes catalyze the degradation of TAG yielding diacylglycerol and an unesterified fatty acid. Both degradation products serve not only as a source of energy, but also as building blocks for membrane lipid formation. To maintain balanced metabolism, it is of utmost importance to adapt the enzyme activity of TAG lipases to cellular requirements. In this project we investigated the fundamental mechanisms regulating the enzyme activity of LD localized TAG lipases. For our studies we used the budding yeast Saccharomyces cerevisiae as model organism. The focus was laid upon the major TAG lipases Tgl3 and Tgl4, which are both located to LDs [1-3]. Tgl4, which is a homolog of mammalian adipose triglyceride lipase ATGL, is post-translationally modified by phosphorylation at numerous amino acids located to the C-terminus of the TAG lipase. By generating a set of mutants expressing Tgl4-variants lacking part of the C-terminus of varying lengths, we could show that the C-terminus of Tgl4 is neither involved in mediating the association with LDs nor in regulating the protein stability. However, this part of the TAG lipase is important to regulate the lipolytic activity of Tgl4. Results of in dept analyses strongly indicate that phosphorylation/de-phosphorylation at an amino acid located to the middle of the cytosolic C-terminus plays a prominent role in regulating the lipolytic activity of this TAG lipase. Similar to Tgl4, the major yeast TAG lipase Tgl3 is post-translationally modified by phosphorylation. Our studies concerning the meaning of phosphorylation in regulating the lipase function of Tgl3 similarly unveiled a formerly uncharacterized phosphorylation site, which is involved in regulating the contribution of this TAG lipase to cell metabolism. However, phosphorylation at this site regulates the contribution of Tgl3 to TAG lipolysis indirectly, as the post-translational modification strongly compromises the protein stability of the lipase. Finally, our studies identified a link between TAG synthesis, LD biogenesis, and the major TAG lipase Tgl3. It is planned to continue these promising studies in the near future. References: 1. Athenstaedt, K., and Daum, G. (2003) J. Biol. Chem. 278(26):23317-23323. DOI: 10.1074/jbc.M302577200 2. Athenstaedt, K., and Daum, G. (2005) J. Biol. Chem. 280:37301-37309. DOI: 10.1074/jbc.M507261200 3. Kurat, C. F., Wolinski, H., Petschnigg, J., Kaluarachchi, S., Andrews, B., Natter, K., and Kohlwein, S. D. (2009) Mol. Cell 33:53-63. DOI: 10.1016/j.molcel.2008.12.019