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. Triglycerides (TG)2 serve different functions in a cell. First, they represent a most efficient way to store energy in the form of fatty acids (FA). Second, diglycerides (DG) liberated from TG by cleavage of a single fatty acyl ester bond, may serve as precursors for re-esterification to membrane phospholipids. Third, TG synthesis may also function as a sink to remove excess free fatty acids from the cellular milieu, in order to prevent FA-induced lipotoxicity. Because TG precursors or degradation products, such as phosphatidic acid or DG species, are also potential second messengers involved in multiple signaling processes, both TG synthesis and breakdown obviously require a stringent spatial and temporal control (1). Fueled by the epidemic dimensions of lipid-associated disorders, such as obesity and type 2 diabetes (2-4), numerous research strategies are focused toward understanding the genetic basis and molecular mechanisms that regulate uptake, synthesis, deposition, and mobilization of lipids, in the context of energy homeostasis (5-7). Because of the complexity of the problem, major input in this endeavor comes from the use of model systems, including mice, flies (Drosophila), worms (Caenorhabditis elegans) or yeast.In yeast, mobilization of fat depots occurs as a consequence of at least three different metabolic stimuli: in stationary phase, upon nutrient depletion, fatty acids are released from TG depots rather slowly and are subjected to peroxisomal ␤-oxidation, providing the metabolic energy for cellular maintenance (8). Alternatively, lipid depots are degraded very rapidly in cells that exit starvation conditions, e.g. from the stationary phase, and enter a vegetative growth cycle upon supplementation with carbohydrates (9). Because peroxisomes are repressed under these conditions, storage lipid compounds including steryl esters (SE) a...

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.

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.In the aqueous cellular environment, fatty acyl chains esterified in glycerophospholipids constitute the hydrophobic barrier of biological membranes. Thus, fatty acid (FA) 3 composition is a crucial determinant of cellular membrane function. Establishment of the specific FA profiles in lipid species of various organelle membranes (1) relies on an intricate balance between endogenous FA synthesis, recycling of FA from lipid breakdown, and perhaps uptake from the exterior. Glycerophospholipids and triacylglycerols (TAG), which serve as the major storage form of FA, share the similar precursors phosphatidic acid (PA) and diacylglycerol (DAG), both generated in the endoplasmic reticulum (ER) membrane. TAG are packaged into lipid droplets and are thus sequestered away from the ER membrane by a mechanism not yet understood. In addition, membranes and lipid storage pools (2, 3) undergo significant turnover and intracellular flux, e.g. during secretion or endocytosis and cellular growth, which must be accounted for by mechanisms that establish and maintain lipid homeostasis in these dynamic membrane systems (4). We have recently shown that TAG degradation provides metabolites that are critical for efficient cell cycle progression at the G 1

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 (ϳ9% 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. Molecular & Cellular Proteomics 4:662-672, 2005.Biological membranes are characterized by a highly complex mixture of lipids that are key determinants of the biophysical parameters of membranes, such as fluidity, permeability, and signaling functions. Thus, lipids affect structure and function of peripheral and integral membrane proteins, and thus play a pivotal role in organelle (membrane) function. In the yeast Saccharomyces cerevisiae, the lipid composition appears rather simple, consisting mainly of glycerophospholipids, sphingolipids, and ergosterol (1); however, recent advances in analytical techniques such as nanoelectrospray ionization tandem mass spectrometry (2) have unveiled a great degree of heterogeneity within the molecular species of phospholipids. Furthermore subcellular membranes differ not only in their lipid composition in terms of lipid classes, they are also characterized by a distinct distribution of molecular species of phospholipids (3). Because only a few membranes harbor enzymes involved in lipid synthesis, this heterogeneity may be established by localized synthesis, localized degradation, and selective trafficking of membrane lipids. Interestingly apparently "linear" pathways for the synthesis of major membrane lipids involve multiple organelles: the synthesis of phosphatidylserine by a synthase (Cho1p) occurs in the endoplasmic reticulum (ER) 1 and is followed by decarboxylation to phosphatidylethanolamine in the mitochondrial inner membrane (by Psd1p) or Golgi/vacuoles (by Psd2p). Subsequently phosphatidyletha...

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