Incorporation and remodeling of phosphatidylethanolamine containing short acyl residues in yeast

Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn− 2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn− 2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.     

Metabolism of phosphatidylcholine and its implications for lipid acyl chain composition in Saccharomyces cerevisiae

Phosphatidylcholine (PC) is a very abundant membrane lipid in most eukaryotes including the model organism Saccharomyces cerevisiae. Consequently, the molecular species profile of PC, i.e. the ensemble of PC molecules with acyl chains differing in number of carbon atoms and double bonds, is important in determining the physical properties of eukaryotic membranes, and should be tightly regulated. In this review current insights in the contributions of biosynthesis, turnover, and remodeling by acyl chain exchange to the maintenance of PC homeostasis at the level of the molecular species in yeast are summarized. In addition, the phospholipid class-specific changes in membrane acyl chain composition induced by PC depletion are discussed, which identify PC as key player in a novel regulatory mechanism balancing the proportions of bilayer and non-bilayer lipids in yeast.     

Regulation of phosphatidylcholine biosynthesis in Saccharomyces cerevisiae

Evidence is presented which indicates that the biosynthesis of phosphatidylcholine by the methylation pathway in growing cultures of Saccharomyces cerevisiae is repressed by the presence of choline in the growth medium. This result, obtained previously for glucose-grown cells, was also observed for lactate-grown cells, of which half of the phosphatidylcholine is mitochondrial. A respiration-deficient mutant of the parent wild-type strain has been studied, and its inability to form functional mitochondria cannot be due to an impaired methylation pathway, as it has been shown to incorporate (14)C-CH(3)-methionine into all of the methylated glycerophosphatides. The incorporation rate is depressed by the inclusion of 1 mm choline in the growth medium, suggesting a regulatory effect similar to that demonstrated for the wild-type strain. The effects of choline on the glycerophospholipid composition of lactate and glucose-grown cells is presented. The repressive effects of the two related bases, mono- and dimethylethanolamine, were examined, and reduced levels of (14)C-CH(3)-methionine incorporation were found for cells grown in the presence of these bases. The effect of choline on the methylation rates is reversible and glucosegrown cells regain the nonrepressed level of methylation activity in 60 to 80 min after removal of choline from the growth medium.     

Cholesteryl ester acyl oxidation and remodeling in murine macrophages: formation of oxidized phosphatidylcholine

Cholesterol is an essential component of eukaryotic cell membranes, regulating fluidity and permeability of the bilayer. Outside the membrane, cholesterol is esterified to fatty acids forming cholesterol esters (CEs). Metabolism of CEs is characterized by recurrent hydrolysis and esterification as part of the CE cycle; however, since recombinant 15-lipoxygenase (15-LO) was shown to oxidize cholesteryl linoleate of LDL, there has been interest in CE oxidation, particularly in the context atherogenesis. Studies of oxidized CE (oxCE) metabolism have focused on hydrolysis and subsequent reverse cholesterol transport with little emphasis on the fate the newly released oxidized fatty acyl component. Here, using mass spectrometry to analyze lipid oxidation products, CE metabolism in murine peritoneal macrophages was investigated. Ex vivo macrophage incubations revealed that cellular 15-LO directly oxidized multiple CE substrates from intracellular stores and from extracellular sources. Freshly harvested murine macrophages also contained 15-LO-specific oxCEs, suggesting the enzyme may act as a CE-oxidase in vivo. The metabolic fate of oxCEs, particularly the hydrolysis and remodeling of oxidized fatty acyl chains, was also examined in the macrophage. Metabolism of deuterated CE resulted in the genesis of deuterated, oxidized phosphatidylcholine (oxPC). Further experiments revealed these oxPC species were formed chiefly from the hydrolysis of oxidized CE and subsequent reacylation of the oxidized acyl components into PC.     

Relationship between ethanol tolerance and fatty acyl composition of Saccharomyces cerevisiae

Summary The effect of ethanol on exponential phase cultures of S. cerevisiae has been examined using l-alanine uptake and proton efflux as indices of ethanol tolerance. Preincubation with 2 M ethanol inhibited l-alanine uptake, proton efflux and fermentation rates. However, the effect of ethanol varied in yeast cells enriched with different fatty acyl residues. It was observed that cells enriched with polyunsaturated fatty acids acquired greater tolerance to ethanol as compared to monounsaturated fatty acids. By varying the degree of unsaturation of supplemented fatty acid, a sequential insertion of double bonds in yeast membrane lipid was achieved. Results demonstrated that S. cerevisiae became more resistant to ethanol with an increase in the degree of unsaturation and that membrane fluidity could be an important determinant of ethanol tolerance.     

Stability of the plasma membrane in Saccharomyces cerevisiae enriched with phosphatidylcholine or phosphatidylethanolamine.

Spheroplasts from Saccharomyces cerevisiae NCYC 366, enriched in phosphatidylethanolamine after growth in medium supplemented with 1 mM ethanolamine, were more resistant to osmotic lysis than were spheroplasts from cells grown in the presence of 1 mM choline and enriched in phosphatidylcholine.     

Production of glutathione in extracellular form by Saccharomyces cerevisiae

The present research was aimed at inducing, in a post fermentative procedure (biotransformation) and by modifying cell permeability, glutathione (GSH) accumulation and subsequent release from cells of Saccharomyces cerevisiae. With the aim of limiting process costs, research considered the possibility of employing baker's yeasts (S. cerevisiae), inexpensive cells source available on the market, in comparison with a collection strain. The tested yeasts showed different sensitivity to the chemical/physical treatments performed to alter cell permeability. Modest effects were evidenced with Triton, active only on Zeus yeast samples (1.7 g GSH/l, near 60% of which in extracellular form). Lauroyl sarcosine showed an interesting action on GB Italy sample (2.8 g GSH/l, near 80% extracellular). Lyophilization evidenced good performance with Lievitalia yeast strain (2.9 g GSH/l, 90% extracellular). The possibility of obtaining GSH directly in extracellular form represents an interesting opportunity of reducing GSH production cost and furthering the range of application of this molecule.     

Identification of Saccharomyces cerevisiae spindle pole body remodeling factors

The Saccharomyces cerevisiae centrosome or spindle pole body (SPB) is a dynamic structure that is remodeled in a cell cycle dependent manner. The SPB increases in size late in the cell cycle and during most cell cycle arrests and exchanges components during G1/S. We identified proteins involved in the remodeling process using a strain in which SPB remodeling is conditionally induced. This strain was engineered to express a modified SPB component, Spc110, which can be cleaved upon the induction of a protease. Using a synthetic genetic array analysis, we screened for genes required only when Spc110 cleavage is induced. Candidate SPB remodeling factors fell into several functional categories: mitotic regulators, microtubule motors, protein modification enzymes, and nuclear pore proteins. The involvement of candidate genes in SPB assembly was assessed in three ways: by identifying the presence of a synthetic growth defect when combined with an Spc110 assembly defective mutant, quantifying growth of SPBs during metaphase arrest, and comparing distribution of SPB size during asynchronous growth. These secondary screens identified four genes required for SPB remodeling: NUP60, POM152, and NCS2 are required for SPB growth during a mitotic cell cycle arrest, and UBC4 is required to maintain SPB size during the cell cycle. These findings implicate the nuclear pore, urmylation, and ubiquitination in SPB remodeling and represent novel functions for these genes.     

L-Asparaginase of Saccharomyces cerevisiae: an extracellular Enzyme.

During recent studies conducted with suspensions of three strains of Saccharomyces cerevisiae, it was observed that ammonia was rapidly liberated when L-asparagine was added to the medium. Subsequent investigation has revealed that these strains of S. cerevisiae have an externally active asparaginase as well as an internally active one. The appearance of the external asparaginase is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The internal enzyme appears to be constitutive. The external activity is relatively insensitive to para-hydroxymercuribenzoate inhibition, whereas the internal activity is highly inhibited by this compound.     

酿酒酵母发酵降解灵芝胞外多糖组分分析及活性研究

本研究采用酿酒酵母发酵的方法对灵芝胞外多糖进行了降解,并对其产物在表观粘度、分子量、多糖得率和含量及单糖组成和生物活性等方面进行了系统比较和分析。结果表明,灵芝发酵胞外液经酿酒酵母培养后,所得胞外液的表观粘度明显降低,其中多糖的分子量也随酵母培养时间的延长出现下降趋势,大分子多糖的分子量从3.55×10 ⁶g/mol下降到1.93×10 ⁶g/mol,低分子多糖的分子量从6.18×10 ⁴g/mol下降到3.11×10 ⁴g/mol。多糖得率和含量测定结果显示,经酵母培养后,灵芝胞外液中20%乙醇沉淀所得20E组分得率明显降低,从2.43g/L下降到0.98g/L,但该组分多糖含量均较高,达到70%以上;而70%乙醇沉淀所得70E组分得率明显增加,达到1.87g/L。单糖组成分析表明,20E组分主要由葡萄糖组成,70E组分主要由甘露糖组成。各组分均表现出较好的与Dectin-1受体结合激活NF-κB增强免疫的活性,且经酿酒酵母发酵24h所得70E组分的活性最好。     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.     

Analysis of acyl CoA ester intermediates of the mevalonate pathway in Saccharomyces cerevisiae

The mevalonate pathway plays an important role in providing the cell with a number of essential precursors for the synthesis of biomass constituents. With respect to their chemical structure, the metabolites of this pathway can be divided into two groups: acyl esters [acetoacetyl CoA, acetyl CoA, hydroxymethylglutaryl (HMG) CoA] and phosphorylated metabolites (isopentenyl pyrophosphate, dimethylallyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate). In this study, we developed a method for the precise analysis of the intracellular concentration of acetoacetyl CoA, acetyl CoA and HMG CoA; and we used this method for quantification of these metabolites in Saccharomyces cerevisiae, both during batch growth on glucose and on galactose and in glucose-limited chemostat cultures operated at three different dilution rates. The level of the metabolites changed depending on the growth phase/specific growth rate and the carbon source, in a way which indicated that the synthesis of acetoacetyl CoA and HMG CoA is subject to glucose repression. In the glucose batch, acetyl CoA accumulated during the growth on glucose and, just after glucose depletion, HMG CoA and acetoacetyl CoA started to accumulate during the growth on ethanol. In the galactose batch, HMG CoA accumulated during the growth on galactose and a high level was maintained into the ethanol growth phase; and the levels of acetyl CoA and HMG CoA were more than two-fold higher in the galactose batch than in the glucose batch.     

Binding of Saccharomyces cerevisiae extracellular proteins to glucane.

Interactions of Saccharomyces cerevisiae cell wall proteins with purified yeast glucane were studied. Using the beta-glucanase (BGL2 gene product) as the model cell wall protein, strong binding to glucane was demonstrated at pH lower than 7, while at pH higher than 8 the reaction did not occur. NaCl (2 M) did not influence the binding, while urea in concentrations higher than 4 M affected the interactions. It was also found that most other cell wall proteins, as well as intracellular proteins, reacted with glucane in the same way, showing that the interactions of proteins with glucane are rather nonspecific. Soluble periplasmic proteins invertase and acid phosphatase failed to react with glucane under the same conditions, indicating that these proteins have certain structural features preventing their interactions with glucane.     

Differential regulation of the N-methyl transferases responsible for phosphatidylcholine synthesis in Saccharomyces cerevisiae

The enzymes catalyzing the conversion of phosphatidylethanolamine to phosphatidylcholine were assayed by measuring the incorporation of label from [¹⁴C-CH₃]-S-adenosyl-methionine into the endogenous phospholipids of particulate, cell-free preparations from S. cerevisiae grown in the presence of N-methylethanolamine, N,N-dimethylethanolamine, or choline. The results indicate that each base in the growth medium results in reduced levels of all the N-methyltransferase activity involved in the formation of the phosphatidyl ester of the given base. By following the conversion of exogenous [³²P]-phosphatidyldimethylethanolamine to [³²P]-phosphatidylcholine it has been shown that the activity of the third methyl transfer is 90% lower in particles prepared from choline grown cells than in particles prepared from cells grown without choline. The results suggest that there are at least two enzymes involved in the conversion of phosphatidylethanolamine to phosphatidylcholine and that their levels can be regulated individually.Supplementing the growth medium with any of the three methylated aminoethanols results in markedly increased cellular levels of their corresponding phosphatidyl esters and decreased levels of the precursor phosphatidyl esters. The fatty acid composition of phosphatidylcholine also changes when the medium is supplemented with choline suggesting that the proportions of the molecular species of this phosphatide depends on whether synthesis is via methylation of phosphatidylethanolamino or from the supplemented aminoethanol.     

Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1. Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end. We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions. Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle. Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle. We provide evidence for dynamic remodeling of these components at telomeres.