A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids.

Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta 1234, 214-220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.

Biochemical characterization and subcellular localization of the sterol C-24(28) reductase, erg4p, from the yeast saccharomyces cerevisiae.

The yeast ERG4 gene encodes sterol C-24(28) reductase which catalyzes the final step in the biosynthesis of ergosterol. Deletion of ERG4 resulted in a complete lack of ergosterol and accumulation of the precursor ergosta-5,7,22,24(28)-tetraen-3beta-ol. An erg4 mutant strain exhibited pleiotropic defects such as hypersensitivity to divalent cations and a number of drugs such as cycloheximide, miconazole, 4-nitroquinoline, fluconazole, and sodium dodecyl sulfate. Similar to erg6 mutants, erg4 mutants are sensitive to the Golgi-destabilizing drug brefeldin A. Enzyme activity measurements with isolated subcellular fractions revealed that Erg4p is localized to the endoplasmic reticulum. This view was confirmed in vivo by fluorescence microscopy of a strain expressing a functional fusion of Erg4p to enhanced green fluorescent protein. We conclude that ergosterol biosynthesis is completed in the endoplasmic reticulum, and the final product is supplied from there to its membranous destinations.

Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains.

The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance.

Lipid composition of subcellular membranes of an FY1679-derived haploid yeast wild-type strain grown on different carbon sources.

The aim of the project EUROFAN (European Functional Analysis Network) is to elucidate the function of unknown genes of the yeast Saccharomyces cerevisiae at a large scale. Functional analysis is based on general and specific tests with yeast deletion strains. A prerequisite for these studies is a profound knowledge of the biochemistry and cell biology of the corresponding wild-type strain FY1679. As a contribution from our laboratory we present here a systematic lipid analysis of the major organelles isolated from FY1679 grown in the presence of different carbon sources. Phospholipid, sterol and fatty acid composition are characteristic for each organelle. Moreover, growth of the yeast on glucose, ethanol or lactate causes in some cases marked changes of the organelle lipid pattern. As the most prominent example, cultivation of the yeast on non-fermentable carbon sources results in an increase of mitochondrial cardiolipin. As another example, the ratio of unsaturated to saturated fatty acids is enhanced in cells grown on ethanol or lactate as compared to glucose. Thus, the lipid composition of yeast subcellular membranes reflects in a significant way the nutrient conditions caused by variation of the carbon source.

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.

Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane.

Systematic analysis of yeast strains with possible defects in lipid metabolism.

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild-type strains, among them FY1679, CEN.PK2-1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism.

YDL142c encodes cardiolipin synthase (Cls1p) and is non-essential for aerobic growth of Saccharomyces cerevisiae.

The unassigned open reading frame YDL142c was identified to code for cardiolipin synthase, Cls1p. A cls1 deletion strain is viable on glucose, galactose, ethanol, glycerol and lactate containing media, although the growth rate on non-fermentable carbon sources is decreased. Mitochondria of the cls1 mutant are devoid of cardiolipin but accumulate the cardiolipin precursor phosphatidylglycerol when grown on non-fermentable carbon sources. Specific activity of phosphatidylglycerolphosphate synthase in cls1 is reduced to 30-75% of the wild-type level. Amounts of mitochondrial cytochromes and activity of cytochrome c oxidase, however, are not affected in the cls1 deletion strain. Collectively, these data indicate that cardiolipin is not essential for aerobic growth of Saccharomyces cerevisiae.

Characterization of a non-specific lipid transfer protein associated with the peroxisomal membrane of the yeast, Saccharomyces cerevisiae.

A lipid transfer protein with a broad substrate specificity is associated with the peroxisomal membrane of the yeast Saccharomyces cerevisiae. The protein catalyzes in vitro the transfer of various phospholipids, phosphatidylinositol and phosphatidylserine being translocated at the highest rates. The transfer protein can be released from peroxisomal membranes by treatment with 0.25 M KCl and highly enriched using conventional chromatographic techniques. It is inactivated by heat, detergents, divalent cations and proteinases. During various steps of purification this lipid transfer protein co-fractionated with peroxisomal acyl-CoA oxidase (Pox1p). In a pox1 disruptant peroxisomal lipid transfer activity was still present, although at a reduced level. The peroxisomal lipid transfer protein from the pox1 mutant exhibited different chromatographic properties as compared to the wild-type strain suggesting that acyl-CoA oxidase and the peroxisomal lipid transfer protein may from a complex.

Phospholipid-synthesizing enzymes in Golgi membranes of the yeast, Saccharomyces cerevisiae.

Golgi membranes of the yeast, Saccharomyces cerevisiae, were isolated by a method similar to the procedure described by Cleves et al. [Cell 64 (1991) 789-800]. Marker proteins of the Golgi, such as Kex2 protease and GDPase, are highly enriched in these preparations. The phospholipid and ergosterol content of Golgi membranes is low. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol are the major phospholipids of this compartment. The amount of phosphatidylserine in the Golgi is significantly higher than in yeast bulk membranes. Inositol-containing sphingolipids, especially inositolphosphorylceramide, are highly enriched in Golgi membranes. Two phospholipid-synthesizing enzymes, namely phosphatidylinositol synthase and sn-1,2-diacylglycerol cholinephosphotransferase, are detected in the Golgi at a specific activity which exceeds that of the endoplasmic reticulum.

Characterization of a microsomal subfraction associated with mitochondria of the yeast, Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria.

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.

Export of steryl esters from lipid particles and release of free sterols in the yeast, Saccharomyces cerevisiae.

Fatty acyl esters of the yeast specific sterol, ergosterol, are exclusively stored in lipid particles. Under conditions of sterol deficiency, e.g., in the presence of terbinafine, an inhibitor of fungal squalene epoxidase, steryl esters are hydrolyzed, and sterols are set free for membrane formation. Lipid particles do not contain steryl-ester hydrolase activity themselves; the highest specific activity of this enzyme is found in the plasma membrane. Therefore, steryl esters have to be exported from lipid particles to their site of hydrolytic cleavage. This process of translocation and metabolic conversion was studied in vivo. Addition of nocodazole to terbinafine-treated cells did not disturb the mobilization of steryl esters, indicating that this process is not mediated by microtubuli-dependent vesicle flux. Under the influence of inhibitors of cellular energy production (azide and fluoride) and protein biosynthesis (cycloheximide) mobilization of steryl esters came to an halt. These results support the view that ongoing membrane proliferation may be a driving force for the release of sterols from steryl esters of lipid particles.

Transport of phospholipids between subcellular membranes of wild-type yeast cells and of the phosphatidylinositol transfer protein-deficient strain Saccharomyces cerevisiae sec 14.

The transfer of glycerophospholipids between microsomes and mitochondria, and from internal membranes to the plasma membrane of Saccharomyces cerevisiae was characterized. Cellular energy production was found to be essential for intracellular translocation of phospholipids, but neither a membrane potential nor an intact cytoskeleton are required for this process. Using the temperature-sensitive mutant strain Saccharomyces cerevisiae sec 14, which is defective in the phosphatidylinositol transfer protein, it could be demonstrated that this protein is not involved in the transport of phosphatidylinositol and phosphatidylcholine from internal membranes to the plasma membrane. Our results also confirm earlier findings that phosphatidylinositol and phosphatidylcholine can be delivered to the plasma membrane in a process independent of the flux of vesicles competent for protein secretion.