The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae

Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.     

The TGL2 Gene of Saccharomyces cerevisiae Encodes an Active Acylglycerol Lipase Located in the Mitochondria

The Saccharomyces cerevisiae Tgl2 protein shows sequence homology to Pseudomonas triacylglycerol (TAG) lipases, but its role in the yeast lipid metabolism is not known. Using hemagglutinin-tagged Tgl2p purified from yeast, we report that this protein carries a significant lipolytic activity toward long-chain TAG. Importantly, mutant hemagglutinin-Tgl2p^S144A, which contains alanine 144 in place of serine 144 in the lipase consensus sequence (G/A)XSXG exhibits no such activity. Although cellular TAG hydrolysis is reduced in the tgl2 deletion mutant, overproduction of Tgl2p in this mutant leads to an increase in TAG degradation in the presence of fatty acid synthesis inhibitor cerulenin, but that of Tgl2p^S144A does not. This result demonstrates the lipolytic function of Tgl2p in yeast. Although other yeast TAG lipases are localized to lipid particles, Tgl2p is enriched in the mitochondria. The mitochondrial fraction purified from the TGL2-overexpressing yeast shows a strong lipolytic activity, which was absent in the tgl2 deletion mutant. Therefore, we conclude that Tgl2p is a functional lipase of the yeast mitochondria. By analyzing phenotypic effects of TGL2-deficient yeast, we also find that lipolysis-competent Tgl2p is required for the viability of cells treated with antimitotic drug. The addition of oleic acid, the product of Tgl2p-catalyzed lipolysis, fully complements the antimitotic drug sensitivity of the tgl2 null mutation. Thus, we propose that the mitochondrial Tgl2p-dependent lipolysis is crucial for the survival of cells under antimitotic drug treatment.     

The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols. In a strain lacking both nonpolar lipids and consequently lipid droplets, Tgl1p as well as Yeh1p were present at low amount, became highly unstable compared to wild-type cells, and lost their enzymatic activity. Under these conditions both steryl ester hydrolases were retained in the endoplasmic reticulum. The lack of steryl esters alone was not sufficient to cause an altered intracellular localization of Tgl1p and Yeh1p. Surprisingly, the stability of Tgl1p and Yeh1p was markedly reduced in a strain lacking triacylglycerols, but their capacity to mobilize steryl esters remained unaffected. We also tested a possible cross-regulation of Tgl1p and Yeh1p by analyzing the behavior of each hydrolase in the absence of its counterpart steryl ester hydrolases. In summary, this study demonstrates a strong regulation of the two lipid droplet associated steryl ester hydrolases Tgl1p and Yeh1p due to the presence/absence of their host organelle.     

Screening for Hydrolytic Enzymes Reveals Ayr1p as a Novel Triacylglycerol Lipase in Saccharomyces cerevisiae

Saccharomyces cerevisiae, as well as other eukaryotes, preserves fatty acids and sterols in a biologically inert form, as triacylglycerols and steryl esters. The major triacylglycerol lipases of the yeast S. cerevisiae identified so far are Tgl3p, Tgl4p, and Tgl5p (Athenstaedt, K., and Daum, G. (2003) YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae. J. Biol. Chem. 278, 23317–23323; Athenstaedt, K., and Daum, G. (2005) Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae, are localized to lipid particles. J. Biol. Chem. 280, 37301–37309). We observed that upon cultivation on oleic acid, triacylglycerol mobilization did not come to a halt in a yeast strain deficient in all currently known triacylglycerol lipases, indicating the presence of additional not yet characterized lipases/esterases. Functional proteome analysis using lipase and esterase inhibitors revealed a subset of candidate genes for yet unknown hydrolytic enzymes on peroxisomes and lipid droplets. Based on the conserved GXSXG lipase motif, putative functions, and subcellular localizations, a selected number of candidates were characterized by enzyme assays in vitro, gene expression analysis, non-polar lipid analysis, and in vivo triacylglycerol mobilization assays. These investigations led to the identification of Ayr1p as a novel triacylglycerol lipase of yeast lipid droplets and confirmed the hydrolytic potential of the peroxisomal Lpx1p in vivo. Based on these results, we discuss a possible link between lipid storage, lipid mobilization, and peroxisomal utilization of fatty acids as a carbon source.     

Dynamics of neutral lipid storage in yeast

Since energy storage is a basic metabolic process, the synthesis of neutral lipids occurs in all kingdoms of life. The yeast, Saccharomyces cerevisiae, widely accepted as a model eukaryotic cell, contains two classes of neutral lipids, namely steryl esters and triacylglycerols. Triacylglycerols are synthesized through two pathways governed by the acyl-CoA diacylglycerol acyltransferase Dga1p and the phospholipid diacylglycerol acyltransferase Lro1p, respectively. Steryl esters are formed by the two steryl ester synthases Are1p and Are2p, two enzymes with overlapping function which also catalyze triacylglycerol formation, although to a minor extent. Storage of neutral lipids is tightly linked to the biogenesis of so called lipid particles. The role of this compartment in lipid homeostasis and its interplay with other organelles involved in neutral lipid dynamics, especially the endoplasmic reticulum and the plasma membrane, are subject of current investigations. In contrast to neutral lipid formation, mobilization of triacylglycerols and steryl esters in yeast are less characterized at the molecular level. Only recently, the triacylglycerol lipase Tgl3p was identified as the first yeast enzyme of this kind by function. Genes and gene products governing steryl ester mobilization still await identification. Besides biochemical properties of enzymes involved in yeast neutral lipid synthesis and degradation, regulatory aspects of these pathways and cell biological consequences of neutral lipid depletion will be discussed in this minireview.     

Regulation of the Yeast Triacylglycerol Lipase Tgl3p by Formation of Nonpolar Lipids

Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, is a component of lipid droplets but is also present in the endoplasmic reticulum in a minor amount. Recently, it was shown that this enzyme can also serve as a lysophospholipid acyltransferase (Rajakumari, S., and Daum, G. (2010) Mol. Biol. Cell 21, 501–510). Here, we describe the effects of the presence/absence of triacylglycerols and lipid droplets on the functionality of Tgl3p. In a dga1Δlro1Δare1Δare2Δ quadruple mutant lacking all four triacylglycerol- and steryl ester-synthesizing acyltransferases and consequently the lipid droplets, the gene expression of TGL3 was only slightly altered. In contrast, protein level and stability of Tgl3p were markedly reduced in the absence of lipid droplets. Under these conditions, the enzyme was localized to the endoplasmic reticulum. Even the lack of the substrate, triacylglycerol, affected stability and localization of Tgl3p to some extent. Interestingly, Tgl3p present in the endoplasmic reticulum seems to lack lipolytic as well as acyltransferase activity as shown by enzymatic analysis and lipid profiling. Thus, we propose that the activity of Tgl3p is restricted to lipid droplets, whereas the endoplasmic reticulum may serve as a parking lot for this enzyme.     

Dynamics of neutral lipid storage and mobilization in yeast

We make use of the yeast Saccharomyces cerevisiae as a flexible experimental system to investigate coordinate pathways of neutral lipid synthesis, storage and mobilization with special emphasis on the role of different organelles in these processes. Recently, a number of new gene products involved in triacylglycerol (TAG) and steryl ester (STE) metabolism were identified in our laboratory and by other groups. STE are synthesized by the two STE synthases Are1p and Are2p, whereas TAG are formed mainly through the action of the two TAG synthases Dga1p and Lro1p with minor contributions of Are1p and Are2p. Once formed, TAG and STE are stored in so-called lipid particles. A dga1Deltalro1Deltaare1Deltaare2Delta quadruple mutant which lacks neutral lipid synthesis and is consequently devoid of lipid particles turned out to be a valuable tool for studying the physiological role of storage lipids and lipid particles. Mobilization of neutral lipid depots occurs through catalysis of TAG lipases and STE hydrolases. Three TAG lipases named Tgl3p, Tgl4p and Tgl5p, and three STE hydrolases named Tgl1p, Yeh1p and Yeh2p were recently identified at the molecular level. Although these hydrolases exhibit overlapping function within the enzyme families, they are specific for TAG and STE, respectively. With the exception of Dga1p, whose activity is partially localized to lipid particles, TAG and STE forming enzymes are restricted to the endoplasmic reticulum. TAG lipases and STE hydrolases are components of lipid particles with the exception of Yeh2p, which is plasma membrane located. Thus, neutral lipid metabolism is not only regulated at the enzyme level but also by the distribution of the components to organelles. The fact that neutral lipid homeostasis is linked to a number of cell biological processes confirms the important role of this class of lipids as cellular modulators or effectors.     

Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle, lipid droplet or oil body. Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis. In the yeast, the major storage lipids are triacylglycerols (TGs) and steryl esters (SEs). An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways. A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases. In this review, we describe our recent knowledge of TG lipases from the yeast based on biochemical, molecular biological and cell biological information. We report about recent findings addressing the versatile role of TG lipases in lipid metabolism, and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.     

Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles

Triacylglycerol (TAG) lipases are required for mobilization of TAG stored in lipid particles. Recently, Tgl3p was identified as a major TAG lipase of the yeast Saccharomyces cerevisiae (Athenstaedt, K., and Daum, G. (2003) J. Biol. Chem. 278, 23317-23323). Here, we report the identification of Tgl4p and Tgl5p as additional TAG lipases of the yeast. Both polypeptides, encoded by open reading frames YKR089c/TGL4 and YOR081c/TGL5, share 30 and 26% homology, respectively, to Tgl3p. Cell fractionation experiments and microscopic inspection of strains bearing Tgl4p-GFP and Tgl5p-GFP hybrids demonstrated that both proteins are localized to lipid particles similar to Tgl3p. A 1.7-fold increased amount of TAG enriched in myristic and palmitic acids and the reduced mobilization rate of TAG from tgl4Delta in the presence of the fatty acid synthesis inhibitor cerulenin demonstrated the lipolytic function of Tgl4p in vivo. In contrast, neither the total amount of TAG nor the TAG mobilization rate after addition of cerulenin was affected in tgl5Delta cells. However, the enrichment of C26:0 esterified to TAG of tgl5Delta, an additional increase of TAG in the tgl4Deltatgl5Delta double deletion mutant compared with tgl4Delta, and the impairment of TAG mobilization in the tgl4Deltatgl5Delta strain in the presence of cerulenin suggested that also Tgl5p functions as a TAG lipase in vivo. Most importantly, the purified His(6)-tagged Tgl4p and Tgl5p hybrids exhibited TAG lipase activity demonstrating their function in vitro. In summary, our data obtained by biochemical, molecular, and cell biological analyses unambiguously identified Tgl4p and Tgl5p as novel TAG lipases of yeast lipid particles with certain enzymatic specificities.     

Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum

Diatoms accumulate triacylglycerols (TAGs) as storage lipids, but the knowledge about the molecular mechanisms of lipid metabolism is still sparse. Starting from a partial sequence for a putative TAG-lipase of the diatom Phaeodactylum tricornutum retrieved from the data bases, we have identified the full length coding sequence, tgl1. The gene encodes an 813 amino acid sequence that shows distinct motifs for so called “true” TAG-lipases [EC 3.1.1.3] that have been functionally characterized in model organisms like Arabidopsis thaliana and Saccharomyces cerevisiae. These lipases mediate the first initial step of TAG breakdown from storage lipids. To test whether Tgl1 can act as a TAG-lipase, a His-tagged version was overexpressed in Escherichia coli and the protein indeed showed esterase activity. To identify the TAG degrading function of Tgl1 in P. tricornutum, knock-down mutant strains were created using an antisense RNA approach. In the mutant cell lines the relative tgl1-mRNA-level was reduced up to 20% of that of the wild type, accompanied by a strong increase of TAG in the lipid extracts. In spite of the TAG accumulation, the polar lipid species pattern appeared to be unchanged, confirming the TAG-lipase function of Tgl1.     

Synthesis and turnover of non-polar lipids in yeast

In the yeast Saccharomyces cerevisiae as in other eukaryotic cells non-polar lipids form a reservoir of energy and building blocks for membrane lipid synthesis. The yeast non-polar lipids, triacylglycerol (TAG) and steryl ester (STE), are synthesized by enzymes with overlapping function. Recently, genes encoding these enzymes were identified and gene products were partially characterized. Once formed, TAG and STE are stored in so-called lipid particles/droplets. This compartment which is reminiscent of mammalian lipoproteins from the structural viewpoint is, however, not only a lipid depot but also an organelle actively contributing to lipid metabolism. Non-polar lipid degrading enzymes, TAG lipases and STE hydrolases, also occur in redundancy in the yeast. These proteins, which are components of the lipid particle surface membrane with the exception of one plasma membrane localized STE hydrolase, mobilize non-polar lipids upon requirement. In this review, we describe the coordinate pathways of non-polar lipid synthesis, storage and mobilization in yeast with special emphasis on the role of the different enzymes and organelles involved in these processes. Moreover, we will discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.     

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.     

Modifications of the C terminus Affect Functionality and Stability of Yeast Triacylglycerol Lipase Tgl3p

Lipid droplets are specific organelles for the storage of triacylglycerols and steryl esters. They are surrounded by a phospholipid monolayer with a small but specific set of proteins embedded. Assembly and insertion of proteins into this surface membrane is an intriguing question of lipid droplet biology. To address this question we studied the topology of Tgl3p, the major triacylglycerol lipase of the yeast Saccharomyces cerevisiae, on lipid droplets. Employing the method of limited proteolysis of lipid droplet surface proteins, we found that the C terminus of Tgl3p faces the inside of the organelle, whereas the N terminus is exposed at the cytosolic side of lipid droplets. Detailed analysis of the C terminus revealed a stretch of seven amino acids that are critical for protein stability and functionality. The negative charge of two aspartate residues within this stretch is crucial for lipase activity of Tgl3p. A portion of Tgl3p, which is located to the endoplasmic reticulum, exhibits a different topology. In the phospholipid bilayer of the endoplasmic reticulum the C terminus faces the cytosol, which results in instability of the protein. Thus, the topology of Tgl3p is important for its function and strongly dependent on the membrane environment.     

Janus-faced Enzymes Yeast Tgl3p and Tgl5p Catalyze Lipase and Acyltransferase Reactions

In the yeast, mobilization of triacylglycerols (TAGs) is facilitated by the three TAG lipases Tgl3p, Tgl4p, and Tgl5p. Motif search analysis, however, indicated that Tgl3p and Tgl5p do not only contain the TAG lipase motif GXSXG but also an H-(X)₄-D acyltransferase motif. Interestingly, lipid analysis revealed that deletion of TGL3 resulted in a decrease and overexpression of TGL3 in an increase of glycerophospholipids. Similar results were obtained with TGL5. Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed, both enzymes not only exhibited lipase activity but also catalyzed acylation of lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two enzymatic activities act independently of each other. We also showed that Tgl3p is important for efficient sporulation of yeast cells, but rather through its acyltransferase than lipase activity. In summary, our results demonstrate that yeast Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and catabolic processes.     

Synthesis, storage and degradation of neutral lipids in yeast

The single cell eukaryote Saccharomyces cerevisiae is an attractive model to study the complex process of neutral lipid (triacylglycerol and steryl ester) synthesis, storage and turnover. In mammals, defects in the metabolism of these lipids are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Since the yeast harbors many counterparts of mammalian enzymes involved in these pathways, conclusions drawn from research with the microorganism can be readily applied to the higher eukaryotic system. Here, we summarize our current knowledge of yeast neutral lipid metabolism, report about pathways and enzymes contributing to formation and degradation of triacylglycerols and steryl esters, and describe storage of these components in lipid particles. The interplay of different subcellular compartments in neutral lipid metabolism, regulatory aspects of this process and cell biological consequences of dysfunctions will be discussed.     

Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris

Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.     

Identification of triacylglycerol and steryl ester synthases of the methylotrophic yeast Pichia pastoris

In yeast like in many other eukaryotes, fatty acids are stored in the biologically inert form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane building blocks. In the present study, we identified gene products catalyzing formation of TG and SE in the methylotrophic yeast Pichia pastoris. Based on sequence homologies to Saccharomyces cerevisiae, the two diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase Are2p from P. pastoris were identified. Mutants bearing single and multiple deletions of the respective genes were analyzed for their growth phenotype, lipid composition and the ability to form lipid droplets. Our results indicate that the above mentioned gene products are most likely responsible for the entire TG and SE synthesis in P. pastoris. Lro1p which has low fatty acid substrate specificity in vivo is the major TG synthase in this yeast, whereas Dga1p contributes less to TG synthesis although with some preference to utilize polyunsaturated fatty acids as substrates. In contrast to S. cerevisiae, Are2p is the only SE synthase in P. pastoris. Also this enzyme exhibits some preference for certain fatty acids as judged from the fatty acid profile of SE compared to bulk lipids. Most interestingly, TG formation in P. pastoris is indispensable for lipid droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1?lro1? double deletion mutant is insufficient to initiate the formation of the storage organelle. In summary, our data provide a first insight into the molecular machinery of non-polar lipid synthesis and storage in P. pastoris and demonstrate specific features of this machinery in comparison to other eukaryotic cells, especially S. cerevisiae.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

Storage lipid synthesis is non-essential in yeast.

Steryl esters and triacylglycerol (TAG) are the main storage lipids in eukaryotic cells. In the yeast Saccharomyces cerevisiae, these storage lipids accumulate during stationary growth phase within organelles known as lipid bodies. We have used single and multiple gene disruptions to study storage lipid synthesis in yeast. Four genes, ARE1, ARE2, DGA1, and LRO1, were found to contribute to TAG synthesis. The most significant contribution is made by DGA1, which encodes a novel acyl-CoA:diacylglycerol acyltransferase. Two of the genes, ARE1 and ARE2, are also involved in steryl ester synthesis. A yeast strain that lacks all four genes is viable and has no apparent growth defects under standard conditions. The strain is devoid of both TAG and steryl esters, and fluorescence microscopy revealed that it also lacks lipid bodies. We conclude that neither storage lipids nor lipid bodies are essential for growth in yeast.