Rapid phase change of lipid microdomains in giant vesicles induced by conversion of sphingomyelin to ceramide

To understand the role of sphingomyelinase (SMase) in the function of biological membranes, we have investigated the effect of conversion of sphingomyelin (SM) to ceramide (Cer) on the assembly of domains in giant unilamellar vesicles (GUVs). The GUVs were prepared from mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-palmitoly-d-erythro-sphingosine (C₁₆Cer), N-palmitoyl-d-erythro-sphingosylphosphorylcholine (C₁₆SM) and cholesterol. The amounts of DOPC, sum of C₁₆Cer and C₁₆SM, and cholesterol were kept constant (the ratio of these four lipids is shown as 1:X:1-X:1 (molar ratio), i.e., X is C₁₆Cer/(C₁₆Cer + C₁₆SM)). Shape and distribution of domains formed in the GUVs were monitored by a fluorescent lipid, Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (0.1 mol%). In GUVs containing low C₁₆Cer (X = 0 and 0.25), round-shaped domains labeled by the fluorescent lipid were present, suggesting coexistence of liquid-ordered and disordered domains. In GUVs containing intermediate Cer concentration (X = 0.5), the fluorescent domain covered most of GUV surface, which was surrounded by gel-like domains. Differential scanning calorimetry of multilamellar vesicles prepared in the presence of higher Cer concentration (X ≥ 0.5) suggested existence of a Cer-enriched gel phase. Video microscopy showed that the enzymatic conversion of SM to Cer caused rapid change in the domain structure: several minutes after the SMase addition, the fluorescent region spread over the GUV surface, within which regions with darker contrast existed. Image-based measurement of generalized polarization (GP) of 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), which is related to the acyl chain ordering of the lipids, was performed. Before the SMase treatment domains with high (0.65) and low (below 0.4) GP values coexisted, presumably reflecting the liquid-ordered and disordered domains; after the SMase treatment regions with intermediate GP values (0.5) and smaller regions with higher GP values (0.65) were present. Generation of Cer thus caused a phase transition from liquid-ordered and disordered phases to a gel and liquid phase.     

Ceramide/long-chain base phosphate rheostat in Saccharomyces cerevisiae: regulation of ceramide synthesis by Elo3p and Cka2p

Sphingolipid precursors, namely, ceramide and long-chain base phosphates (LCBPs), are important growth regulators with often opposite effects on mammalian cells. A set of enzymes that regulate the levels of these precursors, referred to as a ceramide/LCBP rheostat, is conserved in all eukaryotes. In order to gain further insight into the function of the rheostat in Saccharomyces cerevisiae, we searched for mutants that are synthetically lethal with a deletion of the LCB3 gene encoding LCBP phosphatase. In addition to acquiring expected mutants lacking the LCBP lyase, the screen revealed elo3 (sur4) mutants that were defective in fatty acid elongation and cka2 mutants lacking the α′ subunit of the protein kinase CK2 (casein kinase). Both mutations affected the in vivo activity of the acyl coenzyme A (acyl-CoA)-dependent and fumonisin B₁-sensitive ceramide synthase (CS). The Elo3 protein is necessary for synthesis of C₂₆-CoA, which in wild-type yeast is a source of C₂₆ fatty acyls found in the ceramide moieties of all sphingolipids. In the in vitro assay, CS had a strong preference for acyl-CoAs containing longer acyl chains. This finding suggests that a block in the formation of C₂₆-CoA in yeast may cause a reduction in the conversion of LCBs into ceramides and lead to an overaccumulation of LCBPs that is lethal in strains lacking the Lcb3 phosphatase. In fact, elo3 mutants were found to accumulate high levels of LCBs and LCBPs. The cka2 mutants, on the other hand, exhibited only 25 to 30% of the in vitro CS activity found in wild-type membranes, indicating that the α′ subunit of CK2 kinase is necessary for full activation of CS. The cka2 mutants also accumulated high levels of LCBs and had elevated levels of LCBPs. In addition, both the elo3 and cka2 mutants showed increased sensitivity to the CS inhibitors australifungin and fumonisin B₁. Together, our data demonstrate that the levels of LCBPs in yeast are regulated by the rate of ceramide synthesis, which depends on CK2 kinase activity and is also strongly affected by the supply of C₂₆-CoA. This is the first evidence indicating the involvement of protein kinase in the regulation of de novo sphingolipid synthesis in any organism.     

In vivo assessment of hepatic triglycerides in murine non-alcoholic fatty liver disease using magnetic resonance spectroscopy

In vivo¹H magnetic resonance spectroscopy (MRS) was used to examine the progression of fatty liver in two murine models of progressive hepatic steatosis: leptin-deficient obese (ob/ob) mice and mice maintained on a diet deficient in methionine and choline (MCDD). Ob/ob mice displayed high levels of intracellular hepatic triglycerides as early as 9 weeks after birth, as observed with MRS and histopathology. Single voxel spectra of ob/ob liver displayed strong resonances arising from saturated (1.3 ppm) and unsaturated (2.8 and 5.3 ppm) fatty acyl chains that could be resolved in the absence of water suppression. Hepatic inflammation, induced by lipopolysaccharide administration, led to a significant increase in unsaturated and polyunsaturated fatty acyl chain resonances (P < 0.05), indicating a change in the composition of hepatic triglycerides in lipid droplets. Mice maintained on the MCDD displayed histological evidence of hepatic steatosis as early as two weeks, progressing to macrovesicular steatohepatitis at 10 weeks. The histological changes were accompanied by significant increases in saturated and unsaturated fatty acyl chain resonances and a significant decrease in the lipid/(water + lipid) ratio (P < 0.05). These results indicate that in vivo¹H MRS may be a suitable method to monitor the progression of steatohepatitis.     

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.     

Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis

The oleaginous yeast Rhodotorula glutinis has been known to be a potential feedstock for lipid production. In the present study, we investigated the enhancement of expression of malic enzyme (ME; NADP⁺ dependent; EC 1.1.1.40) from Mucor circinelloides as a strategy to improve lipid content inside the yeast cells. The 26S rDNA and 5.8S rDNA gene fragments isolated from Rhodotorula glutinis were used for homologous integration of ME gene into R. glutinis chromosome under the control of the constitutively highly expressed gene phosphoglycerate kinase 1 to achieve stable expression. We demonstrated that by increasing the expression of the foreign ME gene in R. glutinis, we successfully improved the lipid content by more than twofold. At the end of lipid accumulation phrase (96 h) in the transformants, activity of ME was increased by twofold and lipid content of the yeast cells was increased from 18.74 % of the biomass to 39.35 %. Simultaneously, there were no significant differences in fatty acid profiles between the wild-type strain and the recombinant strain. Over 94 % of total fatty acids were C_16:0, C_18:0, C_16:1, C_18:1, and C_18:2. Our results indicated that heterologous expression of NADP⁺-dependent ME involved in fatty acid biosynthesis indeed increased the lipid accumulation in the oleaginous yeast R. glutinis.     

Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2 g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2 l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6 g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (50.6%).     

The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones

The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such “alternative” NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C₁₂ (HDQ) and C₁₄ are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C₅ and C₆) displayed significantly higher IC₅₀ values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.     

Partitioning of Triton X-100, deoxycholate and C10EO8 into bilayers composed of native and hydrogenated egg yolk sphingomyelin

We have used isothermal titration calorimetry (ITC) to study the thermodynamics of Triton X-100 (T_X-100), deoxycholate and decyl octaethylene glycol (C₁₀EO₈) penetration into bilayers composed of native (ESM) and hydrogenated egg yolk sphingomyelin (DHSM). Light scattering measurements were used to study the point of saturation (R_e,sat) and the onset of solubilization of membranes by the detergents. We found that DHSM bilayers at 25 °C were much more resistant to detergent partitioning (lower K) and gave higher reaction enthalpies (ΔH) for all three detergents compared to the ESM bilayer system. Because DHSM lacks double bonds (Δ^4trans and some cis bonds as well), attractive acyl chain interactions are favored in membranes of this lipid class. The high stability and cohesion of DHSM in membranes could be a crucial functional property of this lipid as it is enriched in eye lens membranes.     

Interactions of ciprofloxacin with DPPC and DPPG: Fluorescence anisotropy, ATR-FTIR and P NMR spectroscopies and conformational analysis

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T_m) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and ³¹P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K_app were in the order of 10⁵ M^− 1 and the affinity appeared dependent on the negative charge of liposomes: DPPG > DOPC:DPPG (1:1; M:M) > DPPC > DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Δσ) values determined by ³¹P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T_m of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T_m and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, like phosphatidylglycerol, present at a high content in these membranes.     

3D coordination polymers of [2.2]paracyclophane and in situ silver(I) perfluoro-dicarboxylates: effects of the dicarboxylate spacers and conformations on the formation of complexes

Coordination polymers of [2.2]paracyclophane (pcp) with in situ silver(I) perfluoro-dicarboxylates characterized by single crystal X-ray analysis are described. Structures are found to strongly depend on the dicarboxylate spacer (n). With disilver(I) tetrafluorosuccinate ((CF₂)_n(COOAg)₂, n = 2), 3D network with composition of [Ag₄(pcp)(C₂F₄(CO₂)₂)₂] (1) forms in which silver salts afford infinite double chains and pcp act as linkages between chains. Changing the silver salt to disilver hexafluoroglutarate ((CF₂)_n(COOAg)₂, n = 3) produces 3D pillared-layer structure of composition of [Ag₄(pcp)(C₃F₆(CO₂)₂)₂] · THF (2) (THF = tetrahydrofuran), in which silver salts form 2D sheets and pcp act as pillars between the sheets. With silver octafluoroadipate (HO₂C(CF₂)_nCO₂Ag, n = 4), 2-fold interpenetrated diamond structure, [Ag₂(pcp)₂(HO₂CC₄F₈CO₂)₂]₂ · 2toluene (3), is obtained in which silver-anion chains and silver-pcp chains are connected with each other in the perpendicular manner. The three complexes represent unprecedented metal-organic networks of silver(I) multicarboxylates and polycyclic aromatic compounds. Additionally, the effects of the dicarboxylate conformations as well as the solvents on the resulting structures were discussed.     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Extreme temperature tolerance of a hyperthermophilic protein coupled to residual structure in the unfolded state

Understanding the mechanisms that dictate protein stability is of large relevance, for instance, to enable design of temperature-tolerant enzymes with high enzymatic activity over a broad temperature interval. In an effort to identify such mechanisms, we have performed a detailed comparative study of the folding thermodynamics and kinetics of the ribosomal protein S16 isolated from a mesophilic (S16_meso) and hyperthermophilic (S16_thermo) bacterium by using a variety of biophysical methods. As basis for the study, the 2.0 Å X-ray structure of S16_thermo was solved using single wavelength anomalous dispersion phasing. Thermal unfolding experiments yielded midpoints of 59 and 111 °C with associated changes in heat capacity upon unfolding (ΔC_p⁰) of 6.4 and 3.3 kJ mol^− 1 K^− 1, respectively. A strong linear correlation between ΔC_p⁰ and melting temperature (T_m) was observed for the wild-type proteins and mutated variants, suggesting that these variables are intimately connected. Stopped-flow fluorescence spectroscopy shows that S16_meso folds through an apparent two-state model, whereas S16_thermo folds through a more complex mechanism with a marked curvature in the refolding limb indicating the presence of a folding intermediate. Time-resolved energy transfer between Trp and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide of proteins mutated at selected positions shows that the denatured state ensemble of S16_thermo is more compact relative to S16_meso. Taken together, our results suggest the presence of residual structure in the denatured state ensemble of S16_thermo that appears to account for the large difference in quantified ΔC_p⁰ values and, in turn, parts of the observed extreme thermal stability of S16_thermo. These observations may be of general importance in the design of robust enzymes that are highly active over a wide temperature span.     

Localization of p21-Activated Kinase 1 (PAK1) to Pinocytic Vesicles and Cortical Actin Structures in Stimulated Cells

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.     

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of ¹³C-labeled diC8PC ((methyl-¹³C)₃-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-¹³C)₃-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.     

Molybdenum doubly bridged and chelate nitrosyl complexes incorporating saturated n-alkanediolate ligands

The redox-active doubly bridged species [{Mo(NO)(Tp^Me2)Q}₂] [Tp^Me2 = tris(3,5-dimethylpyrazol-1-yl)hydroborate, Q = O(CH₂)_nO, n = 3, 5, or OCH₂(CF₂)_n−2CH₂O, n = 5, 6], and a chelate complex [Mo(NO)-(Tp^Me2)O(CH₂)₅O] were prepared and characterised by elemental and mass analyses, ¹H NMR and IR spectroscopy. The bimetallic species with C₃, C₅, and C₅(F) bridges exhibit two well-resolved reduction processes in their cyclic voltammograms (ΔE_1/2 values of 290, 170, and 170 mV, respectively). These results indicate that the presence of the second bridge increases the extent of electrochemical interactions (by ca. 90-130 mV) in comparison with their singly bridged analogues. All non-fluorinated and the chelate species were catalytically active in cathodic reduction of chloroform.     

Biosynthesis of triacylglycerol in Thunbergia alata: Additional evidence for involvement of phosphatidylcholine in unusual monoenoic oil production

The seed oil of Thunbergia alata has an unusual fatty acid composition which consists of more than 80 % 16:1^Δ6. This fatty acid is produced in the plastid by the action of a Δ6 palmitoyl (16:0)-ACP desaturase. To examine the biosynthesis of triacylglycerol (TAG) containing high concentrations of this unusual monoenoic fatty acid, endosperm dissected from developing T. alata seeds was labeled with [1-¹⁴C]-acetate. At early time points (5–15 min), the predominant labeled lipid was PC whereas at later time points (greater than 30 min) TAG became the major labeled lipid. Analysis of the acyl group composition of each lipid revealed that radiolabeled 16:1^Δ6 was highest at early time points in PC while at later time points, it was found to be highest in TAG. Further analysis of the distribution of labeled acyl groups within PC indicated that 16:1^Δ6 at the sn-2 position comprised the majority (55–78 %) of total labeled acyl groups whereas 16:1^Δ6 at the sn-1 position constituted only a small fraction (12–15 %) of the total labeled acyl groups. In contrast, unlabeled PC contained lower amounts of 16:1^Δ6 (16 %) at the sn-2 position. These results are consistent with previous studies suggesting a flux of novel monoenoic acids through PC during TAG biosynthesis, and furthermore imply a stereospecific flux through the sn-2 position of PC.     

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10¹⁴ and MW ? 10⁵, respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

Rotation of lipids in membranes: molecular dynamics simulation, 31P spin-lattice relaxation, and rigid-body dynamics

Molecular dynamics simulations and ³¹P-NMR spin-lattice (R₁) relaxation rates from 0.022 to 21.1 T of fluid phase dipalmitoylphosphatidylcholine bilayers are compared. Agreement between experiment and direct prediction from simulation indicates that the dominant slow relaxation (correlation) times of the dipolar and chemical shift anisotropy spin-lattice relaxation are ∼10 ns and 3 ns, respectively. Overall reorientation of the lipid body, consisting of the phosphorus, glycerol, and acyl chains, is well described within a rigid-body model. Wobble, with D_⊥ = 1-2 × 10⁸ s⁻¹, is the primary component of the 10 ns relaxation; this timescale is consistent with the tumbling of a lipid-sized cylinder in a medium with the viscosity of liquid hexadecane. The value for D_|| the diffusion constant for rotation about the long axis of the lipid body, is difficult to determine precisely because of averaging by fast motions and wobble; it is tentatively estimated to be 1 × 10⁷ s⁻¹. The resulting D_||/D_⊥ ≈ 0.1 implies that axial rotation is strongly modulated by interactions at the lipid/water interface. Rigid-body modeling and potential of mean force evaluations show that the choline group is relatively uncoupled from the rest of the lipid. This is consistent with the ratio of chemical shift anisotropy and dipolar correlation times reported here and the previous observations that ³¹P-NMR lineshapes are axially symmetric even in the gel phase of dipalmitoylphosphatidylcholine.     

Ethanol Tolerance in the Yeast Saccharomyces cerevisiae Is Dependent on Cellular Oleic Acid Content

In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (Δ⁹Z-C_16:1) and oleic acid (Δ⁹Z-C_18:1), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen- and NADH-dependent desaturation of palmitic acid (C_16:0) and stearic acid (C_18:0), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., Δ⁹ and Δ¹¹) and substrate chain-length preferences (i.e., C_16:0 and C_18:0); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (Δ¹¹Z-C_18:1), whereas neither Δ¹¹Z-C_16:1 nor palmitoleic acid (Δ⁹Z-C_16:1) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.