improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol A24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDP-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol A24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.Most of the enzymes involved in cellular phospholipid biosynthesis are membrane associated. In mammalian cells, the majority of phospholipids is synthesized in the endoplasmic reticulum (14). Phospholipids specifically required for mitochondrial function (cardiolipin and its precursor phosphatidylglycerol) as well as phosphatidylethanolamine (via decarboxylation of phosphatidylserine) are synthesized in mitochondrial membranes (11).In previous studies, several enzymes of phospholipid biosynthesis of the yeast Saccharomyces cerevisiae (10,26), namely glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine synthase, and phosphatidylinositol synthase, were detected both in the microsomal fraction and in the outer mitochondrial membrane. These observations were based mainly on the separation of subcellular membranes by differential centrifugation and on commonly used marker enzymes for the respective fractions. Motivated by our interest in the mechanisms of lipid flow and membrane assembly in yeasts and by conflicting data concerning the subcellular targeting of phosphatidylserine synthase (38), we reinvestigated the subcellular distribution of lipid-synthesizing enzymes by employing recently developed or improved fractionation procedures for mitochondrial and microsomal membranes, the nuclear membrane (24), the plasma membrane (37) Yeast subcellular membranes were also characterized with respect to their protein-to-lipid ratio, their content of ergosterol and ergosteryl esters, and their pattern of individual glycerophospholipids. Measurements of fluorescence anisotropy revealed significant difference...

Alzheimer’s disease is characterized by accumulation of amyloid plaques and tau aggregates in several cortical brain regions. Tau phosphorylation causes formation of neurofibrillary tangles and neuropil threads. Phosphorylation at tau Ser202/Thr205 is well characterized since labeling of this site is used to assign Braak stage based on occurrence of neurofibrillary tangles. Only little is known about the spatial and temporal phosphorylation profile of other phosphorylated tau (ptau) sites. Here, we investigate total tau and ptau at residues Tyr18, Ser199, Ser202/Thr205, Thr231, Ser262, Ser396, Ser422 as well as amyloid-β plaques in human brain tissue of AD patients and controls. Allo- and isocortical brain regions were evaluated applying rater-independent automated quantification based on digital image analysis. We found that the level of ptau at several residues, like Ser199, Ser202/Thr205, and Ser422 was similar in healthy controls and Braak stages I to IV but was increased in Braak stage V/VI throughout the entire isocortex and transentorhinal cortex. Quantification of ThioS-stained plaques showed a similar pattern. Only tau phosphorylation at Tyr18 and Thr231 was already significantly increased in the transentorhinal region at Braak stage III/IV and hence showed a progressive increase with increasing Braak stages. Additionally, the increase in phosphorylation relative to controls was highest at Tyr18, Thr231 and Ser199. By contrast, Ser396 tau and Ser262 tau showed only a weak phosphorylation in all analyzed brain regions and only minor progression. Our results suggest that the ptau burden in the isocortex is comparable between all analyzed ptau sites when using a quantitative approach while levels of ptau at Tyr18 or Thr231 in the transentorhinal region are different between all Braak stages. Hence these sites could be crucial in the pathogenesis of AD already at early stages and therefore represent putative novel therapeutic targets.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0557-6) contains supplementary material, which is available to authorized users.

Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448) showed that the gene product of YMR313c (named Tgl3p) is a component of yeast lipid particles, and deletion of this gene led to an increase in the cellular level of triacylglycerols (TAG). These observations suggested that TGL3 may encode a TAG lipase of Saccharomyces cerevisiae. Here we demonstrate by cell fractionation and by microscopic inspection of a strain bearing a Tgl3p-GFP hybrid that this polypeptide is highly enriched in the lipid particle fraction but virtually absent from other organelles. The entire TAG lipase activity of lipid particles is attributed to Tgl3p, because the activity in this organelle is completely absent in a ⌬tgl3 deletion mutant, whereas it is significantly enhanced in a strain overexpressing Tgl3p. A His 6 -tagged Tgl3p hybrid purified close to homogeneity from a yeast strain overexpressing this fusion protein exhibited high TAG lipase activity. Most importantly, experiments in vivo using the fatty acid synthesis inhibitor cerulenin demonstrated that deletion of TGL3 resulted in a decreased mobilization of TAG from lipid particles. The amino acid sequence deduced from the open reading frame YMR313c contains the consensus sequence motif GXSXG typical for lipolytic enzymes. Otherwise, Tgl3p has no significant sequence homology to other lipases identified so far. In summary, our data identified Tgl3p as a novel yeast TAG lipase at the molecular level and by function in vivo and in vitro.

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.Fatty acids and sterols are important building blocks of biomembranes. To maintain balanced cellular levels of these components, a certain portion is put on hold in the form of complex neutral lipids. The yeast Saccharomyces cerevisiae similar to other eukaryotic cells stores fatty acids and sterols in the form of triacylglycerols (TAG) 3 and steryl esters (SE). Both TAG and SE are hydrophobic molecules that are not soluble in the cytosol but are also not typical bilayer membrane lipids. Consequently, they need to be stored in specific subcellular compartments that are known as lipid particles (LP), lipid droplets, lipid bodies, or oil bodies. Yeast LP are small spherical organelles with an approximate diameter of 400 nm consisting of 95% neutral lipids, TAG, and SE, and only small amounts of phospholipids and proteins (1). The highly hydrophobic core formed by TAG and SE is surrounded by a phospholipid monolayer containing a well defined set of proteins (2).Identification of LP proteins in various types of cells has changed the view of this organelle. The presence of certain enzymes on the surface of LP suggests that besides lipid storage LP are involved in various metabolic processes. In S. cerevisiae ϳ40 proteins are known to be present on the surface of LP. Strikingly, most of these proteins are enzymes involved in lipid metabolism. As prominent examples, LP from S. cerevisiae harbor enzymes of phosphatidic acid biosynthesis (3), fatty acid activation (4 -6), sterol biosynthesis (7), but also TAG biosynthesis and degradation (8 -11) and SE hydrolysis (12-14).The biogenesis of LP is still a matter of dispute and is under investigation in several laboratories. Whereas little is known about the cell biology of this process, enzymes involved in the formation of major LP components, TAG and SE, have been identified within the last few years (for recent review see Refs. 15 and 16). In the yeast as in other types of cells, the last step of TAG synthesis requires acylation of diacylglycerol. Two different ...

The neutral lipids triacylglycerols (TAG)2 and steryl esters (SE) serve as an energy source and/or a source of building blocks (fatty acids and sterols) needed for membrane biogenesis. In addition, they are required as precursors for the synthesis of specific lipophilic components, e.g. steroid hormones or prostaglandins in higher eukaryotes (1-4). Because TAG and SE are unable to integrate into phospholipid bilayers they cluster and form the hydrophobic core of so-called lipid particles. The structure of this cell compartment is rather simple and reminiscent of lipoproteins in mammals, consisting of a hydrophobic core of neutral lipids that is surrounded by a phospholipid monolayer with a small amount of proteins embedded (reviewed in Ref. 5).Mobilization of neutral lipids from lipid particles is catalyzed by TAG lipases and SE hydrolases. Some proteins associated with the phospholipid monolayer of lipid particles, such as mammalian perilipins or oleosins of plants, were assumed to be involved in this process by serving as docking and/or activating proteins for hydrolytic enzymes. As an example, Londos and co-workers (6, 7) demonstrated that perilipin, the most abundant protein of mammalian lipid particles, accelerated lipolysis catalyzed by the cytosolic hormone-sensitive lipase.Localization of lipolytic enzymes in eukaryotes depends on the type of cell studied. As an example, Lehner et al. (8) reported the presence of a TAG lipase on cytosolic lipid droplets of pig liver cells. Spalinger et al. (9) demonstrated lipase activities in the cytosolic fraction of Caco2 cells, but also in the apical brush border membrane and other organelles. Information about the intracellular localization of plant lipolytic enzymes is rather limited, although many plant TAG lipases were isolated and characterized. It was reported that lipases of maize kernel were associated with oil bodies during germination (10). Similar to perilipins in mammalian cells the major proteins of oil bodies in plants, the oleosins, appear to act as receptors for binding and/or activation of TAG lipases (11). They seem to maximize the surface area of oil bodies, thus accelerating the mobilization of the storage oil from the hydrophobic core by lipases. Analysis of the lipid particle proteome of the yeast Saccharomyces cerevisiae did not identify polypeptides homologous to docking proteins like mammalian perilipins and oleosins of plants (12). Nevertheless, some yeast lipid particle proteins were identified as enzymes catalyzing degradation of TAG and SE (13-15). So far, Tgl3p has been the only TAG lipase identified in the yeast S. cerevisiae. In contrast to the mammalian hormone-sensitive lipase and plant lipases, Tgl3p is permanently associated with lipid particles and exhibits lipolytic activity independent of activator proteins (13). Detection of a small but significant TAG lipase activity in a tgl3⌬ deletion mutant (13) suggested the presence of at least one additional TAG lipase. This observation led us to search for proteins homologous to Tgl3...