Genome editing of a rice CDP-DAG synthase confers multipathogen resistance.

The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes1. Here we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis2. Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). In rice, PtdIns(4,5)P2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor3. By using targeted genome editing, we obtained an allele of RBL1, named RBL1Δ12, which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops.

Arabidopsis trigalactosyldiacylglycerol1 mutants reveal a critical role for phosphtidylcholine remodeling in lipid homeostasis.

Lipid remodeling plays a critical role in plant response to abiotic stress and metabolic perturbations. Key steps in this process involve modifications of phosphatidylcholine (PC) acyl chains mediated by lysophosphatidylcholine: acyl-CoA acyltransferases (LPCATs) and phosphatidylcholine: diacylglycerol cholinephosphotransferase (ROD1). To assess their importance in lipid homeostasis, we took advantage of the trigalactosyldiacylglycerol1 (tgd1) mutant that exhibits marked increases in fatty acid synthesis and fatty acid flux through PC due to a block in inter-organelle lipid trafficking. Here, we showed that the increased fatty acid synthesis in tgd1 is due to posttranslational activation of the plastidic acetyl-coenzyme A carboxylase. Genetic analysis showed that knockout of LPCAT1 and 2 resulted in a lethal phenotype in tgd1. In addition, plants homozygous for lpcat2 and heterozygous for lpcat1 in the tgd1 background showed reduced levels of PC and triacylglycerols (TAG) and alterations in their fatty acid profiles. We further showed that disruption of ROD1 in tgd1 resulted in changes in fatty acid composition of PC and TAG, decreased leaf TAG content and reduced seedling growth. Together, our results reveal a critical role of LPCATs and ROD1 in maintaining cellular lipid homeostasis under conditions, in which fatty acid production largely exceeds the cellular demand for membrane lipid synthesis.

CdsA, a CDP-diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons.

CdsA is a CDP-diacylglycerol synthase essential for phospholipid and glycolipid MPIase biosynthesis, and therefore for growth. The initiation codon of CdsA has been assigned as "TTG," while methionine at the 37th codon was reported to be an initiation codon in the original report. Since a vector containing the open reading frame starting with "TTG" under a controllable promoter complemented the cdsA knockout, "TTG" could function as an initiation codon. However, no evidence supporting that this "TTG" is the sole initiation codon has been reported. We determined the initiation codon by examining the ability of mutants around the N-terminal region to complement cdsA mutants. Even if the "TTG" was substituted with a stop codon, the clear complementation was observed. Moreover, the clones with multiple mutations of stop codons complemented the cdsA mutant up to the 37th codon, indicating that cdsA possesses multiple codons that can function as initiation codons. We constructed an experimental system in which the chromosomal expression of cdsA can be analyzed. By means of this system, we found that the cdsA mutant with substitution of "TTG" with a stop codon is fully functional. Thus, we concluded that CdsA contains multiple initiation codons.

CDS2 expression regulates de novo phosphatidic acid synthesis.

CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the 'PI cycle', leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.

Cytidine diphosphate diacylglycerol synthase is essential for mitochondrial structure and energy production in Arabidopsis thaliana.

Cytidine diphosphate diacylglycerol (CDP-DAG), an important intermediate for glycerolipid biosynthesis, is synthesized under the catalytic activity of CDP-DAG synthase (CDS) to produce anionic phosphoglycerolipids such as phosphatidylglycerol (PG) and cardiolipin (CL). Previous studies showed that Arabidopsis CDSs are encoded by a small gene family, termed CDS1-CDS5, the members of which are integral membrane proteins in endoplasmic reticulum (ER) and in plastids. However, the details on how CDP-DAG is provided for mitochondrial membrane-specific phosphoglycerolipids are missing. Here we present the identification of a mitochondrion-specific CDS, designated CDS6. Enzymatic activity of CDS6 was demonstrated by the complementation of CL synthesis in the yeast CDS-deficient tam41Δ mutant. The Arabidopsis cds6 mutant lacking CDS6 activity showed decreased mitochondrial PG and CL biosynthesis capacity, a severe growth deficiency finally leading to plant death. These defects were rescued partly by complementation with CDS6 or supplementation with PG and CL. The ultrastructure of mitochondria in cds6 was abnormal, missing the structures of cristae. The degradation of triacylglycerol (TAG) in lipid droplets and starch in chloroplasts in the cds6 mutant was impaired. The expression of most differentially expressed genes involved in the mitochondrial electron transport chain was upregulated, suggesting an energy-demanding stage in cds6. Furthermore, the contents of polar glycerolipids in cds6 were dramatically altered. In addition, cds6 seedlings lost the capacity for cell proliferation and showed a higher oxidase activity. Thus, CDS6 is indispensable for the biosynthesis of PG and CL in mitochondria, which is critical for establishing mitochondrial structure, TAG degradation, energy production and seedling development.

Expression of Cds4/5 of Arabidopsis chloroplasts in E. coli reveals the membrane topology of the C-terminal region of CDP-diacylglycerol synthases.

CDP-diacylglycerol synthases (Cds) are conserved from bacteria to eukaryotes. Bacterial CdsA is involved not only in phospholipid biosynthesis but also in biosynthesis of glycolipid MPIase, an essential glycolipid that catalyzes membrane protein integration. We found that both Cds4 and Cds5 of Arabidopsis chloroplasts complement cdsA knockout by supporting both phospholipid and MPIase biosyntheses. Comparison of the sequences of CdsA and Cds4/5 suggests a difference in membrane topology at the C-termini, since the region assigned as the last transmembrane region of CdsA, which follows the conserved cytoplasmic domain, is missing in Cds4/5. Deletion of the C-terminal region abolished the function, indicating the importance of the region. Both 6 × His tag attachment to CdsA and substitution of the C-terminal 6 residues with 6 × His did not affect the function. These 6 × His tags were sensitive to protease added from the cytosolic side in vitro, indicating that this region is not a transmembrane one but forms a membrane-embedded reentrant loop. Thus, the C-terminal region of Cds homologues forms a reentrant loop, of which structure is important for the Cds function.

Elevating Phospholipids Production Yarrowia lipolytica from Crude Glycerol.

Phospholipids (PLs) are a class of lipids with many proven biological functions. They are commonly used in lipid replacement therapy to enrich cell membranes damaged in chronic neurodegenerative diseases, cancer, or aging processes. Due to their amphipathic nature, PLs have been widely used in food, cosmetic, and pharmaceutical products as natural emulsifiers and components of liposomes. In Yarrowia lipolytica, PLs are synthesized through a similar pathway like in higher eukaryotes. However, PL biosynthesis in this yeast is still poorly understood. The key intermediate in this pathway is phosphatidic acid, which in Y. lipolytica is mostly directed to the production of triacylglycerols and, in a lower amount, to PL. This study aimed to deliver a strain with improved PL production, with a particular emphasis on increased biosynthesis of phosphatidylcholine (PC). Several genetic modifications were performed: overexpression of genes from PL biosynthesis pathways as well as the deletion of genes responsible for PL degradation. The best performing strain (overexpressing CDP-diacylglycerol synthase (CDS) and phospholipid methyltransferase (OPI3)) reached 360% of PL improvement compared to the wild-type strain in glucose-based medium. With the substitution of glucose by glycerol, a preferred carbon source by Y. lipolytica, an almost 280% improvement of PL was obtained by transformant overexpressing CDS, OPI3, diacylglycerol kinase (DGK1), and glycerol kinase (GUT1) in comparison to the wild-type strain. To further increase the amount of PL, the optimization of culture conditions, followed by the upscaling to a 2 L bioreactor, were performed. Crude glycerol, being a cheap and renewable substrate, was used to reduce the costs of PL production. In this process 653.7 mg/L of PL, including 352.6 mg/L of PC, was obtained. This study proved that Y. lipolytica is an excellent potential producer of phospholipids, especially from waste substrates.

Differential contributions of choline phosphotransferases CPT1 and CEPT1 to the biosynthesis of choline phospholipids.

Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and also serve as a source of several lipid mediators. These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). However, the contributions of these enzymes to the de novo biosynthesis of lipids remain unknown. Here, we established and characterized CPT1- and CEPT1-deficient human embryonic kidney 293 cells. Immunohistochemical analyses revealed that CPT1 localizes to the trans-Golgi network and CEPT1 to the endoplasmic reticulum. Enzyme assays and metabolic labeling with radiolabeled choline demonstrated that loss of CEPT1 dramatically decreases choline PL biosynthesis. Quantitative PCR and reintroduction of CPT1 and CEPT1 revealed that the specific activity of CEPT1 was much higher than that of CPT1. LC-MS/MS analysis of newly synthesized lipid molecular species from deuterium-labeled choline also showed that these enzymes have similar preference for the synthesis of PC molecular species, but that CPT1 had higher preference for 1-alkyl-2-acyl-sn-glycerophosphocholine with PUFA than did CEPT1. The endogenous level of PC was not reduced by the loss of these enzymes. However, several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium. These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles.

CDP-DAG synthase 1 and 2 regulate lipid droplet growth through distinct mechanisms.

Lipid droplets (LDs) are evolutionarily conserved organelles that play critical roles in mammalian lipid storage and metabolism. However, the molecular mechanisms governing the biogenesis and growth of LDs remain poorly understood. Phosphatidic acid (PA) is a precursor of phospholipids and triacylglycerols and substrate of CDP-diacylglycerol (CDP-DAG) synthase 1 (CDS1) and CDS2, which catalyze the formation of CDP-DAG. Here, using siRNA-based gene knockdowns and CRISPR/Cas9-mediated gene knockouts, along with immunological, molecular, and fluorescence microscopy approaches, we examined the role of CDS1 and CDS2 in LD biogenesis and growth. Knockdown of either CDS1 or CDS2 expression resulted in the formation of giant or supersized LDs in cultured mammalian cells. Interestingly, down-regulation of cell death-inducing DFF45-like effector C (CIDEC), encoding a prominent regulator of LD growth in adipocytes, restored LD size in CDS1- but not in CDS2-deficient cells. On the other hand, reducing expression of two enzymes responsible for triacylglycerol synthesis, diacylglycerol O-acyltransferase 2 (DGAT2) and glycerol-3-phosphate acyltransferase 4 (GPAT4), rescued the LD phenotype in CDS2-deficient, but not CDS1-deficient, cells. Moreover, CDS2 deficiency, but not CDS1 deficiency, promoted the LD association of DGAT2 and GPAT4 and impaired initial LD maturation. Finally, although both CDS1 and CDS2 appeared to regulate PA levels on the LD surface, CDS2 had a stronger effect. We conclude that CDS1 and CDS2 regulate LD dynamics through distinct mechanisms.

Expression of a Lychee PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE with an Escherichia coli CYCLOPROPANE SYNTHASE Enhances Cyclopropane Fatty Acid Accumulation in Camelina Seeds.

Cyclopropane fatty acids (CPAs) are useful feedstocks for biofuels and bioproducts such as lubricants and biodiesel. Our goal is to identify factors that can facilitate the accumulation of CPA in seed triacylglycerol (TAG) storage oil. We hypothesized that the poor metabolism of CPA through the TAG biosynthetic network could be overcome by the addition of enzymes from species that naturally accumulate CPA in their seed oil, such as lychee (Litchi chinensis), which contains approximately 40% CPA in TAG. Our previous work on engineering CPA accumulation in crop and model plants identified a metabolic bottleneck between phosphatidylcholine (PC), the site of CPA biosynthesis, diacylglycerol (DAG), and TAG. Here, we report the cloning and heterologous expression in camelina (Camelina sativa) of a lychee PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL CHOLINEPHOSPHOTRANSFERASE (PDCT), which encodes the enzyme that catalyzes the transfer of the phosphocholine headgroup from PC to DAG. Camelina lines coexpressing LcPDCT and Escherichia coli CYCLOPROPANE SYNTHASE (EcCPS) showed up to a 50% increase of CPA in mature seed, relative to the EcCPS background. Stereospecific lipid compositional analysis showed that the expression of LcPDCT strongly reduced the level of C18:1 substrate at PC-sn-1 and PC-sn-2 (i.e. the sites of CPA synthesis), while the levels of CPA increased in PC-sn-2, DAG-sn-1 and DAG-sn-2, and both sn-1/3 and sn-2 positions in TAG. Taken together, these data suggest that the addition of PDCT facilitates more efficient movement of CPA from PC to DAG and establishes LcPDCT as a useful factor to combine with others to enhance CPA accumulation in plant seed oil.

Cytidinediphosphate-diacylglycerol synthase 5 is required for phospholipid homeostasis and is negatively involved in hyperosmotic stress tolerance.

Cytidinediphosphate diacylglycerol synthase (CDS) uses phosphatidic acid (PA) and cytidinetriphosphate to produce cytidinediphosphate-diacylglycerol, an intermediate for phosphatidylglycerol (PG) and phosphatidylinositol (PI) synthesis. This study shows that CDS5, one of the five CDSs of the Oryza sativa (rice) genome, has multifaceted effects on plant growth and stress responses. The loss of CDS5 resulted in a decrease in PG and PI levels, defective thylakoid membranes, pale leaves in seedlings and growth retardation. In addition, the loss of CDS5 led to an elevated PA level and enhanced hyperosmotic tolerance. The inhibition of phospholipase D (PLD)-derived PA formation in cds5 restored the hyperosmotic stress tolerance of the mutant phenotype to that of the wild type, suggesting that CDS5 functions as a suppressor in PLD-derived PA signaling and negatively affects hyperosmotic stress tolerance.

YnbB is a CdsA paralogue dedicated to biosynthesis of glycolipid MPIase involved in membrane protein integration.

MPIase is a glycolipid involved in protein integration in E. coli. Recently, we identified CdsA, a CDP-diacylglycerol (CDP-DAG) synthase, as a biosynthetic enzyme for MPIase. YnbB is a CdsA paralogue with a highly homologous C-terminal half. Under CdsA-depleted conditions, YnbB overproduction restored MPIase expression, but not phospholipid biosynthesis. YnbB complemented the growth defect of the cdsA knockout when Tam41p, a mitochondrial CDP-DAG synthase, was co-expressed, suggesting that YnbB possesses sufficient activity for MPIase biosynthesis, but not for phospholipid biosynthesis. Consistently, a chimera consisting of the CdsA N-terminal half and the YnbB C-terminal half (CdsA-N-YnbB-C) complemented the cdsA knockout by itself, but a chimera consisting of the YnbB N-terminal half and the CdsA C-terminal half (YnbB-N-CdsA-C) required co-expression of Tam41p for the complementation. The biosynthetic rate for CDP-DAG in CdsA and CdsA-N-YnbB-C was much faster than that in YnbB and YnbB-N-CdsA-C, indicating that the N-terminal half of CdsA accelerates CDP-DAG biosynthesis to give the fast cell growth. Therefore, the role of YnbB seems to be as a backup for MPIase biosynthesis, suggesting that YnbB is dedicated to MPIase biosynthesis. A mutant with a high pH-sensitive CdsA8 was unable to grow even under permissive conditions when the ynbB gene was deleted, supporting its auxiliary role in the CdsA function.

Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization.

Site-specific protein functionalization has become an indispensable tool in modern life sciences. Here, tag-based enzymatic protein functionalization techniques are among the most versatilely applicable approaches. However, many chemo-enzymatic functionalization strategies suffer from low substrate scopes of the enzymes utilized for functional labeling probes. We report on the wide substrate scope of the bacterial enzyme AnkX towards derivatized CDP-choline analogues and demonstrate that AnkX-catalyzed phosphocholination can be used for site-specific one- and two-step protein labeling with a broad array of different functionalities, displaying fast second-order transfer rates of 5×102 to 1.8×104 m-1 s-1 . Furthermore, we also present a strategy for the site-specific dual labeling of proteins of interest, based on the exploitation of AnkX and the delabeling function of the enzyme Lem3. Our results contribute to the wide field of protein functionalization, offering an attractive chemo-enzymatic tag-based modification strategy for in vitro labeling.

Two phylogenetically and compartmentally distinct CDP-diacylglycerol synthases cooperate for lipid biogenesis in Toxoplasma gondii.

Toxoplasma gondii is among the most prevalent protozoan parasites, which infects a wide range of organisms, including one-third of the human population. Its rapid intracellular replication within a vacuole requires efficient synthesis of glycerophospholipids. Cytidine diphosphate-diacylglycerol (CDP-DAG) serves as a major precursor for phospholipid synthesis. Given the peculiarities of lipid biogenesis, understanding the mechanism and physiological importance of CDP-DAG synthesis is particularly relevant in T. gondii Here, we report the occurrence of two phylogenetically divergent CDP-DAG synthase (CDS) enzymes in the parasite. The eukaryotic-type TgCDS1 and the prokaryotic-type TgCDS2 reside in the endoplasmic reticulum and apicoplast, respectively. Conditional knockdown of TgCDS1 severely attenuated the parasite growth and resulted in a nearly complete loss of virulence in a mouse model. Moreover, mice infected with the TgCDS1 mutant became fully resistant to challenge infection with a hyper-virulent strain of T. gondii The residual growth of the TgCDS1 mutant was abolished by consecutive deletion of TgCDS2. Lipidomic analyses of the two mutants revealed significant and specific declines in phosphatidylinositol and phosphatidylglycerol levels upon repression of TgCDS1 and after deletion of TgCDS2, respectively. Our data suggest a "division of labor" model of lipid biogenesis in T. gondii in which two discrete CDP-DAG pools produced in the endoplasmic reticulum and apicoplast are subsequently used for the synthesis of phosphatidylinositol in the Golgi bodies and phosphatidylglycerol in the mitochondria. The essential and divergent nature of CDP-DAG synthesis in the parasite apicoplast offers a potential drug target to inhibit the asexual reproduction of T. gondii.

1,2-Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway.

Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.

Mitochondrial CDP-diacylglycerol synthase activity is due to the peripheral protein, TAMM41 and not due to the integral membrane protein, CDP-diacylglycerol synthase 1.

CDP diacylglycerol synthase (CDS) catalyses the conversion of phosphatidic acid (PA) to CDP-diacylglycerol, an essential intermediate in the synthesis of phosphatidylglycerol, cardiolipin and phosphatidylinositol (PI). CDS activity has been identified in mitochondria and endoplasmic reticulum of mammalian cells apparently encoded by two highly-related genes, CDS1 and CDS2. Cardiolipin is exclusively synthesised in mitochondria and recent studies in cardiomyocytes suggest that the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1α and ß) serve as transcriptional regulators of mitochondrial biogenesis and up-regulate the transcription of the CDS1 gene. Here we have examined whether CDS1 is responsible for the mitochondrial CDS activity. We report that differentiation of H9c2 cells with retinoic acid towards cardiomyocytes is accompanied by increased expression of mitochondrial proteins, oxygen consumption, and expression of the PA/PI binding protein, PITPNC1, and CDS1 immunoreactivity. Both CDS1 immunoreactivity and CDS activity were found in mitochondria of H9c2 cells as well as in rat heart, liver and brain mitochondria. However, the CDS1 immunoreactivity was traced to a peripheral p55 cross-reactive mitochondrial protein and the mitochondrial CDS activity was due to a peripheral mitochondrial protein, TAMM41, not an integral membrane protein as expected for CDS1. TAMM41 is the mammalian equivalent of the recently identified yeast protein, Tam41. Knockdown of TAMM41 resulted in decreased mitochondrial CDS activity, decreased cardiolipin levels and a decrease in oxygen consumption. We conclude that the CDS activity present in mitochondria is mainly due to TAMM41, which is required for normal mitochondrial function.

Functional assessment of plant and microalgal lipid pathway genes in yeast to enhance microbial industrial oil production.

As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks.

Lipid droplet growth and adipocyte development: mechanistically distinct processes connected by phospholipids.

The differentiation of preadipocytes into mature adipocytes is accompanied by the growth and formation of a giant, unilocular lipid droplet (LD). Mechanistically however, LD growth and adipogenesis are two different processes. Recent studies have uncovered a number of proteins that are able to regulate both LD dynamics and adipogenesis, such as SEIPIN, LIPIN and CDP-Diacylglycerol Synthases. It appears that phospholipids, phosphatidic acid in particular, play a critical role in both LD budding/growth and adipocyte development. This review summarizes recent advances, and aims to provide a better understanding of LD growth as well as adipogenesis, two critical aspects in mammalian fat storage. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.

Features of the Phosphatidylinositol Cycle and its Role in Signal Transduction.

The phosphatidylinositol cycle (PI-cycle) has a central role in cell signaling. It is the major pathway for the synthesis of phosphatidylinositol and its phosphorylated forms. In addition, some lipid intermediates of the PI-cycle, including diacylglycerol and phosphatidic acid, are also important lipid signaling agents. The PI-cycle has some features that are important for the understanding of its role in the cell. As a cycle, the intermediates will be regenerated. The PI-cycle requires a large amount of metabolic energy. There are different steps of the cycle that occur in two different membranes, the plasma membrane and the endoplasmic reticulum. In order to complete the PI-cycle lipid must be transferred between the two membranes. The role of the Nir proteins in the process has recently been elucidated. The lipid intermediates of the PI-cycle are normally highly enriched with 1-stearoyl-2-arachidonoyl molecular species in mammals. This enrichment will be retained as long as the intermediates are segregated from other lipids of the cell. However, there is a significant fraction (>15 %) of lipids in the PI-cycle of normal cells that have other acyl chains. Phosphatidylinositol largely devoid of arachidonoyl chains are found in cancer cells. Phosphatidylinositol species with less unsaturation will not be as readily converted to phosphatidylinositol-3,4,5-trisphosphate, the lipid required for the activation of Akt with resulting effects on cell proliferation. Thus, the cyclical nature of the PI-cycle, its dependence on acyl chain composition and its requirement for lipid transfer between two membranes, explain many of the biological properties of this cycle.

Modulation of Rab GTPase function by a protein phosphocholine transferase.

The intracellular pathogen Legionella pneumophila modulates the activity of host GTPases to direct the transport and assembly of the membrane-bound compartment in which it resides. In vitro studies have indicated that the Legionella protein DrrA post-translationally modifies the GTPase Rab1 by a process called AMPylation. Here we used mass spectrometry to investigate post-translational modifications to Rab1 that occur during infection of host cells by Legionella. Consistent with in vitro studies, DrrA-mediated AMPylation of a conserved tyrosine residue in the switch II region of Rab1 was detected during infection. In addition, a modification to an adjacent serine residue in Rab1 was discovered, which was independent of DrrA. The Legionella effector protein AnkX was required for this modification. Biochemical studies determined that AnkX directly mediates the covalent attachment of a phosphocholine moiety to Rab1. This phosphocholine transferase activity used CDP-choline as a substrate and required a conserved histidine residue located in the FIC domain of the AnkX protein. During infection, AnkX modified both Rab1 and Rab35, which explains how this protein modulates membrane transport through both the endocytic and exocytic pathways of the host cell. Thus, phosphocholination of Rab GTPases represents a mechanism by which bacterial FIC-domain-containing proteins can alter host-cell functions.