Neutral lipid storage and mobilization in yeast

All types of cells including the model eukaryote baker`s yeast contain subcellular structures named lipid particles (lipid droplets), which represent a depot for the neutral lipids, triacylglycerols (TAG) and steryl esters (STE). Neutral lipids are highly dynamic components which serve as a source of energy and as a store of building blocks (fatty acids, sterols) required for the formation of biological membranes. During the last few years initial attempts were made to investigate the problem of lipid storage at the molecular level. The yeast Saccharomyces cerevisiae as an experimental system has proven as a valuable tool for these studies which resulted in the identification of most enzymes of neutral lipid (TAG and STE) formation and hydrolysis. Thus, the next step to understand neutral lipid storage and mobilization in more detail has to be the elucidation of mechanisms involved in these processes. The present project application describes strategies (i) to study the physiological role of lipid depots (lipid particles), (ii) to characterize the molecular function of gene products contributing to TAG and STE storage and mobilization in the yeast Saccharomyces cerevisiae, and (iii) to identify novel genes involved in these pathways. Based on previous and ongoing work, we will focus on the biochemistry and cell biology of neutral lipid synthesizing and hydrolyzing enzymes. Using single and multiple deletion mutants, we will study the contribution of individual neutral lipid synthesizing enzymes to the formation of lipid particles. Lipid profiles of total cell extracts or isolated subcellular fractions from single and multiple mutants compromised in this process will allow us to understand the complex network of neutral lipid homeostasis. The interplay of the endoplasmic reticulum and lipid particles, which share the set of neutral lipid synthesizing enzymes, will be of specific interest. Similar to the different contributions of TAG and STE synthases to lipid particle formation, the different TAG lipases and STE hydrolases appear to contribute differently to the mobilization of lipid depots. Also in this case, multiple mutants will be used to study the physiological relevance of individual reactions catalyzed by these proteins. Moreover, we wish to identify helper proteins on the surface of lipid particles, which facilitate access of TAG and STE hydrolytic enzymes to their substrates stored in the core of the droplets. Such novel gene products will be detected through a proteome analysis of highly purified lipid particles, bioinformatic evaluation of the data and functional analysis of the proteins. Finally, we intend to study cell biological consequences of TAG and/or STE shortage or overproduction caused by disturbed synthesis or mobilization. Possible roles of neutral lipids to protect certain proteins from degradation or as a sink for unwanted membrane constituents, e.g., membrane bilayer disturbing fatty acids, will be addressed. All together, these experiments will broaden our fundamental knowledge of the role of neutral lipids in the yeast and also open parallels to higher eukaryotes including man.

Yeast cells have the capacity to store neutral lipids TAG (triacylglycerols) and STE (steryl esters) in subcellular structures named lipid particles/droplets. Upon requirement, TAG and STE can be mobilized and fatty acids set free serve as building blocks for the biosynthesis of membrane phospholipids. The aim of the present project was to investigate enzymatic steps which lead to the mobilization of TAG and STE depots in more detail. For these experiments biochemical, molecular biological and cell biological methods were employed. One of the major findings of this project was that the three yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids from TAG depots to the site of sphingolipid and glycerophospholipid synthesis. Through this mechanism the TAG lipases contribute to synthesis and remodelling of complex membrane lipids. We also found that Tgl3p, Tgl4p and Tgl5p did not only catalyze TAG lipolysis but also act as lysophospholipid acyltransferases. Consequently, these proteins are not only lipolytic enzymes but also involved in the anabolic network of lipid synthesis. During these investigations we also characterized proteome and lipidome of lipid particles, the organelle of non-polar lipid storage, in more detail. Using a mass spectrometric approach we identified novel proteins of this compartment and studied the impact of changing culture conditions on the molecular equipment of lipid particles. Biophysical experiments revealed that lipid particles have a distinct internal structure. Under a phospholipid surface monolayer several shells consisting of STE stabilize the particle. In the core of the lipid particle TAG is randomly arranged. We also showed that under lipotoxic stress caused by growth of yeast cells on oleic acid the storage mechanism for fatty acids in the form of TAG and STE can adapt. Specific regulatory mechanisms affecting TAG and STE synthesis which led to morphological changes of intracellular membranes and altered lipid composition became evident. Finally, we demonstrated that under certain condition squalene, a precursor of sterol synthesis can accumulate as a storage lipid. Surprisingly, this lipid is not only stored in lipid particles but can also be incorporated in cellular membranes at substantial amounts. In summary, this project shed light on the processes of lipid storage and mobilization and provided molecular evidence for the role of enzymes involved.