Defects in triacylglycerol lipolysis affect synthesis of triacylglycerols and steryl esters in the yeast

Tgl3p, Tgl4p and Tgl5p are the major triacylglycerol lipases of the yeast Saccharomyces cerevisiae catalyzing degradation of triacylglycerols stored in lipid droplets. Previous results from our laboratory (Athenstaedt and Daum, 2005, J. Biol. Chem. 280, 37301–37309) demonstrated that a yeast strain lacking all three triacylglycerol lipases accumulates not only triacylglycerols at high amount, but also steryl esters. Here we show a metabolic link between synthesis and mobilization of non-polar lipids. In particular, we demonstrate that a block in tri-acylglycerol degradation in a tgl3?tgl4?tgl5? triple mutant lacking all major triacylglycerol lipases causes marked changes in non-polar lipid synthesis. Under these conditions formation of triacylglycerols is reduced, whereas steryl ester synthesis is enhanced as shown by quantification of non-polar lipids, in vivo labeling of lipids using [¹⁴C]oleic acid and [¹⁴C]acetic acid as precursors, and enzyme analyses in vitro. In summary, this study demonstrates that triacylglycerol metabolism and steryl ester metabolism are linked processes. The importance of balanced storage and degradation of these components for lipid homeostasis in the yeast is highlighted.     

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.     

YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae

Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448) showed that the gene product of YMR313c (named Tgl3p) is a component of yeast lipid particles, and deletion of this gene led to an increase in the cellular level of triacylglycerols (TAG). These observations suggested that TGL3 may encode a TAG lipase of Saccharomyces cerevisiae. Here we demonstrate by cell fractionation and by microscopic inspection of a strain bearing a Tgl3p-GFP hybrid that this polypeptide is highly enriched in the lipid particle fraction but virtually absent from other organelles. The entire TAG lipase activity of lipid particles is attributed to Tgl3p, because the activity in this organelle is completely absent in a Deltatgl3 deletion mutant, whereas it is significantly enhanced in a strain overexpressing Tgl3p. A His6-tagged Tgl3p hybrid purified close to homogeneity from a yeast strain overexpressing this fusion protein exhibited high TAG lipase activity. Most importantly, experiments in vivo using the fatty acid synthesis inhibitor cerulenin demonstrated that deletion of TGL3 resulted in a decreased mobilization of TAG from lipid particles. The amino acid sequence deduced from the open reading frame YMR313c contains the consensus sequence motif GXSXG typical for lipolytic enzymes. Otherwise, Tgl3p has no significant sequence homology to other lipases identified so far. In summary, our data identified Tgl3p as a novel yeast TAG lipase at the molecular level and by function in vivo and in vitro.     

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.     

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 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.     

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.     

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.     

A defect of the vacuolar putative lipase Atg15 accelerates degradation of lipid droplets through lipolysis

Lipid droplets (LDs) are the conserved organelles for the deposit of neutral lipids, and function as reservoirs of membrane and energy sources. To date, functional links between autophagy and LD dynamics have not been fully elucidated. Here, we report that a vacuolar putative lipase, Atg15, required for degradation of autophagic bodies, is crucial for the maintenance of LD amount in the yeast Saccharomyces cerevisiae in the stationary phase. Mutant analyses revealed that the putative lipase motif and vacuolar localization of Atg15 are important for the maintenance of LD amount. Loss of autophagosome formation by simultaneous deletion of core ATG genes cancelled the reduction in the LD amount in ATG15-deleted cells, indicating that degradation of autophagic bodies accounts for the functional involvement of Atg15 in LD dynamics. The reduced level of LDs in the mutant strain was dependent on Tgl3 and Tgl4, major lipases for lipolysis in S. cerevisiae. An altered phosphorylation status of Tgl3, higher accumulation of Tgl4, and closer associations of Tgl3 and Tgl4 with LDs were detected in the ATG15-deleted cells. Furthermore, increased levels of downstream metabolites of lipolysis in the mutant strain strongly suggested enhanced lipolytic activity caused by loss of ATG15. Our data provide evidence for a novel link between autophagic flux and LD dynamics integrated with Atg15 activity.     

Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae

In the yeast as in other eukaryotes, formation and hydrolysis of steryl esters (SE) are processes linked to lipid storage. In Saccharomyces cerevisiae, the three SE hydrolases Tgl1p, Yeh1p and Yeh2p contribute to SE mobilization from their site of storage, the lipid particles/droplets. Here, we provide evidence for enzymatic and cellular properties of these three hydrolytic enzymes. Using the respective single, double and triple deletion mutants and strains overexpressing the three enzymes, we demonstrate that each SE hydrolase exhibits certain substrate specificity. Interestingly, disturbance in SE mobilization also affects sterol biosynthesis in a type of feedback regulation. Sterol intermediates stored in SE and set free by SE hydrolases are recycled to the sterol biosynthetic pathway and converted to the final product, ergosterol. This recycling implies that the vast majority of sterol precursors are transported from lipid particles to the endoplasmic reticulum, where sterol biosynthesis is completed. Ergosterol formed through this route is then supplied to its subcellular destinations, especially the plasma membrane. Only a minor amount of sterol precursors are randomly distributed within the cell after cleavage from SE. Conclusively, SE storage and mobilization although being dispensable for yeast viability contribute markedly to sterol homeostasis and distribution.     

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.     

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.     

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.     

A homolog of Arabidopsis SDP1 lipase in Nannochloropsis is involved in degradation of de novo-synthesized triacylglycerols in the endoplasmic reticulum

Organisms of the microalgal genus Nannochloropsis produce high levels of triacylglycerols (TAGs), an efficient raw material for biofuels. A complete understanding of the TAG-breakdown pathway is critical for improving the productivity of TAGs to meet future needs. Among a number of lipases annotated as TAG lipase in the genomes of every organism, Arabidopsis SUGAR-DEPENDENT 1 (AtSDP1) lipases are characterized as a type of crucial TAG lipase in plants, similar to ScTgl3–5 in Saccharomyces cerevisiae. Homologs of the AtSDP1 TAG lipases are universally found in the genomes of plants, fungi, and algae. Here we identified two homologs of AtSDP1 TAG lipases in the oleaginous microalga species Nannochloropsis oceanica, NoTGL1 and NoTGL2. We generated single- and double-knockout strains for these lipases by homologous recombination. Whereas overall TAG content in the NoTGL2 single-knockout mutant was identical to that of wild type, the NoTGL1 knockout showed a two-fold increase in TAG content per cell in early log phase under nutrient-sufficient conditions without affecting growth. Homologs of AtSDP1 in S. cerevisiae are localized to the surface of lipid droplets, and AtSDP1 is transported from peroxisomes to the surface of lipid droplets. In contrast, NoTGL1 localized to the endoplasmic reticulum in both Nannochloropsis and yeast. We suggest that homologs of AtSDP1 lipases in Nannochloropsis modulate de novo TAG biosynthesis in the endoplasmic reticulum, unlike the roles of these lipases in other organisms. These results provide important insights into the mechanisms of TAG metabolism catalyzed by homologs of AtSDP1 lipase, which are highly conserved across species.     

An Energy-Independent Pro-longevity Function of Triacylglycerol in Yeast

Intracellular triacylglycerol (TAG) is a ubiquitous energy storage lipid also involved in lipid homeostasis and signaling. Comparatively, little is known about TAG’s role in other cellular functions. Here we show a pro-longevity function of TAG in the budding yeast Saccharomyces cerevisiae. In yeast strains derived from natural and laboratory environments a correlation between high levels of TAG and longer chronological lifespan was observed. Increased TAG abundance through the deletion of TAG lipases prolonged chronological lifespan of laboratory strains, while diminishing TAG biosynthesis shortened lifespan without apparently affecting vegetative growth. TAG-mediated lifespan extension was independent of several other known stress response factors involved in chronological aging. Because both lifespan regulation and TAG metabolism are conserved, this cellular pro-longevity function of TAG may extend to other organisms.     

Characterization of neutral lipases revealed the tissue-specific triacylglycerol hydrolytic activity in Nilaparvata lugens

The lipid metabolism plays an essential role in the development and reproduction of insects, and lipases are important enzymes in lipid metabolism. In Nilaparvata lugens, an important insect pest on rice, triacylglycerol hydrolytic activities were different among tissues, with high activity in integument, ovary, and fat body, but low activity in intestine. To figure out the tissue-specific triacylglycerol hydrolytic activity, we identified 43 lipases in N. lugens. Of these 43 lipases, 23 belonged to neutral lipases, so this group was selected to perform further experiments on triacylglycerol hydrolysis. The complete motifs of catalytic triads, β9 loop, and lid motif, are required for the triacylglycerol hydrolytic activity in neutral lipases, which were found in some neutral lipases with high gene expression levels in integument and ovary, but not in intestine. The recombinant proteins of 3 neutral lipases with or without 3 complete motifs were obtained, and the activity determination confirmed the importance of 3 motifs. Silencing XM_022331066.1, which is highly expressed in ovary and with 3 complete motifs, significantly decreased the egg production and hatchability of N. lugens, partially through decline of the lipid metabolism. In summary, at least one-third of important motifs were incomplete in all neutral lipases with high gene expression in intestine, which could partially explain why the lipase activity in intestine was much lower than that in other tissues. The low activity to hydrolyze triacylglycerol in N. lugens intestine might be associated with its food resource and nutrient components, and the ovary-specific neutral lipases were important for N. lugens reproduction.