Good Fat, Essential Cellular Requirements for Triacylglycerol Synthesis to Maintain Membrane Homeostasis in Yeast

Storage triacylglycerols (TAG) and membrane phospholipids share common precursors, i.e. phosphatidic acid and diacylglycerol, in the endoplasmic reticulum. In addition to providing a biophysically rather inert storage pool for fatty acids, TAG synthesis plays an important role to buffer excess fatty acids (FA). The inability to incorporate exogenous oleic acid into TAG in a yeast mutant lacking the acyltransferases Lro1p, Dga1p, Are1p, and Are2p contributing to TAG synthesis results in dysregulation of lipid synthesis, massive proliferation of intracellular membranes, and ultimately cell death. Carboxypeptidase Y trafficking from the endoplasmic reticulum to the vacuole is severely impaired, but the unfolded protein response is only moderately up-regulated, and dispensable for membrane proliferation, upon exposure to oleic acid. FA-induced toxicity is specific to oleic acid and much less pronounced with palmitoleic acid and is not detectable with the saturated fatty acids, palmitic and stearic acid. Palmitic acid supplementation partially suppresses oleic acid-induced lipotoxicity and restores carboxypeptidase Y trafficking to the vacuole. These data show the following: (i) FA uptake is not regulated by the cellular lipid requirements; (ii) TAG synthesis functions as a crucial intracellular buffer for detoxifying excess unsaturated fatty acids; (iii) membrane lipid synthesis and proliferation are responsive to and controlled by a balanced fatty acid composition.     

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

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

Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion

Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1p(D398E) had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion.     

Triacylglycerol biosynthesis in yeast

Triacylglycerol (TAG) is the major storage component for fatty acids, and thus for energy, in eukaryotic cells. In this mini-review, we describe recent progress that has been made with the yeast Saccharomyces cerevisiae in understanding formation of TAG and its cell biological role. Formation of TAG involves the synthesis of phosphatidic acid (PA) and diacylglycerol (DAG), two key intermediates of lipid metabolism. De novo formation of PA in yeast as in other types of cells can occur either through the glycerol-3-phosphate- or dihydroxyacetone phosphate-pathways-each named after its respective precursor. PA, formed in two steps of acylation, is converted to DAG by phosphatidate phosphatase. Acylation of DAG to yield TAG is catalyzed mainly by the two yeast proteins Dga1p and Lro1p, which utilize acyl-CoA or phosphatidylcholine, respectively, as acyl donors. In addition, minor alternative routes of DAG acylation appear to exist. Endoplasmic reticulum and lipid particles (LP), the TAG storage compartment in yeast, are the major sites of TAG synthesis. The interplay of these organelles, formation of LP, and enzymatic properties of enzymes catalyzing the synthesis of PA, DAG, and TAG in yeast are discussed in this communication.     

Incorporation of specific exogenous fatty acids into membrane lipids modulates protonophore resistance in Bacillus subtilis.

Attempts to manipulate the level of C16:1 fatty acids in membrane phospholipids were made by using Bacillus subtilis and its protonophore-resistant mutants to test the hypothesis that C16:1 fatty acid levels relate to the bioenergetic properties of the mutant strains. Growth of the three mutants in the presence of palmitoleic acid restored the level of C16:1 fatty acids in the membrane lipids to somewhat above those found in the wild type. The palmitoleic acid was preferentially incorporated into diphosphatidylglycerol (cardiolipin) and phosphatidylethanolamine and was associated with increased levels of these phospholipids. These membrane preparations showed no increase in the levels of free fatty acids. The increase in C16:1 fatty acids achieved by growth in the presence of palmitoleic acid was accompanied by secondary changes in membrane lipids as well as a pronounced diminution in the protonophore resistance of growth and ATP synthesis. Other membrane-associated properties that had been observed in these mutants, e.g., elevated ATPase levels, were not altered coordinately with protonophore resistance and C16:1 fatty acid levels. Growth of the wild type in the presence of palmitic acid caused a modest elevation of the C16:0 of the membrane lipids and a modest increase in the protonophore resistance of growth and ATP synthesis. Growth of the wild type at elevated temperatures, in the absence of fatty acid supplementation, also enhanced its resistance to protonophores. The results support the hypothesis that specific changes in membrane lipid composition underlie the bioenergetic changes associated with protonophore resistance.     

The Saccharomyces cerevisiae Lipin homolog is a Mg2+-dependent phosphatidate phosphatase enzyme

Mg(2+)-dependent phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of PA to yield diacylglycerol and P(i). In this work, we identified the Saccharomyces cerevisiae PAH1 (previously known as SMP2) gene that encodes Mg(2+)-dependent PA phosphatase using amino acid sequence information derived from a purified preparation of the enzyme (Lin, Y.-P., and Carman, G. M. (1989) J. Biol. Chem. 264, 8641-8645). Overexpression of PAH1 in S. cerevisiae directed elevated levels of Mg(2+)-dependent PA phosphatase activity, whereas the pah1Delta mutation caused reduced levels of enzyme activity. Heterologous expression of PAH1 in Escherichia coli confirmed that Pah1p is a Mg(2+)-dependent PA phosphatase enzyme and showed that its enzymological properties were very similar to those of the enzyme purified from S. cerevisiae. The PAH1-encoded enzyme activity was associated with both the membrane and cytosolic fractions of the cell, and the membrane-bound form of the enzyme was salt-extractable. Lipid analysis showed that mutants lacking PAH1 accumulated PA and had reduced amounts of diacylglycerol and its derivative triacylglycerol.ThePAH1-encoded Mg(2+)-dependent PA phosphatase shows homology to mammalian lipin, a fat-regulating protein whose molecular function is unknown. Heterologous expression of human LPIN1 in E. coli showed that lipin 1 is also a Mg(2+)-dependent PA phosphatase enzyme.     

Mutant phosphatidate phosphatase Pah1-W637A exhibits altered phosphorylation,membrane association,and enzyme function in yeast

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1–Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by AlphaFold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis.     

Fatty acid alterations in Saccharomyces cerevisiae exposed to ethanol stress

The influence of ethanol concentration on fatty acid alterations in total phospholipids (PL), phosphatidylcholine (PCH), phosphatidylethanolamine (PE), phosphatidylinositol (PI), sterol esters (ES) and triacylglycerols (TAG) of Saccharomyces cerevisiae was studied. Ethanol induced the elevation of palmitic and oleic acid level in major membrane phospholipids (PCH and PE) and also the palmitoleic acid content in ES and TAG.     

Coordination of storage lipid synthesis and membrane biogenesis: evidence for cross-talk between triacylglycerol metabolism and phosphatidylinositol synthesis

Despite the importance of triacylglycerols (TAG) and steryl esters (SE) in phospholipid synthesis in cells transitioning from stationary-phase into active growth, there is no direct evidence for their requirement in synthesis of phosphatidylinositol (PI) or other membrane phospholipids in logarithmically growing yeast cells. We report that the dga1Δlro1Δare1Δare2Δ strain, which lacks the ability to synthesize both TAG and SE, is not able to sustain normal growth in the absence of inositol (Ino(-) phenotype) at 37 °C especially when choline is present. Unlike many other strains exhibiting an Ino(-) phenotype, the dga1Δlro1Δare1Δare2Δ strain does not display a defect in INO1 expression. However, the mutant exhibits slow recovery of PI content compared with wild type cells upon reintroduction of inositol into logarithmically growing cultures. The tgl3Δtgl4Δtgl5Δ strain, which is able to synthesize TAG but unable to mobilize it, also exhibits attenuated PI formation under these conditions. However, unlike dga1Δlro1Δare1Δare2Δ, the tgl3Δtgl4Δtgl5Δ strain does not display an Ino(-) phenotype, indicating that failure to mobilize TAG is not fully responsible for the growth defect of the dga1Δlro1Δare1Δare2Δ strain in the absence of inositol. Moreover, synthesis of phospholipids, especially PI, is dramatically reduced in the dga1Δlro1Δare1Δare2Δ strain even when it is grown continuously in the presence of inositol. The mutant also utilizes a greater proportion of newly synthesized PI than wild type for the synthesis of inositol-containing sphingolipids, especially in the absence of inositol. Thus, we conclude that storage lipid synthesis actively influences membrane phospholipid metabolism in logarithmically growing cells.     

Effects of different fatty acids on BRL3A rat liver cell damage

To evaluate the effects of fatty acids on endoplasmic reticulum (ER) stress, oxidative stress, and lipid damage. We treated BRL3A rat liver cells with, linoleic (LA), linolenic, oleic (OA), palmitic (PA), palmitoleic (POA), or stearic (SA) acid for 12 hr. The characteristics of cell lipid deposition, oxidative stress indexes, ER stress markers, nuclear factor κB p65 (NF-κB p65), lipid synthesis and transport regulators, and cholesterol metabolism regulators were analyzed. Endoplasmic chaperones like glucose-regulated protein 78, CCAAT-enhancer-binding protein, NF-κB p65, hydrogen peroxide, and malonaldehyde in PA- and SA-treated cells were significantly higher than in other treated cells. Deposition of fatty acids especially LA and POA were significantly increased than in other treated cells. De novo lipogenesis regulators sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-coenzyme A carboxylase 1 (ACC1) expression were significantly increased in all fatty acid stimulation groups, and PA- and SA-treated cells showed lower p-ACC1 expression and higher scd1 expression than other fatty acid groups. Very low-density lipoprotein synthesis and apolipoprotein B100 expression in free fatty acids treated cells were significantly lower than control. PA, SA, OA, and POA had shown significantly increased cholesterol synthesis than other treated cells. PA and SA showed the lower synthesis of cytochrome P7A1 and total bile acids than other fatty acids treated cells. Excess of saturated fatty acids led to severe ER and oxidative stress. Excess unsaturated fatty acids led to increased lipid deposition in cultured hepatocytes. A balanced fatty acid intake is needed to maintain lipid homeostasis.     

发菜膜脂及其脂肪酸组成

对野生发菜(Nostocflagelliforme Bom.et Flab)的膜脂(主要成分为类囊体膜脂)及其脂肪酸组成进行了测定分析.发菜的膜脂由单半乳糖甘油二酯(MGDG)、双半乳糖甘油二酯(DGDG)、磷酯酰甘油(PG)和硫代异鼠李糖甘油二酯(SQDG)组成,其酯酰基连接有棕榈酸(16:0)、十六碳烯酸(16:1)、硬脂酸(18:0)、油酸(18:1)、亚油酸(18:2)和亚麻酸(18:3)6种脂肪酸.发菜的不饱和脂肪酸含量可达总脂的73%,特别是16:1和18:3分别高达29%和34%,远远高于已报道的其他蓝藻,说明了发菜类囊体膜具有较强的抗逆性特点.同时还对复水30 min和复水后生长24 h的发菜膜脂及其脂肪酸组成进行了分析.结果表明,复水对野生发菜的膜脂及其脂肪酸组成没有显著影响,说明发菜的膜脂和脂肪酸组成在干燥-吸水过程中能保持很高的稳定性.     

发菜类囊体膜色素蛋白复合物分离及其谱性质的研究

Nostoc flagelliforme Born. et Flah is highly adapted to drought stress, cold and light stresses, and suitable for growing in the unfavorable areas. This paper presents the results of the analysis of the membrane (mainly thylakoid membrane) lipids from N. flagelliforme in order to investigate the relationship between membrane lipid composition and stress resistance to this cyanobacteria. The membrane lipids are composed of monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG) and phosphatidylglycerol (PG). The major fatty acids in these lipids are palmitic (16∶0), palmitoleic (16∶1), stearic (18∶0), oleic (18∶1), linoleic (18∶2) and linolenic (18∶3) acids. In N. flagelliforme, polyunsaturated fatty acids account for 73% of the total fatty acids, much higher than that of the other cyanobacteria reported so far. Among which 16∶1 and 18∶3 are as high as 28.9% and 34.3% respectively. The high resistance of N. flagelliforme to abnormal conditions may be associated with the extent of unsaturation of fatty acids. In addition, the wild N. flagelliforme treated with water for 30 min and cultured for 24 h and the lipid and fatty acid composition were found to be not affected by water-absorption.     

The phosphatidylcholine diacylglycerol cholinephosphotransferase is required for efficient hydroxy fatty acid accumulation in transgenic Arabidopsis

We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds.     

Age-dependent modifications in membrane lipids: lipid composition, fluidity and palmitoyl-CoA desaturase in Tetrahymena membranes

The membrane lipid composition of Tetrahymena pyriformis NT-I was observed to change in a manner markedly dependent on the progress of culture age. The pellicular, mitochondrial and microsomal membranes were isolated from cell harvested at various growth phases (I, early exponential; II, mid-exponential; III, late exponential; IV, early stationary; V, late stationary) and their lipid composition was analyzed by thin-layer and gas-liquid chromatography. Although the phospholipid composition varied somewhat among membrane fractions, the most general age-dependent alteration was a considerable decrease in the content of phosphatidylethanolamine accompanied by a small increase in phosphatidylcholine. The 2-aminoethylphosphonolipid, enriched in the surface membrane pellicle, did not undergo a consistent change. As for fatty acid composition the most notable variation occurred in unsaturated fatty acids; a great increase in oleic and linoleic acids and a compensatory decrease in palmitoleic acid. This resulted in an augmented unsaturation of the overall phospholipid fatty acid profile of the aged membranes. The age-associated drastic decline in the palmitoleic acid content in membrane phospholipids could be accounted for by the markedly lowered activity of palmitoyl-CoA desaturase. The microsomes from the early exponential phase cells possess a 4-fold higher activity of the desaturase as compared to that of the late stationary phase microsomes. The decreased desaturase activity associated with the culture age was also reflected in the corresponding decrease in the conversion rate of [14C]palmitate to [14C]palmitoleate in cells labelled in vivo. The ESR spectra of the spin-labeled phospholipids extracted from the pellicular and microsomal membranes have led to the suggestion that these types of membrane would become more fluid with the age of growth.