Redundant systems of phosphatidic acid biosynthesis via acylation of glycerol-3-phosphate or dihydroxyacetone phosphate in the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611-7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate.     

Synthesis of Triacylglycerols by the Acyl-Coenzyme A:Diacyl-Glycerol Acyltransferase Dga1p in Lipid Particles of the Yeast Saccharomyces cerevisiae

The terminal step of triacylglycerol (TAG) formation in the yeast Saccharomyces cerevisiae is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase (DAGAT). In this study we demonstrate that the gene product of YOR245c, Dga1p, catalyzes a major yeast DAGAT activity which is localized to lipid particles. Enzyme measurements employing a newly established assay containing radioactively labeled diacylglycerol (DAG) as a substrate and unlabeled palmitoyl-CoA as a cosubstrate revealed a 70- to 90-fold enrichment of DAGAT in lipid particles over the homogenate but also a 2- to 3-fold enrichment in endoplasmic reticulum fractions. In a dga1 deletion strain, the DAGAT activity in lipid particles is dramatically reduced, whereas the activity in microsomes is affected only to a minor extent. Thus, we propose the existence of DAGAT isoenzymes in the microsomal fraction. Furthermore, we unveiled an acyl-CoA-independent TAG synthase activity in lipid particles which is distinct from Dga1p and the phosphatidylcholine:DAGAT Lro1p. This acyl-CoA-independent TAG synthase utilizes DAG as an acceptor and free fatty acids as cosubstrates and occurs independently of the acyl-CoA synthases Faa1p to Faa4p. Based on lipid analysis of the respective deletion strains, Lro1p and Dga1p are the major contributors to total cellular TAG synthesis, whereas other TAG synthesizing systems appear to be of minor importance. In conclusion, at least three different pathways are involved in the formation of storage TAG in the yeast.     

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.     

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

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.     

Glycosylphosphatidylinositol (GPI) proteins of Saccharomyces cerevisiae contain ethanolamine phosphate groups on the alpha1,4-linked mannose of the GPI anchor

In humans and Saccharomyces cerevisiae the free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater in gpi7Delta sec18 double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion of GPI7) influences the stability of the one on the first mannose. The phosphodiester-linked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomes in vitro but cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels in gpi7Delta cells by overexpression of PSD1 restore cell growth at 37 degrees C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test.     

Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae.

Lipid particles of the yeast Saccharomyces cerevisiae harbor two enzymes that stepwise acylate glycerol-3-phosphate to phosphatidic acid, a key intermediate in lipid biosynthesis. In lipid particles of the s1c1 disruptant YMN5 (M. M. Nagiec et al., J. Biol. Chem. 268:22156-22163, 1993) acylation stops after the first step, resulting in the accumulation of lysophosphatidic acid. Two-dimensional gel electrophoresis confirmed that S1c1p is a component of lipid particles. Lipid particles of a second mutant strain, TTA1 (T. S. Tillman and R. M. Bell, J. Biol. Chem. 261:9144-9149, 1986), which harbors a point mutation in the GAT gene, are essentially devoid of glycerol-3-phosphate acyltransferase activity in vitro. Synthesis of phosphatidic acid is reconstituted by combining lipid particles from YMN5 and TTA1. These results indicate that two distinct enzymes are necessary for phosphatidic acid synthesis in lipid particles: the first step, acylation of glycerol-3-phosphate, is catalyzed by a putative Gat1p; the second step, acylation of lysophosphatidic acid, requires S1c1p. Surprisingly, YMN5 and TTA1 mutants grow like the corresponding wild types because the endoplasmic reticulum of both mutants has the capacity to form a reduced but significant amount of phosphatidic acid. As a consequence, an s1c1 gat1 double mutant is also viable. Lipid particles from this double mutant fail completely to acylate glycerol-3-phosphate, whereas endoplasmic reticulum membranes harbor residual enzyme activities to synthesize phosphatidic acid. Thus, yeast contains at least two independent systems of phosphatidic acid biosynthesis.     

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.     

Involvement of the Saccharomyces cerevisiae hydrolase Ldh1p in lipid homeostasis

Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Saccharomyces cerevisiae Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.     

Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae

Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.     

Yeast oxidosqualene cyclase (Erg7p) is a major component of lipid particles.

Oxidosqualene cyclase of the yeast encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study (Athenstaedt, K., Zweytick, D., Jandrositz, A, Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448), Erg7p was identified as a component of yeast lipid particles. Here, we present evidence that Erg7p is almost exclusively associated with this compartment as shown by analysis of enzymatic activity, Western blot analysis, and in vivo localization of Erg7p-GFP. Occurrence of oxidosqualene cyclase in other organelles including the endoplasmic reticulum is negligible. In an erg7 deletion strain or in wild-type cells treated with an inhibitor of oxidosqualene cyclase, the substrate of Erg7p, oxidosqualene, accumulated mostly in lipid particles. Storage in lipid particles of this intermediate produced in excess may provide a possibility to exclude this membrane-perturbing component from other organelles. Thus, our data provide evidence that lipid particles are not only a depot for neutral lipids, but also participate in coordinate sterol metabolism and trafficking and serve as a storage site for compounds that may negatively affect membrane integrity.     

Effect of Lipid Particle Biogenesis on the Subcellular Distribution of Squalene in the Yeast Saccharomyces cerevisiae

Squalene belongs to the group of isoprenoids and is a precursor for the synthesis of sterols, steroids, and ubiquinons. In the yeast Saccharomyces cerevisiae, the amount of squalene can be increased by variation of growth conditions or by genetic manipulation. In this report, we show that a hem1Δ mutant accumulated a large amount of squalene, which was stored almost exclusively in cytoplasmic lipid particles/droplets. Interestingly, a strain bearing a hem1Δ deletion in a dga1Δlro1Δare1Δare2Δ quadruple mutant background (QMhem1Δ), which is devoid of the classical storage lipids, triacylglycerols and steryl esters, and lacks lipid particles, accumulated squalene at similar amounts as the hem1Δ mutant in a wild type background. In QMhem1Δ, however, increased amounts of squalene were found in cellular membranes, especially in microsomes. The fact that QMhem1Δ did not form lipid particles indicated that accumulation of squalene solely was not sufficient to initiate proliferation of lipid particles. Most importantly, these results also demonstrated that (i) squalene was not lipotoxic under the conditions tested, and (ii) organelle membranes in yeast can accommodate relatively large quantities of this non-polar lipid without compromising cellular functions. In summary, localization of squalene as described here can be regarded as an unconventional example of non-polar lipid storage in cellular membranes.     

SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1Delta cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Delta and slc4Delta cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Delta and slc4Delta cells incubated with [14C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1Delta and slc4Delta cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg2+-dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.     

Organella-targeted expression of rat liver cytochrome P450c27 in yeast. Genetically engineered alteration of mitochondrial P450 into a microsomal form creates a novel functional electron transport chain.

A modified rat cytochrome P450c27, whose mitochondrial targeting signal had been replaced by a possible microsomal targeting signal of bovine cytochrome P450c17, was expressed in yeast. The modified P450c27 hemoprotein was correctly localized on yeast microsomes and exhibited the P450c27-dependent monooxygenase activity by addition of bovine adrenodoxin (ADX) and NADPH-adrenodoxin reductase (ADR). Considering the previous observation that P450c27 with its own mitochondrial targeting signal was imported into yeast mitochondria (Akiyoshi-Shibata, M., Usui, E., Sakaki, T., Yabusaki, Y., Noshiro, M., Okuda, K., and Ohkawa, H. (1991) FEBS Lett. 280, 367-370), it is now suggested that the destination of P450c27 to either mitochondria or microsomes in yeast depends solely on the amino-terminal targeting signal. In addition, the modified P450c27 was simultaneously expressed in yeast with mature forms of bovine ADX and ADR. The recombinant yeast produced the P450 on the microsomes and mature forms of ADX and ADR in the cytoplasm, and showed the monooxygenase activity. Accordingly, a novel type of functional electron transport chain has been established between the cytoplasm and the microsomes in yeast.     

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.     

Reg1p targets protein phosphatase 1 to dephosphorylate hexokinase II in Saccharomyces cerevisiae: characterizing the effects of a phosphatase subunit on the yeast proteome.

Protein phosphatase 1 (Glc7p) and its binding protein Reg1p are essential for the regulation of glucose repression pathways in Saccharomyces cerevisiae. In order to identify physiological substrates for the Glc7p-Reg1p complex, we examined the effects of deletion of the REG1 gene on the yeast phosphoproteome. Analysis by two-dimensional phosphoprotein mapping identified two distinct proteins that were greatly increased in phosphate content in reg1Delta mutants. Mixed peptide sequencing identified these proteins as hexokinase II (Hxk2p) and the E1alpha subunit of pyruvate dehydrogenase. Consistent with increased phosphorylation of Hxk2p in response to REG1 deletion, fractionation of yeast extracts by anion-exchange chromatography identified Hxk2p phosphatase activity in wild-type strains that was selectively lost in the reg1Delta mutant. The phosphorylation state of Hxk2p and Hxk2p phosphatase activity was restored to wild-type levels in the reg1Delta mutant by expression of a LexA-Reg1p fusion protein. In contrast, expression of LexA-Reg1p containing mutations at phenylalanine in the putative PP-1C-binding site motif (K/R)(X)(I/V)XF was unable to rescue Hxk2p dephosphorylation in intact yeast or restore Hxk2p phosphatase activity. These results demonstrate that Reg1p targets PP-1C to dephosphorylate Hxk2p in vivo and that the motif (K/R)(X) (I/V)XF is necessary for its PP-1 targeting function.