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.     

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.     

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 Saccharomyces cerevisiae Actin Patch Protein App1p Is a Phosphatidate Phosphatase Enzyme

Phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. In the yeast Saccharomyces cerevisiae, PAP is encoded by PAH1, DPP1, and LPP1. The presence of PAP activity in the pah1Δ dpp1Δ lpp1Δ triple mutant indicated another gene(s) encoding the enzyme. We purified PAP from the pah1Δ dpp1Δ lpp1Δ triple mutant by salt extraction of mitochondria followed by chromatography with DE52, Affi-Gel Blue, phenyl-Sepharose, MonoQ, and Superdex 200. Liquid chromatography/tandem mass spectrometry analysis of a PAP-enriched sample revealed multiple putative phosphatases. By analysis of PAP activity in mutants lacking each of the proteins, we found that APP1, a gene whose molecular function has been unknown, confers ∼30% PAP activity of wild type cells. The overexpression of APP1 in the pah1Δ dpp1Δ lpp1Δ mutant exhibited a 10-fold increase in PAP activity. The PAP activity shown by App1p heterologously expressed in Escherichia coli confirmed that APP1 is the structural gene for the enzyme. Introduction of the app1Δ mutation into the pah1Δ dpp1Δ lpp1Δ triple mutant resulted in a complete loss of PAP activity, indicating that distinct PAP enzymes in S. cerevisiae are encoded by APP1, PAH1, DPP1, and LPP1. Lipid analysis of cells lacking the PAP genes, singly or in combination, showed that Pah1p is the only PAP involved in the synthesis of triacylglycerol as well as in the regulation of phospholipid synthesis. App1p, which shows interactions with endocytic proteins, may play a role in vesicular trafficking through its PAP activity.     

Membrane Protein Rim21 Plays a Central Role in Sensing Ambient pH in Saccharomyces cerevisiae

External alkalization activates the Rim101 pathway in Saccharomyces cerevisiae. In this pathway, three integral membrane proteins, Rim21, Dfg16, and Rim9, are considered to be the components of the pH sensor machinery. However, how these proteins are involved in pH sensing is totally unknown. In this work, we investigated the localization, physical interaction, and interrelationship of Rim21, Dfg16, and Rim9. These proteins were found to form a complex and to localize to the plasma membrane in a patchy and mutually dependent manner. Their cellular level was also mutually dependent. In particular, the Rim21 level was significantly decreased in dfg16Δ and rim9Δ cells. Upon external alkalization, the proteins were internalized and degraded. We also demonstrate that the transient degradation of Rim21 completely suppressed the Rim101 pathway but that the degradation of Dfg16 or Rim9 did not. This finding strongly suggests that Rim21 is the pH sensor protein and that Dfg16 and Rim9 play auxiliary functions through maintaining the level of Rim21 and assisting in its plasma membrane localization. Even without external alkalization, the Rim101 pathway was activated in a Rim21-dependent manner by either protonophore treatment or depletion of phosphatidylserine in the inner leaflet of the plasma membrane, both of which caused plasma membrane depolarization like the external alkalization. Therefore, plasma membrane depolarization seems to be one of the key signals for the pH sensor molecule Rim21.     

DGK1-encoded diacylglycerol kinase activity is required for phospholipid synthesis during growth resumption from stationary phase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, triacylglycerol mobilization for phospholipid synthesis occurs during growth resumption from stationary phase, and this metabolism is essential in the absence of de novo fatty acid synthesis. In this work, we provide evidence that DGK1-encoded diacylglycerol kinase activity is required to convert triacylglycerol-derived diacylglycerol to phosphatidate for phospholipid synthesis. Cells lacking diacylglycerol kinase activity (e.g. dgk1Δ mutation) failed to resume growth in the presence of the fatty acid synthesis inhibitor cerulenin. Lipid analysis data showed that dgk1Δ mutant cells did not mobilize triacylglycerol for membrane phospholipid synthesis and accumulated diacylglycerol. The dgk1Δ phenotypes were partially complemented by preventing the formation of diacylglycerol by the PAH1-encoded phosphatidate phosphatase and by channeling diacylglycerol to phosphatidylcholine via the Kennedy pathway. These observations, coupled to an inhibitory effect of dioctanoyl-diacylglycerol on the growth of wild type cells, indicated that diacylglycerol kinase also functions to alleviate diacylglycerol toxicity.     

Proteomic Analysis Reveals a Novel Function of the Kinase Sat4p in Saccharomyces cerevisiae Mitochondria

The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.     

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

NAD⁺ is an essential metabolic cofactor involved in various cellular biochemical processes. Nicotinamide riboside (NR) is an endogenously produced key pyridine metabolite that plays important roles in the maintenance of NAD⁺ pool. Using a NR-specific cell-based screen, we identified mutants that exhibit altered NR release phenotype. Yeast cells lacking the ORF YCL047C/POF1 release considerably more NR compared with wild type, suggesting that POF1 plays an important role in NR/NAD⁺ metabolism. The amino acid sequence of Pof1 indicates that it is a putative nicotinamide mononucleotide adenylyltransferase (NMNAT). Unlike other yeast NMNATs, Pof1 exhibits NMN-specific adenylyltransferase activity. Deletion of POF1 significantly lowers NAD⁺ levels and decreases the efficiency of NR utilization, resistance to oxidative stress, and NR-induced life span extension. We also show that NR is constantly produced by multiple nucleotidases and that the intracellular NR pools are likely to be compartmentalized, which contributes to the regulation of NAD⁺ homeostasis. Our findings may contribute to the understanding of the molecular basis and regulation of NAD⁺ metabolism in higher eukaryotes.     

Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the Δ⁹-acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an ∼75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.     

Protein phosphatase type-1 regulatory subunits Reg1p and Reg2p act as signal transducers in the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae

The REG1 gene encodes a regulatory subunit of the type-1 protein phosphatase (PP1) Glc7 in Saccharomyces cerevisiae, which directs the catalytic subunit to substrates involved in glucose repression. Loss of REG1 relieves glucose repression of many genes, including the MAL structural genes that encode the maltose fermentation enzymes. In this report, we explore the role of Reg1p and its homolog Reg2p in glucose-induced inactivation of maltose permease. Glucose stimulates the proteolysis of maltose permease and very rapid loss of maltose transport activity – more rapid than can be explained by loss of the permease protein alone. In a reg1Δ strain we observe a significantly reduced rate of glucose-induced proteolysis of maltose permease, and the rapid loss of maltose transport activity does not occur. Instead, surprisingly, the slow rate of proteolysis of maltose permease is accompanied by an increase in maltose transport activity. Loss of Reg2p modestly reduces the rates of both glucose-induced proteolysis of maltose permease and inactivation of maltose transport activity. Overexpression of Reg2p in a reg1Δ strain suppresses the effect on maltose permease proteolysis and partially restores the inactivation of maltose transport activity, but does not affect the insensitivity of MAL gene expression to repression by glucose observed in this strain. Thus, protein phosphatase type-1 (Glc7p-Reg1p and Glc7p-Reg2p) plays a role in transduction of the glucose signal during glucose-induced proteolysis of maltose permease, but only Glc7p-Reg1p is involved in glucose-induced inactivation of maltose transport activity and glucose repression of MAL gene expression. Overexpression of REG1 partially restores proteolysis of maltose permease in a grr1Δ strain, which lacks glucose signaling, but does not rescue rapid inactivation of maltose transport activity or sensitivity to glucose repression. A model for the role of Reg1p and Reg2p in glucose signaling pathways is discussed. We also uncovered a previously unrecognized G2/M delay in the grr1Δ but not the reg1Δ strains, and this delay is suppressed by REG1 overexpression. The G1/S delay seen in grr1Δ mutants is slightly suppressed as well, but REG1 overexpression does not suppress other grr1Δ phenotypes such as insensitivity to glucose repression. Received: 21 October 1999 / Accepted: 28 December 1999     

Identification of CBS2 as a mitochondrial protein in Saccharomyces cerevisiae

Summary The nuclear genome encoded yeast protein CBS2 is required for translational activation of mitochondrial cytochrome b RNA. Genetic studies have shown that the target sequence of the CBS2 protein is the 5 untranslated leader sequence of cytochrome b RNA. Here we report on the intracellular localization of CBS2. CBS2 protein, expressed in Escherichia coli and prepared from inclusion bodies, was used as an antigen to raise a polyclonal rabbit antiserum. Affinity-purified CBS2 antibodies detect a 45 kDa protein in mitochondrial lysates of wild-type cells, which is absent in a strain in which the CBS2 gene has been deleted. The protein is overexpressed in mitochondrial extracts of a transformant carrying the CBS2 gene on a high copy number plasmid, but undetectable in the post-mitochondrial supernatant. Intramitochondrial localization of CBS2 was verified by in vitro import of CBS2 protein that had been synthesized in a reticulocyte lysate programmed with CBS2 mRNA transcribed in vitro. Mitochondrial import of CBS2 is not accompanied by any detectable proteolytic processing.     

para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable ¹³C₆-isotope of pABA (p- amino[aromatic-¹³C₆]benzoic acid ([¹³C₆]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[¹³C₆]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [¹³C₆]pABA or [¹³C₆]4HB generate both ¹³C₆-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product ¹³C₆-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ₆. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ₆ quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.     

Yarrowia lipolytica as a model for bio-oil production

The yeast Yarrowia lipolytica has developed very efficient mechanisms for breaking down and using hydrophobic substrates. It is considered an oleaginous yeast, based on its ability to accumulate large amounts of lipids. Completion of the sequencing of the Y. lipolytica genome and the existence of suitable tools for genetic manipulation have made it possible to use the metabolic function of this species for biotechnological applications. In this review, we describe the coordinated pathways of lipid metabolism, storage and mobilization in this yeast, focusing in particular on the roles and regulation of the various enzymes and organelles involved in these processes. The physiological responses of Y. lipolytica to hydrophobic substrates include surface-mediated and direct interfacial transport processes, the production of biosurfactants, hydrophobization of the cytoplasmic membrane and the formation of protrusions. We also discuss culture conditions, including the mode of culture control and the culture medium, as these conditions can be modified to enhance the accumulation of lipids with a specific composition and to identify links between various biological processes occurring in the cells of this yeast. Examples are presented demonstrating the potential use of Y. lipolytica in fatty-acid bioconversion, substrate valorization and single-cell oil production. Finally, this review also discusses recent progress in our understanding of the metabolic fate of hydrophobic compounds within the cell: their terminal oxidation, further degradation or accumulation in the form of intracellular lipid bodies.     

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1delta mutant

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase (meu1delta). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1delta cells. By comparing the results with this mutant with a meu1delta spe2delta mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1+ SPE2+ cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15delta mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.     

Controlling lipid fluxes at glycerol-3-phosphate acyltransferase step in yeast: unique contribution of Gat1p to oleic acid-induced lipid particle formation

The ability to channel excess fatty acids into neutral lipids like triacylglycerol (TAG) is a critical strategy used by cells to maintain lipid homeostasis. Upon activation to acyl-CoA, fatty acids become readily available as substrates for acyltransferases involved in neutral lipid synthesis. Neutral lipids are then packed into organelles derived from the endoplasmic reticulum called lipid particles (LPs). The first acylation step in the de novo pathway for TAG synthesis is catalyzed by glycerol-3-phosphate acyltransferases (GPATs). Two isoforms, Gat1p/Gpt2p and Gat2p/Sct1p, are present in the yeast Saccharomyces cerevisiae. Previous evidence indicated that these enzymes contribute differentially to the synthesis of TAG in actively growing cells. In this work we studied the role of the yeast GPATs in the formation of LPs induced by a surplus of oleic acid. Yeast lacking Gat1p (but not Gat2p) were sensitive to oleate and failed to accumulate LPs induced by this unsaturated fatty acid. It is shown that oleate induces dephosphorylation of Gat1p as well as an increment in its levels. Most importantly, we identified novel Gat1p crescent structures that are formed in the presence of oleate. These structures are connected with the endoplasmic reticulum and are intimately associated with LPs. No such structures were observed for Gat2p. A crucial point of control of lipid fluxes at the GPAT step is proposed.