Phosphatidic Acid Biosynthesis Research Articles

High-level extracellular expression of phospholipase D by combinatorial fine-tuning strategy in Bacillus subtilis for efficient biosynthesis of phosphatidic acid

Although Bacillus subtilis shows promise as a potential microbial cell factory for phospholipase D (PLD) expression, its production capacity remains insufficient. In this study, a secretory expression system, by co-optimization the promoter and signal peptides and employing a fed-batch fermentation strategy, was constructed to enhance expression of PLD from separate sources. The highest PLD production of 4056.9 U/mL was observed using this system, with a PLD production efficiency of 52.0 U/mL/h. Finally, a phosphatidic acid (PA) biosynthesis system was established using the constructed PLD as a catalyst, which achieved a PA yield of 219.1 g/L. This is the highest PLD production and PA yield reported globally to date. The protocol has significant potential for application for industrial PLD production as well as enzymatic phospholipids modification and also provides a valuable reference for overexpressing proteins in B. subtilis.

Fine-tuning phosphatidic acid production for optimal plant stress responses

Phosphatidic acid (PA) is involved in biotic and abiotic stress responses in plants. Here, we summarize quantitative lipidomics and real-time imaging used in PA studies and highlight recent studies of diacylglycerol (DAG) kinase (DGK) 5, an enzyme involved in PA biosynthesis, facilitating fine-tuning PA production for optimal stress responses in plants.

Discovery of Salifungin as a Repurposed Antibiotic against Methicillin-Resistant Staphylococcus aureus with Limited Resistance Development.

Exploring novel antimicrobial drugs and strategies has become essential to the fight MRSA-associated infections. Herein, we found that membrane-disrupted repurposed antibiotic salifungin had excellent bactericidal activity against MRSA, with limited development of drug resistance. Furthermore, adding salifungin effectively decreased the minimum inhibitory concentrations of clinical antibiotics against Staphylococcus aureus. Evaluations of the mechanism demonstrated that salifungin disrupted the level of H+ and K+ ions using hydrophilic and lipophilic groups to interact with bacterial membranes, causing the disruption of bacterial proton motive force followed by impacting on bacterial the function of the respiratory chain and adenosine 5'-triphosphate, thereby inhibiting phosphatidic acid biosynthesis. Moreover, salifungin also significantly inhibited the formation of bacterial biofilms and eliminated established bacterial biofilms by interfering with bacterial membrane potential and inhibiting biofilm-associated gene expression, which was even better than clinical antibiotics. Finally, salifungin exhibited efficacy comparable to or even better than that of vancomycin in the MRSA-infected animal models. In conclusion, these results indicate that salifungin can be a potential drug for treating MRSA-associated infections.

Carbon dioxide treatment modulates phosphatidic acid signaling and stress response to improve chilling tolerance and postharvest quality in paprika

IntroductionPaprika (Capsicum annuum L.) is prone to chilling injury (CI) during low-temperature storage. Although recent findings suggest that CO2 treatment may protect against CI, the effects of short-term CO2 treatment on CI and the underlying molecular mechanisms in paprika remain unknown. Therefore, this study aimed to examine the effect of short-term CO2 treatment on CI and postharvest quality in paprika during storage at cold storage and retail condition at physio-biochemical-molecular level.MethodsPaprika was treated with 20 and 30% CO2 for 3 h and stored at 4°C for 14 days, followed by additional storage for 2 days at 20°C (retail condition). Fruit quality parameters, including weight loss, firmness, color, and pitting were assessed, and the molecular mechanism of the treatment was elucidated using transcriptomic and metabolomic analyses.ResultsShort-term treatment with 20 and 30% CO2 effectively maintained paprika quality during cold storage and retailer conditions, with reduced surface pitting, a common symptom of CI. Additionally, transcriptomic and metabolomic analyses revealed that 20% CO2 treatment induced genes associated with biosynthesis of phosphatidic acid (PA), diacylglycerol, triacylglycerol, and stress response, metabolites associated with phasphatidyl inositol signaling, inositol phosphate metabolism, and starch and sucrose metabolism.ConclusionCO2 treatment activates PA biosynthesis through PLD and PLC-DGK pathways, and induces inositol phosphate, starch, and sucrose metabolism, thereby regulating chilling stress response via the ICE-CBF pathway. These findings suggest that short-term CO2 treatment enhances resistance to cold-induced injury and preserves postharvest quality in non-climacteric fruits, such as paprika, through activation of PA signaling, which improves membrane stability during cold storage and distribution.

Membrane lipid metabolism is involved in regulating γ‐aminobutyric acid‐mediated cold tolerance in peach fruit

AbstractThe cold‐sensitive peach fruits are prone to chilling injury (CI). γ‐aminobutyric acid (GABA) has been confirmed to effectively improve peaches’ cold tolerance. This research aimed to probe the role of dynamic changes in membrane lipid metabolism in GABA‐mediated peaches’ cold tolerance and reveal the underlying mechanisms based on lipid metabolomics. GABA treatment improved peaches’ cold tolerance by reducing phosphatidic acid (PA) accumulation via inhibiting phospholipid degradation, attenuating PA biosynthesis, and maintaining higher phosphatidylcholine (PC) and phosphatidylethanolamine (PE) levels. Under chilling conditions, GABA treatment protected peaches against low‐temperature stress and delayed CI by maintaining higher PC and PE levels and enhancing the massive accumulation of diacylglycerol (DAG) and triacylglycerol (TAG). This study provides a novel mechanism regulating cold tolerance of postharvest peach fruits.

Recent insights into cell responses to cold stress in plants: Signaling, defence, and potential functions of phosphatidic acid

Upon exposure to cold stress (CS), crop plants face an array of detrimental effects on growth, development, and final yield. Seed germination, seedling emergence, leaf number, root morphology, photosynthetic activity and metabolism are all disrupted by CS. Plants have evolved various mechanisms to avoid or induce cold tolerance from the cellular level to the whole plant level. Phosphatidic acid (PA) is considered a prime signaling phospholipid in plant membranes that plays an important role in signal transduction in stress avoidance responses. Recent research studies have advanced our understanding of the functions of PA in plant response to abiotic stress. As a second messenger, PA can bind to target proteins to enhance plant cold response and adaption. However, there is a paucity of information on PA-mediated alterations in lipid metabolism as a plant cold tolerance mechanism. Firstly, this review presents plant response to CS at morphological, physiological, biochemical, and molecular levels. The authors also discuss the roles of protective proteins, osmo-protectants, antioxidant systems and lipid metabolisms in plant cold tolerance mechanisms. Furthermore, the current review documents state of the art literature on the effects of CS on plant PA biosynthesis pathways (PLD and PLC-DGK pathways) in plants and its promising role in improving cold tolerance. Finally, we summarized the PA signaling in plant cold tolerance and suggested research directions in CS studies in the future. • Cold stress (CS) significantly reduces seedling emergence, photosynthetic rate, plant biomass and enhances ROS accumulation. • Plants increase cold tolerance by triggering COR genes and producing protective proteins, osmo-protectants, and antioxidants. • Lipid remodeling is a key strategy to maintain membrane stability and functions in plants under CS. • Phosphatidic acid (PA) is an emerging phospholipid that plays an essential role in lipid metabolism and CS response. • Under CS conditions, PA accumulates rapidly through Phospholipase D and Phospholipase C – Diacylglycerol kinase pathways. • PA can bind to target proteins to enhance plant CS response and adaptation.

Physiological and proteomics analyses reveal the resistance response mechanism to alkali stress in the early seedlings (cotyledons vs. roots) of castor plant (Ricinus communis L.)

Soil alkalinity is a major environmental problem influencing plant growth and productivity in northeastern China. Castor plant (Ricinus communis) is one of the most important oilseed crops worldwide, and has good salt tolerance. The early seedling stage is extremely vulnerable to abiotic stresses, but little is known about the physiological and molecular mechanisms involved in the cotyledons and roots of castor seedlings under alkali stress. In this study, biomass, photosynthesis, root vitality, inorganic ions, organic solutes and antioxidant enzyme activities were measured. Two-dimensional gel electrophoresis-based proteomic approaches were also applied to identify differentially abundant proteins in alkali-treated seedlings. The results showed that alkali stress strongly inhibited seedling growth, especially of the roots. Na+ content increased with increasing alkalinity, and the roots had much higher Na+ and lower K+ than cotyledons. The proline and soluble sugars were both increased in cotyledons, but kept unchanged in the roots under alkali stress. Moreover, proteomic analysis revealed a total of 15 and 25 alkali-responsive proteins in the cotyledons and roots, respectively. For cotyledons, most of the identified proteins were involved in photosynthesis, stress and defense (＞10 %), while those for roots were mainly involved in carbohydrate and energy metabolism, genetic information, stress and defense (＞10 %). These results suggest that cotyledons and roots respond differently to alkali stress. Cotyledons retain efficiency of photosynthetic electron transport and accumulation of osmolytes, while the roots enhance transfer efficiency of the mitochondrial electron transport chain, adjust fatty acid biosynthesis, and promote phosphatidic acid biosynthesis under alkali stress. Additionally, the co-increased abundance of chaperonins indicated that both cotyledons and roots maintained folding of proteins into their proper 3D structures in response to alkali stress. These findings provide new insights into different physiological mechanisms in cotyledon and root responses to alkali stress during the early seedling stage in castor.

Lipid Signaling through G Proteins.

N-Acylethanolamine (NAE) signaling has received considerable attention in vertebrates as part of the endocannabinoid signaling system, where anandamide acts as a ligand for G protein-coupled cannabinoid receptors. Recent studies indicate that G proteins also are required for some types of NAE signaling in plants. The genetic ablation of the Gβγ dimer or loss of the full set of extra-large G proteins strongly attenuated NAE-induced chloroplast responses in seedlings. Intriguing parallels and distinct differences have emerged between plants and animals in NAE signaling, despite the conserved use of these lipid mediators to modulate cellular processes. Here we compare similarities and differences and identify open questions in a fundamental lipid signaling pathway in eukaryotes with components that are both conserved and diverged in plants.

Metabolic engineering for glycoglycerolipids production in E. coli: Tuning phosphatidic acid and UDP-glucose pathways

Glycolipids are target molecules in biotechnology and biomedicine as biosurfactants, biomaterials and bioactive molecules. An engineered E. coli strain for the production of glycoglycerolipids (GGL) used the MG517 glycolipid synthase from M. genitalium for glucosyl transfer from UDPGlc to diacylglycerol acceptor (Mora-Buyé et al., 2012). The intracellular diacylglycerol pool proved to be the limiting factor for GGL production. Here we designed different metabolic engineering strategies to enhance the availability of precursor substrates for the glycolipid synthase by modulating fatty acids, acyl donor and phosphatidic acid biosynthesis. Knockouts of tesA, fadE and fabR genes involved in fatty acids degradation, overexpression of the transcriptional regulator FadR, the acyltransferases PlsB and C, and the pyrophosphatase Cdh for phosphatidic acid biosynthesis, as well as the phosphatase PgpB for conversion to diacylglycerol were explored with the aim of improving GGL titers. Among the different engineered strains, the ΔtesA strain co-expressing MG517 and a fusion PlsCxPgpB protein was the best producer, with a 350% increase of GGL titer compared to the parental strain expressing MG517 alone. Attempts to boost UDPGlc availability by overexpressing the uridyltransferase GalU or knocking out the UDP-sugar diphosphatase encoding gene ushA did not further improve GGL titers. Most of the strains produced GGL containing a variable number of glucosyl units from mono-to tetra-saccharides. Interestingly, the strains co-expressing Cdh showed a shift in the GGL profile towards the diglucosylated lipid (up to 80% of total GGLs) whereas the strains with a fadR knockout presented a higher amount of unsaturated acyl chains. In all cases, GGL production altered the lipidic composition of the E. coli membrane, observing that GGL replace phosphatidylethanolamine to maintain the overall membrane charge balance.

Destruction of the cell membrane and inhibition of cell phosphatidic acid biosynthesis in Staphylococcus aureus: an explanation for the antibacterial mechanism of morusin.

Morusin is a prenylated flavonoid found in mulberry that shows antimicrobial activity against foodborne pathogens. The MIC values of morusin toward S. aureus ATCC 6538 and S. aureus ATCC 25923 were both 14.9 μmol L-1. This study further investigated the antimicrobial mechanism of morusin in inhibiting the growth of Staphylococcus aureus ATCC 6538. Scanning electron microscopy and transmission electron microscopy revealed that morusin disrupted the integrity of the bacterial cell membrane. Morusin may also affect the phospholipid-repair system of bacteria, which repairs membrane structures. To test this hypothesis, quantitative real-time PCR was used to examine the effect of morusin treatment of S. aureus on the regulation of genes associated with the cell phosphatidic acid biosynthesis pathway. Gas chromatography-mass spectrometry was used to investigate the fatty acid components, which are used to synthesize bacterial phosphatidic acids. In summary, the results revealed that morusin showed a potent antibacterial effect by disrupting the cell membrane architecture and inhibiting the phosphatidic acid biosynthesis pathway of S. aureus.

Location, location, location: lipid metabolism varies in different parts of the seed.

Location, location, location: lipid metabolism varies in different parts of the seed.

Calcium deprivation enhances non-selective fluid-phase endocytosis and modifies membrane lipid profiles in Arabidopsis roots

Ecological studies have revealed significant decreases in calcium (Ca) levels in various soils, and widely occurring physiological Ca deficits worldwide. These changes may cause decreases in plant diversity and increases in plant vulnerability to environmental stress, but the underlying cellular mechanism is not well understood. In this study, we found that in Arabidopsis thaliana roots, deprivation of Ca2+, but not other minerals, dramatically enhanced plasma membrane invagination, endosome formation, and trafficking to the vacuole through the trans-Golgi network and pre-vacuole compartment, a typical pathway of endocytosis. Antagonist and cellular tracing analyses using non-bioactive, membrane-impermeable fluorescent probes indicated that this type of endocytosis is not regulated by receptors, instead representing a non-selective, non-specific fluid phase-based process. We performed lipid-profiling analysis of roots in response to Ca2+ deprivation, finding increased phosphatidylcholine (PC), Lyso-PC, phosphatidylethanolamine (PE), Lyso-PE, phosphatidylinositol (PI) and triacylglycerols (TAG) biosynthesis but deceased phosphatidic acid (PA) and diacylglycerols (DAG) biosynthesis. The increased TAG contents and molar ratio of PC/PE in these roots might reflect a cellular response to maintain membrane stability and the balance between the membrane and storage lipids. This study demonstrates the essential role of Ca in maintaining plasma membrane stability and the selectivity of plant root cells, and it highlights the potential deleterious effect of decreased Ca levels in surface soil on plant growth.

Draft genome sequence of Actinotignum schaalii DSM 15541T: Genetic insights into the lifestyle, cell fitness and virulence.

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Overexpression of AtGolS3 and CsRFS in poplar enhances ROS tolerance and represses defense response to leaf rust disease.

Plants respond to pathogens through an orchestration of signaling events that coordinate modifications to transcriptional profiles and physiological processes. Resistance to necrotrophic pathogens often requires jasmonic acid, which antagonizes the salicylic acid dependent biotrophic defense response. Recently, myo-inositol has been shown to negatively impact salicylic acid (SA) levels and signaling, while galactinol enhances jasmonic acid (JA)-dependent induced systemic resistance to necrotrophic pathogens. To investigate the function of these compounds in biotrophic pathogen defense, we characterized the defense response of Populus alba × grandidentata overexpressing Arabidopsis GALACTINOL SYNTHASE3 (AtGolS) and Cucumber sativus RAFFINOSE SYNTHASE (CsRFS) challenged with Melampsora aecidiodes, a causative agent of poplar leaf rust disease. Relative to wild-type leaves, the overexpression of AtGolS3 and CsRFS increased accumulation of galactinol and raffinose and led to increased leaf rust infection. During the resistance response, inoculated wild-type leaves displayed reduced levels of galactinol and repressed transcript abundance of two endogenous GolS genes compared to un-inoculated wild-type leaves prior to the up-regulation of NON-EXPRESSOR OF PR1 and PATHOGENESIS-RELATED1. Transcriptome analysis and qRT-PCR validation also revealed the repression of genes participating in calcium influx, phosphatidic acid biosynthesis and signaling, and salicylic acid signaling in the transgenic lines. In contrast, enhanced tolerance to H2O2 and up-regulation of antioxidant biosynthesis genes were exhibited in the overexpression lines. Thus, we conclude that overexpression of AtGolS and CsRFS antagonizes the defense response to poplar leaf rust disease through repressing reactive oxygen species and attenuating calcium and phosphatidic acid signaling events that lead to SA defense.

Diacylglycerol Kinases Are Widespread in Higher Plants and Display Inducible Gene Expression in Response to Beneficial Elements, Metal, and Metalloid Ions.

Diacylglycerol kinases (DGKs) are pivotal signaling enzymes that phosphorylate diacylglycerol (DAG) to yield phosphatidic acid (PA). The biosynthesis of PA from phospholipase D (PLD) and the coupled phospholipase C (PLC)/DGK route is a crucial signaling process in eukaryotic cells. Next to PLD, the PLC/DGK pathway is the second most important generator of PA in response to biotic and abiotic stresses. In eukaryotic cells, DGK, DAG, and PA are implicated in vital processes such as growth, development, and responses to environmental cues. A plethora of DGK isoforms have been identified so far, making this a rather large family of enzymes in plants. Herein we performed a comprehensive phylogenetic analysis of DGK isoforms in model and crop plants in order to gain insight into the evolution of higher plant DGKs. Furthermore, we explored the expression profiling data available in public data bases concerning the regulation of plant DGK genes in response to beneficial elements and other metal and metalloid ions, including silver (Ag), aluminum (Al), arsenic (As), cadmium (Cd), chromium (Cr), mercury (Hg), and sodium (Na). In all plant genomes explored, we were able to find DGK representatives, though in different numbers. The phylogenetic analysis revealed that these enzymes fall into three major clusters, whose distribution depends on the composition of structural domains. The catalytic domain conserves the consensus sequence GXGXXG/A where ATP binds. The expression profiling data demonstrated that DGK genes are rapidly but transiently regulated in response to certain concentrations and time exposures of beneficial elements and other ions in different plant tissues analyzed, suggesting that DGKs may mediate signals triggered by these elements. Though this evidence is conclusive, further signaling cascades that such elements may stimulate during hormesis, involving the phosphoinositide signaling pathway and DGK genes and enzymes, remain to be elucidated.

Metabolomic shifts in Brassica napus lines with enhanced BnPLC2 expression impact their response to low temperature stress and plant pathogens

Phosphatidylinositol-specific phospholipase C2 (PLC2) is a signaling enzyme with hydrolytic activity against membrane-bound phosphoinositides. It catalyzes the cleavage of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) into two initial second messengers, myo-inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). The former, as well as its fully phosphorylated derivative, myo-inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), play a major role in calcium signaling events within the cell, while DAG may be used in the regeneration of phospholipids or as a precursor for phosphatidic acid (PA) biosynthesis, an important signaling molecule involved in both biotic and abiotic types of stress tolerance. Overexpression of the gene for Brassica napus phospholipase C2 (BnPLC2) in Brassica napus has been shown to enhance drought tolerance, modulate multiple genes involved in different processes and favorably affect hormonal levels in different tissues. We, therefore, undertook the current study with a view to examining, at the metabolome level, its effect on both abiotic (low temperature) and biotic (stem white rot disease) types of stress in canola. Thus, while transgenic plants exhibited a significant rise in maltose levels and a concomitant elevation in some unsaturated free fatty acids (FFAs), glycerol, and glycerol 3-phosphate under subzero temperatures, they accumulated high levels of raffinose, stachyose and other sugars as well as some flavonoids under acclimatization conditions. Collectively, overexpression of BnPLC2 appears to have triggered different metabolite patterns consistent with its abiotic and, to a limited extent, biotic stress tolerance phenotypes.

Phosphatidic acid is required for the constitutive ruffling and macropinocytosis of phagocytes

Macrophages and dendritic cells continuously survey their environment in search of foreign particles and soluble antigens. Such surveillance involves the ongoing extension of actin-rich protrusions and the consequent formation of phagosomes and macropinosomes. The signals inducing this constitutive cytoskeletal remodeling have not been defined. We report that, unlike nonphagocytic cells, macrophages and immature dendritic cells have elevated levels of phosphatidic acid (PA) in their plasma membrane. The plasmalemmal PA is synthesized by phosphorylation of diacylglycerol, which is in turn generated by a G protein-stimulated phospholipase C. Inhibition of diacylglycerol kinase activity results in the detachment of T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1)-a Rac guanine exchange factor-from the plasma membrane, thereby depressing Rac activity and abolishing the constitutive ruffling and macropinocytosis that characterize macrophages and immature dendritic cells. Accumulation of PA and binding of TIAM1 to the membrane require the activity of phosphatidylinositol-4,5-bisphosphate 3-kinase. Thus a distinctive, constitutive pathway of PA biosynthesis promotes the actin remodeling required for immune surveillance.

Effect of haloxyfop and cerulenin on de novo biosynthesis of lipids in roots of wheat and maize.

The study examines the effects of haloxyfop (herbicide) and cerulenin (antibiotic) on de novo biosynthesis of fatty acids and complex lipids in roots of two sensitive species: wheat and maize. Seedlings were grown in hydroponic cultures with addition of [1-(14)C]acetate (control) and [1-(14)C]acetate together with one of the tested substances. Neither haloxyfop nor cerulenin prevented the uptake of [1-(14)C]acetate by the roots of tested species. In contrast, a strong inhibition of de novo biosynthesis of fatty acids was observed after a 4-h treatment. This phenomenon, however, tended to disappear with treatment time. After a 24-h incubation, the amount of radioactivity in de novo biosynthesized fatty acids in 1-cm-long root tips was up to three times higher than in the untreated control. In the "rest of roots", restoration of fatty acid biosynthesis capacity was less pronounced. Besides the effect on fatty acid biosynthesis, both tested inhibitors strongly suppressed the de novo biosynthesis of non-fatty acid-containing lipids. Analyses of radioactivity in individual lipid classes showed that after a 4-h treatment with haloxyfop or cerulenin the biosynthesis of most of the lipid classes was inhibited, although not to the same extent. After a 24-h treatment, an inhibition of de novo biosynthesis of some of the lipids was still observable, whereas the biosynthesis of others, especially phosphatidylethanolamine and phosphatidic acid, was strongly up-regulated. Contrary to the mainstream view that inhibition of fatty acid biosynthesis is the cause of haloxyfop and cerulenin phytotoxicity, the obtained results suggest multidirectional effects of both inhibitors.

Topology of the microsomal glycerol‐3‐phosphate acyltransferase Gpt2p/Gat1p of Saccharomyces cerevisiae

All glycerophospholipids are made from phosphatidic acid, which, according to the traditional view, is generated at the cytosolic surface of the ER. In yeast, phosphatidic acid is synthesized de novo by two acyl-CoA-dependent acylation reactions. The first is catalysed by one of the two homologous glycerol-3-phosphate acyltransferases Gpt2p/Gat1p and Sct1p/Gat2p, the second by one of the two 1-acyl-sn-glycerol-3-phosphate acyltransferases Slc1p and Ale1p/Slc4p. To study the biogenesis and topology of Gpt2p we observed the location of dual topology reporters inserted after various transmembrane helices. Moreover, using microsomes, we probed the accessibility of natural and substituted cysteine residues to a membrane impermeant alkylating agent and tested the protease sensitivity of various epitope tags inserted into Gpt2p. Finally, we assayed the sensitivity of the acyltransferase activity to membrane impermeant agents targeting lysine residues. By all these criteria we find that the most conserved motifs of Gpt2p and its functionally relevant lysines are oriented towards the ER lumen. Thus, the first step in biosynthesis of phosphatidic acid in yeast seems to occur in the ER lumen and substrates may have to cross the ER membrane.

Stoichiometric network reconstruction and analysis of yeast sphingolipid metabolism incorporating different states of hydroxylation

The first elaborate metabolic model of Saccharomyces cerevisiae sphingolipid metabolism was reconstructed in silico. The model considers five different states of sphingolipid hydroxylation, rendering it unique among other models. It is aimed to clarify the significance of hydroxylation on sphingolipids and hence to interpret the preferences of the cell between different metabolic pathway branches under different stress conditions. The newly constructed model was validated by single, double and triple gene deletions with experimentally verified phenotypes. Calcium sensitivity and deletion mutations that may suppress calcium sensitivity were examined by CSG1 and CSG2 related deletions. The model enabled the analysis of complex sphingolipid content of the plasma membrane coupled with diacylglycerol and phosphatidic acid biosynthesis and ATP consumption in in silico cell. The flux data belonging to these critically important key metabolites are integrated with the fact of phytoceramide induced cell death to propose novel potential drug targets for cancer therapeutics. In conclusion, we propose that IPT1, GDA1, CSG and AUR1 gene deletions may be novel candidates of drug targets for cancer therapy according to the results of flux balance and variability analyses coupled with robustness analysis.