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...
Emerging evidence indicates that disruption of the gut microbial community (dysbiosis) impairs mental health. Germ-free mice and antibiotic-induced gut dysbiosis are two approaches to establish causality in gut microbiota-brain relationships. However, both models have limitations, as germ-free mice display alterations in blood-brain barrier and brain ultrastructure and antibiotics may act directly on the brain. We hypothesized that the concerns related to antibiotic-induced gut dysbiosis can only adequately be addressed if the effect of intragastric treatment of adult mice with multiple antibiotics on (i) gut microbial community, (ii) metabolite profile in the colon, (iii) circulating metabolites, (iv) expression of neuronal signaling molecules in distinct brain areas and (v) cognitive behavior is systematically investigated. Of the antibiotics used (ampicillin, bacitracin, meropenem, neomycin, vancomycin), ampicillin had some oral bioavailability but did not enter the brain. 16S rDNA sequencing confirmed antibiotic-induced microbial community disruption, and metabolomics revealed that gut dysbiosis was associated with depletion of bacteria-derived metabolites in the colon and alterations of lipid species and converted microbe-derived molecules in the plasma. Importantly, novel object recognition, but not spatial, memory was impaired in antibiotic-treated mice. This cognitive deficit was associated with brain region-specific changes in the expression of cognition-relevant signaling molecules, notably brain-derived neurotrophic factor, N-methyl-d-aspartate receptor subunit 2B, serotonin transporter and neuropeptide Y system. We conclude that circulating metabolites and the cerebral neuropeptide Y system play an important role in the cognitive impairment and dysregulation of cerebral signaling molecules due to antibiotic-induced gut dysbiosis.
Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol A4-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.Lipid transport in eukaryotic cells is an essential process, because synthesis of lipids is restricted to certain organelles, whereas lipids are required as constitutive components of all subcellular membranes (3, 37). Lipid migration must be efficiently regulated, because lipids are not randomly distributed among subcellular membranes. In fact, certain lipids are characteristic for specific membranes, e.g., cardiolipin for the inner mitochondrial membrane (6) and sterols (15, 41) and sphingolipids (15, 24) for the plasma membrane. Possible mechanisms of lipid transport are spontaneous or proteincatalyzed transfer of lipid monomers between membranes, vesicle flow, and membrane contact and fusion (3).Sterols are essential components of the eukaryotic plasma membrane. The mechanism of their transport from internal membranes, where they are synthesized, to the periphery of the cell is still obscure. Vesicle flow as a possible mechanism seems very likely, but the vesicles involved need not be identical to protein secretory vesicles (36). Sterol carrier proteins, which have been shown to stimulate translocation of sterols in vitro, have not been proven to catalyze this process in vivo (1).We have chosen the yeast Saccharomyces cerevisiae as a model cell to study intracellular transport of sterols. The yeast-specific sterol ergosterol is structurally and functionally related to sterols found in higher eukaryotes. Under conditions in which yeast cells cannot produce their own ergosterol, e.g., under anaerobiosis, in auxotrophic mutants, or in the presence of inhibitors of sterol biosynthesis, addition of ergosterol to the growth medium and uptake into cells are essential for cellular growth and pro...
Lipid particles of the yeast, Saccharomyces cerevisiae, were isolated to high purity and their components were analysed. The hydrophobic core of this organelle consists of triacylglycerols and steryl esters, which are almost exclusively located to that compartment. Lipid particles are stabilized by a surface membrane consisting of phospholipids and proteins. Electron microscopy confirmed the purity of the preparations and the proposed structure deduced from biochemical experiments. Major proteins of lipid particles have molecular weights of 72, 52, 43 and 34 kDa, respectively. The 43 kDa protein reacts with an antiserum against human apolipoprotein AII. In lipid particles of the yeast mutant strain S. cerevisiae erg6, which is deficient in sterol delta 24-methyltransferase, this protein is missing thereby identifying the protein and confirming our previous finding (Zinser et al., 1993) that sterol delta 24-methylation is associated with lipid particles. A possible involvement of surface proteins of lipid particles in the interaction with other organelles is discussed with respect to sterol translocation in yeast.
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