Phosphatidylcholine Biosynthesis Research Articles

Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis

The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.

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.

Membrane glycerolipid equilibrium under endoplasmic reticulum stress in Arabidopsis thaliana

Endoplasmic reticulum (ER) is an indispensable organelle for secretory protein synthesis as well as metabolism of phospholipids and their derivatives in eukaryotic cells. Various external and internal factors may cause an accumulation of aberrant proteins in the ER, which causes ER stress and activates cellular ER stress responses to cope with the stress. In animal research, molecular mechanisms for protein quality control upon ER stress are well documented; however, how cells maintain lipid homeostasis under ER stress is an emerging issue. The ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE), two major phospholipid classes, is important under ER stress in animal cells. However, in seed plants, no study has reported on the changes in membrane lipid content under ER stress, although a number of physiologically important environmental stresses, such as heat and salinity, induce ER stress. Here, we investigated membrane glycerolipid metabolism under ER stress in Arabidopsis. ER stress transcriptionally affected PC and PE biosynthesis pathways differentially, with no significant changes in membrane glycerolipid content. Our results suggest that higher plants maintain membrane lipid equilibrium during active transcription of phospholipid biosynthetic genes under ER stress.

Evolution of the Phosphatidylcholine Biosynthesis Pathways in Green Algae: Combinatorial Diversity of Methyltransferases.

Phosphatidylcholine (PC) is one of the most common phospholipids in eukaryotes, although some green algae such as Chlamydomonas reinhardtii are known to lack PC. Recently, we detected PC in four species in the genus Chlamydomonas: C. applanata NIES-2202, C. asymmetrica NIES-2207, C. debaryana NIES-2212, and C. sphaeroides NIES-2242. To reveal the PC biosynthesis pathways in green algae and the evolutionary scenario involved in their diversity, we analyzed the PC biosynthesis genes in these four algae using draft genome sequences. Homology searches suggested that PC in these species is synthesized by phosphoethanolamine-N-methyltransferase (PEAMT) and/or phosphatidylethanolamine-N-methyltransferase (PEMT), both of which are absent in C. reinhardtii. Recombinant PEAMTs from these algae showed methyltransferase activity for phosphoethanolamine but not for monomethyl phosphoethanolamine in vitro, in contrast to land plant PEAMT, which catalyzes the three methylations from phosphoethanolamine to phosphocholine. This suggested an involvement of other methyltransferases in PC biosynthesis. Here, we characterized the putative phospholipid-N-methyltransferase (PLMT) genes of these species by genetic and phylogenetic analysis. Complementation assays using a PC biosynthesis-deficient yeast suggested that the PLMTs of these algae can synthesize PC from phosphatidylethanolamine. These results indicated that the PC biosynthesis pathways in green algae differ from those of land plants, although the enzymes involved are homologous. Phylogenetic analysis suggested that the PEAMTs and PLMTs in these algae were inherited from the common ancestor of green algae. The absence of PC biosynthesis in many Chlamydomonas species is likely a result of parallel losses of PEAMT and PLMT in this genus.

Diverse effect of phosphatidylcholine biosynthetic genes on phospholipid homeostasis, cell autophagy and fungal developments in Metarhizium robertsii.

Phosphatidylcholine (PC) plays an important role in maintaining membrane integrity and functionality. In this study, two key genes (Mrpct and Mrpem) putatively involved in the cytidine diphosphate (CDP)-choline and phosphatidylethanolamine N-methyltransferase (PEMT) pathways for PC biosynthesis were characterized in the insect pathogenic fungus Metarhizium robertsii. The results indicated that disruption of Mrpct did not lead to any reduction of total PC content but impaired fungal virulence and increased cellular accumulation of triacylglycerol. Deletion of Mrpem reduced PC content and impaired fungal conidiation and infection structure differentiation but did not result in virulence defects. Lipidomic analysis revealed that deletion of Mrpct and Mrpem resulted in dissimilar effects on increase and decrease of PC moieties and other phospholipid species accumulations. Interestingly, we found that these two genes played opposite roles in activation of cell autophagy when the fungi were grown in a nutrient-rich medium. The connection between PC metabolism and autophagy was confirmed because PC content was drastically reduced in Mratg8Δ and that the addition of PC could rescue null mutant sporulation defect. The results of this study facilitate the understanding of PC metabolism on fungal physiology.

Serum Metabolic Profile Alteration Reveals Response to Platinum-Based Combination Chemotherapy for Lung Cancer: Sensitive Patients Distinguished from Insensitive ones

Most lung cancers are diagnosed at fairly advanced stages due to limited clinical symptoms. Platinum-based chemotherapy, either as single regimen or in combination with radiation, is one of the major recommendations for the patients. Earlier evaluation of the effectiveness of the chemotherapies is critical for developing better treatment plan given the toxicity of the chemotherapeutic reagents. Drug efficacy could be reflected in the systemic metabolism characteristics though knowledge about which remains scarce. In this study, serum metabolism influence of three types of commonly used platinum-based combination chemotherapy regimens, namely cisplatin with gemcitabine, vinorelbine or docetaxel, were studied using pattern recognition coupled with nuclear magnetic resonance techniques. The treated patients were divided into sensitive or insensitive subgroups according to their response to the treatments. We found that insensitive subjects can be identified from the sensitive ones with up-regulation of glucose and taurine but reduced alanine and lactate concentrations in serum. The combination chemotherapy of lung cancer is accompanied by disturbances of multiple metabolic pathways such as energy metabolism, phosphatidylcholine biosynthesis, so that the treated patients were marginally discriminated from the untreated. Serum metabolic profile of patients shows potential as an indicator of their response to platinum-based combination chemotherapy.

Lysophosphatidylcholine Regulates Sexual Stage Differentiation in the Human Malaria Parasite Plasmodium falciparum

SummaryTransmission represents a population bottleneck in the Plasmodium life cycle and a key intervention target of ongoing efforts to eradicate malaria. Sexual differentiation is essential for this process, as only sexual parasites, called gametocytes, are infective to the mosquito vector. Gametocyte production rates vary depending on environmental conditions, but external stimuli remain obscure. Here, we show that the host-derived lipid lysophosphatidylcholine (LysoPC) controls P. falciparum cell fate by repressing parasite sexual differentiation. We demonstrate that exogenous LysoPC drives biosynthesis of the essential membrane component phosphatidylcholine. LysoPC restriction induces a compensatory response, linking parasite metabolism to the activation of sexual-stage-specific transcription and gametocyte formation. Our results reveal that malaria parasites can sense and process host-derived physiological signals to regulate differentiation. These data close a critical knowledge gap in parasite biology and introduce a major component of the sexual differentiation pathway in Plasmodium that may provide new approaches for blocking malaria transmission.

Ex Uno Plura: Differential Labeling of Phospholipid Biosynthetic Pathways with a Single Bioorthogonal Alcohol.

Imaging approaches that track biological molecules within cells are essential tools in modern biochemistry. Lipids are particularly challenging to visualize, as they are not directly genetically encoded. Phospholipids, the most abundant subgroup of lipids, are structurally diverse and accomplish many cellular functions, acting as major structural components of membranes and as signaling molecules that regulate cell growth, division, apoptosis, cytoskeletal dynamics, and numerous other physiological processes. Cells regulate the abundance, and therefore bioactivity, of phospholipids by modulating the activities of their biosynthetic enzymes. Thus, techniques that enable monitoring of flux through individual lipid biosynthetic pathways can provide key functional information. For example, the choline analogue propargylcholine (ProCho) can report on de novo biosynthesis of phosphatidylcholine by conversion to an alkynyl lipid that can be imaged following click chemistry tagging with an azido fluorophore. We report that ProCho is also a substrate of phospholipase D enzymes-which normally hydrolyze phosphatidylcholine to generate the lipid second messenger phosphatidic acid-in a transphosphatidylation reaction, generating the identical alkynyl lipid. By controlling the activities of phosphatidylcholine biosynthesis and phospholipase D enzymes, we establish labeling conditions that enable this single probe to selectively report on two different biosynthetic pathways. Just as nature exploits the economy of common metabolic intermediates to efficiently diversify biosynthesis, so can biochemists in interrogating such pathways with careful probe design. We envision that ProCho's ability to report on multiple metabolic pathways will enable studies of membrane dynamics and improve our understanding of the myriad roles that lipids play in cellular homeostasis.

Growth factors regulate phospholipid biosynthesis in human fibroblast-like synoviocytes obtained from osteoarthritic knees

Elevated levels of growth factors and phospholipids (PLs) have been found in osteoarthritic synovial fluid (SF), although the metabolic regulation of PLs is currently unknown. This study aimed to determine the effects of growth factors on the biosynthesis of PLs by fibroblast-like synoviocytes (FLS) obtained from human osteoarthritic knee joints. Electrospray ionization tandem mass spectrometry was applied to analyse the newly synthesized PLs. In the presence of stable isotope-labelled PL precursors, cultured FLS were treated with either transforming growth factor-β1 (TGF-β1), bone morphogenetic protein (BMP)-2, BMP-4, BMP-7 or insulin-like growth factor-1 (IGF-1) alone or in combination with specific inhibitors of cell signalling pathways. TGF-β1 and IGF-1 markedly stimulated the biosynthesis of phosphatidylcholine (PC) before sphingomyelin (SM) and lysophosphatidylcholine (LPC) species were stimulated. BMPs elaborated less pronounced effects. The BMPs tested have different potentials to induce the biosynthesis of phosphatidylethanolamine (PE) and PE-based plasmalogens. Our study shows for the first time that TGF-β1 and IGF-1 substantially regulate the biosynthesis of PC, SM and LPC in human FLS. The functional consequences of elevated levels of PLs require additional study. The BMPs tested may be joint protective in that they upregulate PE-based plasmalogens that function as endogenous antioxidants against reactive oxygen species.

From masochistic enzymology to mechanistic physiology and disease

The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt-/- mice are protected from weight gain and insulin resistance.

Four-week dietary supplementation with 10- and/or 15-fold basal choline caused decreased body weight in Sprague Dawley rats.

Choline is an essential nutrient utilized for phosphatidylcholine biosynthesis and lipoprotein packaging and secretion. Recently, choline supplementation has been used by athletes and the public for weight loss. However, the potential toxicological impact of choline dietary supplementation requires further investigation. This study examined the effects of choline dietary supplementation in Sprague Dawley rats for 4 weeks. Rats were fed diets containing basal choline levels (control) or 5-, 10-, or 15-fold (5×, 10×, or 15×) basal diet concentration. In groups fed choline-supplemented diets, there were no toxicologically relevant findings in clinical observations, food intake, clinical chemistry, liver weights, or liver histopathology. However, decreased mean body weights (8.5-10.2%) and body weight gains (24-31%) were noted for the 10× choline-supplemented (females only) and 15× choline-supplemented (both sexes) groups relative to the control groups from day 3 onward. These body weight effects were not related to a persistent reduction in average food intake. Serum cholesterol was increased in the 15× choline-supplemented male rats relative to the controls, an expected effect of choline supplementation; however, there were no changes in the serum cholesterol of female rats. Serum choline concentrations were increased in female rats relative to the male rats across all treatment groups. The maximum tolerated dose for male and female rats were the 15× and 10× choline supplements, respectively, based on decreased mean body weight and body weight gains. This study supported the conclusions of a clinical trial that showed a high choline diet can decrease body weight in humans.

Insights into the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in Leishmania parasites and characterization of a choline kinase from Leishmania infantum.

The protozoan parasite Leishmania infantum is a causative agent of the disease visceral leishmaniasis, which can be fatal if not properly treated. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) biosynthesis pathways are attractive targets for new antileishmanial compounds since these Leishmania cell membrane phospholipids are important for parasite morphology and physiology. In this work we observed Leishmania synthesize PC and PE from extracellular choline and ethanolamine, respectively, suggesting the presence of CDP-choline and CDP-ethanolamine pathways. In addition, Leishmania converted PE to PC, indicating the parasite possesses phosphatidylethanolamine N-methyltransferase (PEMT) activity. The first step in the biosynthesis of PC or PE requires the phosphorylation of choline or ethanolamine by a kinase. We cloned the gene encoding a putative choline/ethanolamine kinase from Leishmania infantum and expressed and purified the encoded recombinant protein. The enzyme possesses choline kinase activity with a Vmax of 3.52μmol/min/mg and an apparent Km value of 0.089mM with respect to choline. The enzyme can also phosphorylate ethanolamine in vitro, but the apparent Km for ethanolamine is 850-fold greater than for choline. In an effort to probe requirements for small molecule inhibition of Leishmania choline kinase, the recombinant enzyme was evaluated for the ability to be inhibited by novel quaternary ammonium salts. The most effective inhibitor was N-iodomethyl-N,N,-dimethyl-N-(6,6-diphenyl hex-5-en-1-yle) ammonium iodide, denoted compound C6. In the presence of 4mM compound C6, the Vmax/Km decreased to approximately 1% of the wild-type catalytic efficiency. In addition, in Leishmania cells treated with compound C6 choline transport was inhibited.

Genes essential for phototrophic growth by a purple alphaproteobacterium

Tn-seq was used to identify genes essential for phototrophic growth by the purple bacterium Rhodopseudomonas palustris. About 167 genes required for anaerobic growth on acetate in light were identified, 35 of which are annotated as photosynthesis genes. The essentiality of many of these genes by analysing the phenotypes of independently generated mutants that had altered pigmentation was verified. Three genes were identified, two possibly involved in biogenesis of the membrane-bound photosynthetic apparatus and one for phosphatidylcholine biosynthesis, that were not known to be essential for phototrophic growth. Site-directed mutagenesis was used to show that the NADH:quinone oxidoreductase complex IE was essential for phototrophic growth under strictly anaerobic conditions and appeared to play a role in reverse electron transport to generate NADH. A homologous NADH:quinone oxidoreductase complex IA likely operates in the opposite direction to oxidize NADH. The operation of the two enzymes in opposition would allow R.palustris to maintain redox balance. As a complement to the genetic data, proteomics experiments were carried out in which it was found that 408 proteins were present in significantly higher amounts in cells grown anaerobically in light compared with aerobically. Among these were proteins encoded by subset of the phototrophic growth-essential genes.

Membrane Lipids Speak to Histones

In this issue of Molecular Cell, Ye etal. (2017) use a combination of genetic, metabolomic, and transcriptomic approaches to explore the potential for methionine metabolism to influence signal transduction and gene expression in budding yeast.

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.

Effects of dietary phospholipids on growth performance and expression of key genes involved in phosphatidylcholine metabolism in larval and juvenile large yellow croaker, Larimichthys crocea

The objective of this study was to investigate the effects of dietary phospholipids (PLs) on survival rate, growth performance and expression of key genes related with phosphatidylcholine (PC) metabolism in larval and juvenile large yellow croaker, Larimichthys crocea. Three isonitrogenous and isolipidic diets with graded levels of PLs (2.53%, 6.32% and 12.7%) were randomly allocated to triplicate groups of larvae (initial body weight: 3.86±0.24mg) eight times daily for 30days. Five isonitrogenous and isolipidic diets with graded levels of PLs (1.32%, 3.05%, 5.86%, 7.4% and 9.63%) were randomly allocated to triplicate groups of juveniles (initial body weight: 7.36±0.33g) twice daily to apparent satiation for 60days. Results showed that survival rate and specific growth rate of larvae fed the diet with 2.53% PLs were significantly lower compared with those of larvae in other groups (P<0.05). However, specific growth rate and survival rate were not significantly different among dietary treatments in juveniles. No significant difference was observed in the transcript levels of key genes involved in PC de novo synthesis, including CTP: choline phosphate cytidylyltransferase and phosphatidylethanolamine N-methyltransferase, in both the whole larvae body and the livers of juveniles fed diets with different concentrations of PLs. Although no significant difference was found, the mRNA expression levels of 1, 2-diacylglycerol choline phosphotransferase increased with the increasing levels of dietary PLs in the livers of juveniles, with acyl-CoA: diacylglycerol acyltransferase 2 showing the opposite trend. These results confirmed that the beneficial effects of dietary PLs on survival and growth performance of large yellow croaker were restricted to fish larvae. Dietary PLs might reduce lipid deposition in the liver of juvenile large yellow croaker through utilizing more diacylglycerol for PC synthesis, rather than triglyceride synthesis. Statement of relevanceThe present study was conducted to investigate the effects of dietary phospholipids (PLs) on survival rate, growth performance and expression of key genes related to phosphatidylcholine (PC) biosynthesis in larval and juvenile large yellow croaker, (Larimichthys crocea). The results are reliable and of both theoretical and practical importance. For “theoretical importance”, our study provided the first evidence that dietary PLs might reduce lipid deposition in the liver of juvenile large yellow croaker through utilizing more diacylglycerol for PC synthesis, rather than triglyceride synthesis, which could contribute to better understanding the mechanisms underlying the discrepancy in requirements for phospholipids between larval and juvenile large yellow croaker. For “practical importance”, our study confirmed that the beneficial effects of dietary PLs on growth performance of large yellow croaker were restricted to larvae rather than juveniles (at least 7.36g) and these findings could be applied to reduce the supplementation of PLs in commercial feeds for large yellow croaker, which in turn might reduce the cost of the commercial feeds for large yellow croaker.The work described has not been submitted elsewhere for publication, in whole or in part, and all the authors listed have approved the manuscript that is enclosed. I have read and have abided by the statement of ethical standards for manuscripts submitted to Aquaculture.

Cyclin-dependent kinase activity enhances phosphatidylcholine biosynthesis in Arabidopsis by repressing phosphatidic acid phosphohydrolase activity.

SummaryCoordination of endomembrane biogenesis with cell cycle progression is considered to be important in maintaining cell function during growth and development. We previously showed that the disruption of PHOSPHATIDIC ACID PHOSPHOHYDROLASE (PAH) activity in Arabidopsis thaliana stimulates biosynthesis of the major phospholipid phosphatidylcholine (PC) and causes expansion of the endoplasmic reticulum. Here we show that PC biosynthesis is repressed by disruption of the core cell cycle regulator CYCLIN‐DEPENDENT KINASE A;1 (CDKA;1) and that this repression is reliant on PAH. Furthermore, we show that cyclin‐dependent kinases (CDKs) phosphorylate PAH1 at serine 162, which reduces both its activity and membrane association. Expression of a CDK‐insensitive version of PAH1 with a serine 162 to alanine substitution represses PC biosynthesis and also reduces the rate of cell division in early leaf development. Together our findings reveal a physiologically important mechanism that couples the rate of phospholipid biosynthesis and endomembrane biogenesis to cell cycle progression in Arabidopsis.

Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants: A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS

Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).

StarD7 Protein Deficiency Adversely Affects the Phosphatidylcholine Composition, Respiratory Activity, and Cristae Structure of Mitochondria

Phosphatidylcholine (PC) is a major phospholipid of mitochondria, comprising 40-50% of both the outer and the inner membranes. However, PC must be imported from its production organelles because mitochondria lack the enzymes essential for PC biosynthesis. In a previous study, we found that StarD7 mediates the intracellular transfer of PC to mitochondria. Therefore, in this study, we analyzed the contribution of StarD7 to the maintenance of mitochondrial phospholipid content and function using siRNA-mediated knockdown and knock-out (KO) of the StarD7 gene in HEPA-1 cells. Real time analysis of respiratory activity demonstrated that the oxygen consumption rate and activity of mitochondrial complexes were impaired in StarD7-KD cells. To confirm these results, we established StarD7-KO HEPA-1 cells by double nicking using CRISPR/Cas9n. As expected, StarD7-KD and -KO cells showed a significant reduction in mitochondrial PC content. The ATP level and growth rate of KO cells were notably lower compared with wild-type cells when cultured in glucose-free galactose-containing medium to force cells to rely on mitochondrial ATP production. In KO cells, the level of the MTCO1 protein, a primary subunit of complex IV, was reduced without a concomitant decrease in its mRNA, but the level was restored when StarD7-I was overexpressed. StarD7-KO cells showed impaired formation of the mitochondrial supercomplexes and exhibited a disorganized cristae structure, with no changes in optic atrophy 1 protein. These findings indicate that StarD7 plays important roles in maintaining the proper composition of mitochondrial phospholipids as well as mitochondrial function and morphogenesis.

Impaired biosynthesis of the non-bilayer lipids phosphatidylethanolamine or cardiolipin does not affect peroxisome biogenesis and proliferation in Saccharomyces cerevisiae

The non-bilayer forming lipids cardiolipin (CL) and phosphatidylethanolamine (PE) modulate membrane curvature, facilitate membrane fusion and affect the stability and function of membrane proteins. Yeast peroxisomal membranes contain significant amounts of CL and PE.We analysed the effect of CL deficiency and PE depletion on peroxisome biogenesis and proliferation in Saccharomyces cerevisiae. Our data indicate that deletion of CRD1, which encodes cardiolipin synthase, does not affect peroxisome biogenesis or abundance, both at peroxisome repressing (glucose) or inducing (oleate) growth conditions. Analysis of strains deficient in one of the three PE biosynthesis pathways (psd1, psd2 or the triple deletion strain eki1 cki1 dpl1) revealed that in all three strains peroxisome numbers were reduced upon growth of cells on oleic acid, whereas the psd1 strain also showed a reduction in peroxisome abundance upon growth on glucose. Because PE is an intermediate of the phosphatidylcholine (PC) biosynthesis pathway, PE depletion affects PC formation. PC however can be synthesized by an alternative pathway when choline is supplemented to the growth medium. Because the addition of choline resulted in suppression of the peroxisome phenotypes in phosphatidylserine decarboxylase mutant strains, we conclude that peroxisome biogenesis and proliferation are not crucially dependent on CL or PE.