Klaus Natter

Obese Yeast: Triglyceride Lipolysis Is Functionally Conserved from Mammals to Yeast

Storage and degradation of triglycerides are essential processes to ensure energy homeostasis and availability of precursors for membrane lipid synthesis. Recent evidence suggests that an emerging class of enzymes containing a conserved patatin domain are centrally important players in lipid degradation. Here we describe the identification and characterization of a major triglyceride lipase of the adipose triglyceride lipase/Brummer family, Tgl4, in the yeast Saccharomyces cerevisiae. Elimination of Tgl4 in a tgl3 background led to fat yeast, rendering growing cells unable to degrade triglycerides. Tgl4 and Tgl3 lipases localized to lipid droplets, independent of each other. Serine 315 in the GXSXG lipase active site consensus sequence of the patatin domain of Tgl4 is essential for catalytic activity. Mouse adipose triglyceride lipase (which also contains a patatin domain but is otherwise highly divergent in primary structure from any yeast protein) localized to lipid droplets when expressed in yeast, and significantly restored triglyceride breakdown in tgl4 mutants in vivo. Our data identify yeast Tgl4 as a functional ortholog of mammalian adipose triglyceride lipase.

Cdk1/Cdc28-dependent activation of the major triacylglycerol lipase Tgl4 in yeast links lipolysis to cell-cycle progression.

Triacylglycerols (TGs) serve essential cellular functions as reservoirs for energy substrates (fatty acids) and membrane lipid precursors (diacylglycerols and fatty acids). Here we show that the major yeast TG lipase Tgl4, the functional ortholog of murine adipose TG lipase ATGL, is phosphorylated and activated by cyclin-dependent kinase 1 (Cdk1/Cdc28). Phospho-Tgl4-catalyzed lipolysis contributes to early bud formation in late G1 phase of the cell cycle. Conversely, lack of lipolysis delays bud formation and cell-cycle progression. In the absence of beta-oxidation, lipolysis-derived metabolites are thus required to support cellular growth. TG homeostasis is the only metabolic process identified as yet that is directly regulated by Cdk1/Cdc28-dependent phosphorylation of key anabolic and catabolic enzymes, highlighting the importance of FA storage and mobilization during the cell cycle. Our data provide evidence for a direct link between cell-cycle-regulatory kinases and TG degradation and suggest a general mechanism for coordinating membrane synthesis with cell-cycle progression.

The Spatial Organization of Lipid Synthesis in the Yeast Saccharomyces cerevisiae Derived from Large Scale Green Fluorescent Protein Tagging and High Resolution Microscopy

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins (∼9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.

Good fat, essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in yeast.

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.

A novel strategy for constructing N-terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae.

A novel rapid polymerase chain reaction (PCR)‐based technique for N‐terminal attachment of green fluorescent protein (GFP) to a yeast protein is described. Genomic integration of a PCR‐generated loxPkanMX4loxP‐yEGFP fusion cassette immediately upstream of the open reading frame (ORF) allows for selection of G418 resistant transformants carrying GFP fused N‐terminally to the protein of interest. In a subsequent step, the loxPkanMX4loxP selection marker that is inserted between the tagged ORF and the endogenous promoter is excised upon site‐specific recombination between the loxP sites by Cre recombinase, leaving behind in the promoter one loxP site, immediately upstream of the GFP start codon. The essential protein Ydl193wp of unknown function and the oleate‐inducible fatty acid activation protein, encoded by FAA2, were N‐terminally tagged using the novel technique. Both experiments yielded viable haploid strains with growth phenotypes indistinguishable from the wild type strain. The subcellular localization pattern for the chromosomally expressed GFP‐Ydl193wp to the endoplasmic reticulum and lipid particles was identical to the pattern observed for a plasmid‐borne GFP construct expressed under control of the MET25p promoter, albeit at a lower level and with a more homogeneous distribution among the cell population. GFP‐FAA2 was inducible by oleate, as is the wild type gene, demonstrating that specific expression patterns are not grossly affected by the promoter manipulation. In agreement with previous reports, GFP‐Faa2p was found to localize to peroxisomes.

YPL.db: the Yeast Protein Localization database

The Yeast Protein Localization database (YPL.db) contains information about the localization patterns of yeast proteins resulting from microscopic analyses. The data and parameters of the experiments to obtain the localization information, together with images from confocal or video microscopy, are stored in a relational database, building an archive of, and the documentation for, all experiments. The database can be queried based on gene name, protein localization, growth conditions and a number of additional parameters. All experiment parameters are selectable from predefined lists to ensure database integrity and conformity across different investigators. The database provides a structure reference resource to allow for better characterization of unknown or ambiguous localization patterns. Links to MIPS, YPD and SGD databases are provided to allow fast access to further information not contained in the localization database itself. YPL.db is available at http://ypl.tugraz.at.

Lipid Droplet-associated Proteins Are Involved in the Biosynthesis and Hydrolysis of Triacylglycerol in Mycobacterium bovis Bacillus Calmette-Guérin

Mycobacteria store triacylglycerols (TGs) in the form of intracellular lipid droplets (LDs) during hypoxia-induced nonreplicating persistence. These bacteria are phenotypically drug-resistant and therefore are believed to be the cause for prolonged tuberculosis treatment. LDs are also associated with bacilli in tuberculosis patient sputum and hypervirulent strains. Although proteins bound to LDs are well characterized in eukaryotes, the identities and functions of such proteins have not been described in mycobacteria. Here, we have identified five proteins: Tgs1 (BCG3153c), Tgs2 (BCG3794c), BCG1169c, BCG1489c, and BCG1721, which are exclusively associated with LDs purified from hypoxic nonreplicating Mycobacterium bovis bacillus Calmette-Guérin (BCG). Disruption of genes tgs1, tgs2, BCG1169c, and BCG1489c in M. bovis BCG revealed that they are indeed involved in TG metabolism. We also characterized BCG1721, an essential bi-functional enzyme capable of promoting buildup and hydrolysis of TGs, depending on the metabolic state. Nonreplicating mycobacteria overexpressing a BCG1721 construct with an inactive lipase domain displayed a phenotype of attenuated TG breakdown and regrowth upon resuscitation. In addition, by heterologous expression in bakers yeast, these mycobacterial proteins also co-localized with LDs and complemented a lipase-deficient yeast strain, indicating that neutral lipid deposition and homeostasis in eukaryotic and prokaryotic microorganisms are functionally related. The demonstrated functional role of BCG1721 to support growth upon resuscitation makes this novel LD-associated factor a potential new target for therapeutic intervention.

Yeast and cancer cells - common principles in lipid metabolism.

One of the paradigms in cancer pathogenesis is the requirement of a cell to undergo transformation from respiration to aerobic glycolysis – the Warburg effect – to become malignant. The demands of a rapidly proliferating cell for carbon metabolites for the synthesis of biomass, energy and redox equivalents, are fundamentally different from the requirements of a differentiated, quiescent cell, but it remains open whether this metabolic switch is a cause or a consequence of malignant transformation. One of the major requirements is the synthesis of lipids for membrane formation to allow for cell proliferation, cell cycle progression and cytokinesis. Enzymes involved in lipid metabolism were indeed found to play a major role in cancer cell proliferation, and most of these enzymes are conserved in the yeast, Saccharomyces cerevisiae. Most notably, cancer cell physiology and metabolic fluxes are very similar to those in the fermenting and rapidly proliferating yeast. Both types of cells display highly active pathways for the synthesis of fatty acids and their incorporation into complex lipids, and imbalances in synthesis or turnover of lipids affect growth and viability of both yeast and cancer cells. Thus, understanding lipid metabolism in S. cerevisiae during cell cycle progression and cell proliferation may complement recent efforts to understand the importance and fundamental regulatory mechanisms of these pathways in cancer.

Identification of Yju3p as functional orthologue of mammalian monoglyceride lipase in the yeast Saccharomycescerevisiae.

Monoacylglycerols (MAGs) are short-lived intermediates of glycerolipid metabolism. Specific molecular species, such as 2-arachidonoylglycerol, which is a potent activator of cannabinoid receptors, may also function as lipid signaling molecules. In mammals, enzymes hydrolyzing MAG to glycerol and fatty acids, resembling the final step in lipolysis, or esterifying MAG to diacylglycerol, are well known; however, despite the high level of conservation of lipolysis, the corresponding activities in yeast have not been characterized yet. Here we provide evidence that the protein Yju3p functions as a potent MAG hydrolase in yeast. Cellular MAG hydrolase activity was decreased by more than 90% in extracts of Yju3p-deficient cells, indicating that Yju3p accounts for the vast majority of this activity in yeast. Loss of this activity was restored by heterologous expression of murine monoglyceride lipase (MGL). Since yju3Δ mutants accumulated MAG in vivo only at very low concentrations, we considered the possibility that MAGs are re-esterified into DAG by acyltransferases. Indeed, cellular MAG levels were further increased in mutant cells lacking Yju3p and Dga1p or Lro1p acyltransferase activities. In conclusion, our studies suggest that catabolic and anabolic reactions affect cellular MAG levels. Yju3p is the functional orthologue of mammalian MGL and is required for efficient degradation of MAG in yeast.

Quantitative modeling of triacylglycerol homeostasis in yeast--metabolic requirement for lipolysis to promote membrane lipid synthesis and cellular growth.

Triacylglycerol metabolism in Saccharomyces cerevisiae was analyzed quantitatively using a systems biological approach. Cellular growth, glucose uptake and ethanol secretion were measured as a function of time and used as input for a dynamic flux‐balance model. By combining dynamic mass balances for key metabolites with a detailed steady‐state analysis, we trained a model network and simulated the time‐dependent degradation of cellular triacylglycerol and its interaction with fatty acid and membrane lipid synthesis. This approach described precisely, both qualitatively and quantitatively, the time evolution of various key metabolites in a consistent and self‐contained manner, and the predictions were found to be in excellent agreement with experimental data. We showed that, during pre‐logarithmic growth, lipolysis of triacylglycerol allows for the rapid synthesis of membrane lipids, whereas de novo fatty acid synthesis plays only a minor role during this growth phase. Progress in triacylglycerol hydrolysis directly correlates with an increase in cell size, demonstrating the importance of lipolysis for supporting efficient growth initiation.