Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae

Abstract

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...

Identification of a novel, Ca2+‐dependent phospholipase D with preference for phosphatidylserine and phosphatidylethanolamine in Saccharomyces cerevisiae

“…Since PtdSer (33% of total phospholipid,0 and PtdEtn (20%) are highly enriched in the plasma membrane [26], this preference for aminophospholipids might be of physiological significance. Activity towards PtdEtn has been reported for PLsD from mammalian and plant sources [27,28], while PtdSer has not yet been described to be a substrate for eukaryotic PLsD.…”

Import of sterols into mitochondria of the yeast Saccharomyces cerevisiae

“…A driving force for the import of ergosterol in vitro may be the imbalance of the sterol concentration between donor vesicles and the outer mitochondrial membrane caused by the lipid transfer protein. Since the concentration of ergosterol in the inner membrane of yeast mitochondria is much higher than in the outer membrane [4,7], ergosterol has to be imported against a concentration gradient. Cholesterol is not a component of both yeast mitochondrial membranes and partitioning effects may lead to an equilibrium between donor vesicles, the outer and the inner mitochondrial membrane.…”

“…Radioactivity was measured by liquid scintillation counting using Safety Cocktail (Baker). Sterols [3,4] and protein [21] were quantified by published procedures. SDS-PAGE was carried out by the method of Laemmli [22] and Western blotting using antibodies against mitochondrial porin was performed as described by Haid and Suissa [23].…”

“…Sterol synthesis is considered to be a microsomal process. Recent results from our laboratory obtained with the yeast Saecharomyces cerevisiae demonstrated the participation of so-called lipid particles [4][5][6] in the biosynthesis of sterols. The fact that site(s) of synthesis and subcellular destination(s) of sterols are not identical, necessitates intracellular translocation of these lipids.…”

“…In higher eukaryotes and Neurospora crassa, most of the mitochondrial sterol is located in the outer membrane, whereas in mitochondria of the yeast S. cerevisiae the majority of sterols is inner membrane associated [4]. The mechanisms of import into mitochondria and intramitochondrial translocation of sterols are poorly understood.…”