Guenther Daum

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 Δtgl3 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.

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 tgl4Δ 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 tgl5Δ cells. However, the enrichment of C26:0 esterified to TAG of tgl5Δ, an additional increase of TAG in the tgl4Δtgl5Δ double deletion mutant compared with tgl4Δ, and the impairment of TAG mobilization in the tgl4Δtgl5Δ strain in the presence of cerulenin suggested that also Tgl5p functions as a TAG lipase in vivo. Most importantly, the purified His6-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.

Structural and Biochemical Properties of Lipid Particles from the Yeast Saccharomyces cerevisiae

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.

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

The yeast triacylglycerol (TAG) lipases Tgl3p and Tgl5p not only contain a TAG lipase, but also an acyltransferase motif. We show that purified Tgl3p and Tgl5p indeed exhibit lysophospholipid acyltransferase activity. Both Tgl3p and Tgl5p affect the level of glycerophospholipids, and Tgl3p is also important for efficient sporulation of yeast.

Lipid topology and physical properties of the outer mitochondrial membrane of the yeast, Saccharomyces cerevisiae.

The outer membrane of yeast mitochondria was studied with respect to its lipid composition, phospholipid topology and membrane fluidity. This membrane is characterized by a high phospholipid to protein ratio (1.20). Like other yeast cellular membranes the outer mitochondrial membrane contains predominantly phosphatidylcholine (44% of total phospholipids), phosphatidylethanolamine (34%) and phosphatidylinositol (14%). Cardiolipin, the characteristic phospholipid of the inner mitochondrial membrane (13% of total phospholipids) is present in the outer membrane only to a moderate extent (5%). The ergosterol to phospholipid ratio is higher in the inner (7.0 wt%) as compared to the outer membrane (2.1 wt.%). Attempts to study phospholipid asymmetry by selective degradation of phospholipids of the outer leaflet of the outer mitochondrial membrane failed, because isolated right-side-out vesicles of this membrane became leaky upon treatment with phospholipases. Selective removal of phospholipids of the outer leaflet with the aid of phospholipid transfer proteins and chemical modification with trinitrobenzenesulfonic acid on the other hand, gave satisfactory results. Phosphatidylcholine and phosphatidylinositol are more or less evenly distributed between the two sides of the outer mitochondrial membrane, whereas the majority of phosphatidylethanolamine is oriented towards the intermembrane space. The fluidity of mitochondrial membranes was determined by measuring fluorescence anisotropy using diphenylhexatriene (DPH) as a probe. The lower anisotropy of DPH in the outer as compared to the inner membrane, which is an indication for an increased lipid mobility in the outer membrane, was attributed to the higher phospholipid to protein and the lower ergosterol to phospholipid ratio. The data presented here show, that the outer mitochondrial membrane, in spite of its close contact to the inner membrane, is distinct not only with respect to its protein pattern, but also with respect to its lipid composition and physical membrane properties.

Multiple functions as lipase, steryl ester hydrolase, phospholipase, and acyltransferase of Tgl4p from the yeast Saccharomyces cerevisiae.

Triacylglycerol (TAG) hydrolysis, membrane lipid biosynthesis, and lipid turnover are largely interlinked processes. In yeast, TAG is mobilized by three TAG lipases named Tgl3p, Tgl4p, and Tgl5p, which are localized to lipid particles/droplets. These TAG lipases posses a conserved GXSXG motif that is characteristic of hydrolytic enzymes. Here, we demonstrated that the yeast TAG lipase Tgl4p, the functional ortholog of the adipose TAG lipase, ATGL, catalyzes multiple functions in lipid metabolism. An extended domain and motif search analysis revealed that Tgl4p bears not only a lipase consensus domain but also a conserved motif for calcium-independent phospholipase A2. We show that Tgl4p exhibits TAG lipase, steryl ester hydrolase, and phospholipase A2 activities, but surprisingly it also catalyzed the acyl-CoA-dependent acylation of lysophosphatidic acid to phosphatidic acid (PA). Heterologous overexpression of Tgl4p in Pichia pastoris increased total phospholipid and specifically PA synthesis. Moreover, deletion of TGL4 in Saccharomyces cerevisiae showed an altered pattern of phosphatidylcholine and PA molecular species. Altogether, our data suggest that yeast Tgl4p functions as a hydrolytic enzyme in lipid degradation but also contributes to fatty acid channeling and phospholipid remodeling.

Oleate Inhibits Steryl Ester Synthesis and Causes Liposensitivity in Yeast

In the yeast Saccharomyces cerevisiae, neutral lipids can be synthesized by four acyltransferases, namely Dga1p and Lro1p producing triacylglycerols (TAG) and Are1p and Are2p forming steryl esters (SE). TAG and SE are stored in an organelle called lipid particles/droplet. Growth of yeast cells on oleate-supplemented media strongly induced proliferation of lipid particles and specifically the synthesis of TAG, which serve as the major pool for the excess of fatty acids. Surprisingly, SE synthesis was strongly inhibited under these conditions. Here, we show that this effect was not due to decreased expression of ARE2 encoding the major yeast SE synthase at the transcriptional level but to competitive enzymatic inhibition of Are2p by free oleate. Consequently, a triple mutant dga1Δlro1Δare1ΔARE2+ grown on oleate did not form substantial amounts of SE and exhibited a growth phenotype similar to the dga1Δlro1Δare1Δare2Δ quadruple mutant, including lack of lipid particles. Growth of these mutants on oleate was strongly delayed, and cell viability was decreased but rescued by adaptation. In these strains, oleate stress caused morphological changes of intracellular membranes, altered phospholipid composition and formation of an additional lipid class, ethyl esters of fatty acids. In summary, our data showed that exposure to oleate led to disturbed lipid and membrane homeostasis along with liposensitivity of the yeast.

At4g24160, a Soluble Acyl-Coenzyme A-Dependent Lysophosphatidic Acid Acyltransferase

Human CGI-58 (for comparative gene identification-58) and YLR099c, encoding Ict1p in Saccharomyces cerevisiae, have recently been identified as acyl-CoA-dependent lysophosphatidic acid acyltransferases. Sequence database searches for CGI-58 like proteins in Arabidopsis (Arabidopsis thaliana) revealed 24 proteins with At4g24160, a member of the α/β-hydrolase family of proteins being the closest homolog. At4g24160 contains three motifs that are conserved across the plant species: a GXSXG lipase motif, a HX4D acyltransferase motif, and V(X)3HGF, a probable lipid binding motif. Dendrogram analysis of yeast ICT1, CGI-58, and At4g24160 placed these three polypeptides in the same group. Here, we describe and characterize At4g24160 as, to our knowledge, the first soluble lysophosphatidic acid acyltransferase in plants. A lipidomics approach revealed that At4g24160 has additional triacylglycerol lipase and phosphatidylcholine hydrolyzing enzymatic activities. These data establish At4g24160, a protein with a previously unknown function, as an enzyme that might play a pivotal role in maintaining the lipid homeostasis in plants by regulating both phospholipid and neutral lipid levels.

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the yeast Saccharomyces cerevisiae

Previous work from our laboratory had demonstrated that deletion of TGL3 encoding the major yeast triacylglycerol (TAG) lipase resulted in decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG fraction [K. Athenstaedt and G. Daum, J. Biol. Chem. 278 (2003) 23317-23323]. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we carried out metabolic labeling experiments with wild type and deletion strains bearing defects in the three major yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Using [(3)H]inositol, [(32)P]orthophosphate, [(3)H]palmitate and [(14)C]acetate as precursors for complex lipids we demonstrated that tgl mutants had a lower level of sphingolipids and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG accumulation in these mutant cells resulted in reduced amounts of phospholipids and sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected the metabolic flux of long chain fatty acids and very long chain fatty acids required for sphingolipid and glycerophospholipid synthesis. Activity and expression level of fatty acid elongases, Elo1p and Elo2p were enhanced as a consequence of reduced TAG lipolysis. Finally, the pattern of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine molecular species was altered in tgl deletion strain underlining the important role of TAG turnover in maintaining the pool size of these compounds and the remodeling of complex membrane lipids.