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...

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...

In the yeast, Saccharomyces cerevisiae, similar to higher eukaryotes most phospholipids are synthesized in microsomes. Mitochondria contribute to the cellular biosynthesis of phospholipids insofar as they harbor phosphatidylethanolamine decarboxylase, and enzymes of phosphatidylglycerol and cardiolipin synthesis. In this paper we present evidence that certain enzymes of phospholipid biosynthesis, namely phosphatidylserine and phosphatidylinositol synthase, are enriched in a special microsomal fraction associated with mitochondria, which we named MAM. This fraction was isolated and characterized with respect to marker enzymes, protein and phospholipid composition, and enzymes of phospholipid synthesis. According to these analyses MAMs are a specialized subfraction of the endoplasmic reticulum, which is distinct from other microsomal subfractions. Phosphatidylserine synthesized in MAMs can be readily imported into mitochondria and converted to phosphatidylethanolamine. Reassociation of MAMs with purified mitochondria led to reconstitution of the import of phosphatidylserine into mitochondria. Organelle contact is suggested as a possible mechanism of this process.

Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparentiy located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.In eucaryotic cells, phospholipids required for the biogenesis and maintenance of membranes are synthesized in distinct subcellular compartments. In mammalian cells, the bulk of cellular phospholipids is synthesized in the endoplasmic reticulum (13), with specific contributions from mitochondria (phosphatidylglycerol and cardiolipin [18]; phosphatidylethanolamine via decarboxylation of phosphatidylserine [13]) and peroxisomes (acyldihydroxyacetonephosphate [25] and its alkyl analog [15]). In contrast to mammalian cells, in Saccharomyces cerevisiae most of the reactions involved in phospholipid biosynthesis have been reported to occur both in mitochondria and in the endoplasmic reticulum (9).The present study on the subcellular and submitochondrial lpcalization of phospholipid synthesizing enzymes in S. cerevisiae was motivated mainly by our interest in the mechanism(s) underlying intracellular phospholipid transport. Knowledge of the origin of the various phospholipids is a prerequisite for an understanding of the process of their transfer across and between subcellular membranes.There are only few reports dealing with the subcellular localization of phospholipid-synthesizing enzymes in S. cerevisiae. Some are at variance, e.g., with respect to ...

No abstract