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 ∼± 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 elo3Δ 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.

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.

The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipase B/lysophospholipase in yeast (Lee, S. K., Patton, J. L., Fido, M., Hines, L. K., Kohlwein, S. D., Paltauf, F., Henry, S. A., and Levin, D. E. (1994) J. Biol. Chem. 269, 19725-19730). Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases. In cell free extracts of a plb1 plb2 plb3 triple mutant, no phospholipase B activity was detectable. Upon overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant. Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids including phosphatidylinositol 3,4-bisphosphate and phosphatidic acid. Consistent with a function as an extracellular lysophospholipase, PLB2 overexpression conferred resistance to lyso-phosphatidylcholine. Deletion of Plb2p function had no effect on glycerophosphoinositol or glycerophosphocholine release in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoinositol release from the cells. In vitro, Plb3p hydrolyzes only phosphatidylinositol and phosphatidylserine and, to a lesser extent, their lysoanalogs. Plb3p activity in a plb1 plb2 mutant background was observed in periplasmic space extracts. Both Plb3p and Plb2p display transacylase activity in vitro, in the presence or absence, respectively, of detergent.Phospholipases B catalyze the hydrolytic cleavage of both acylester bonds of glycerophospholipids. Products of phospholipase B activity are fatty acids and water-soluble glycerophosphodiesters. In yeast, soluble degradation products are released to some extent into the culture medium, and are thus an indicator of cellular phospholipase B activity. The only acylester-hydrolyzing enzyme from yeast characterized so far at the molecular level is a phospholipase B encoded by the PLB1 gene (1). The highly glycosylated enzyme of about 220 kDa (73 kDa for the protein part, predicted from the sequence) is enriched in the yeast plasma membrane but was also found in the periplasmic space and in the culture supernatant. The lysophospholipase activity of Plb1p greatly exceeds the activity catalyzing the first step of hydrolysis; thus, lyso-phospholipids do not accumulate as intermediate products of Plb1p activity (2-4). In addition, this enzyme has transacylase activity, catalyzing the synthesis of phosphatidylcholine (PtdCho) 1 from two molecules of lyso-phosphatidylcholine. The physiological function of yeast phospholipase B is still unclear; neither disruption of the PLB1 gene nor its overexpression result in detectable growth phenotypes. However, the amount of glycerophosphocholine (GroPCho) and glycerophosphoethanolamine released into the culture su...

Acyl chains linked to phospholipids of the yeast, Saccharomyces cerevisiae, are mainly C16:1 and C18:1 accompanied by minor amounts of C14:0, C16:0 and C18:0. In view of this rather simple fatty acid composition, the question arose whether in yeast, as in higher eukaryotes, fatty acyl groups were characteristically distributed among the sn-1 and sn-2 positions of distinct phospholipid classes. Analysis of fatty acids linked to the sn-1 and sn-2 positions of the major phospholipids showed that indeed saturated fatty acyl groups predominated in the sn-1 positions. While the percentage of saturated fatty acids was low (10%) in phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) from cells grown on rich medium, it was higher in phosphatidylserine (PtdSer) (25%) and highest in phosphatidylinositol (PtdIns) (41%). Oleate was mainly linked to position sn-2, while palmitoleate predominated in position sn-1. Striking differences in the fatty acid distribution of phospholipids that are metabolically closely related (e.g. PtdSer and PtdEtn, PtdEtn and PtdCho, and PtdIns and PtdSer) suggest that pathways must exist for the generation of distinct phospholipid molecular species within the different phospholipid classes. The highly selective incorporation of exogenous [14C]palmitic acid (90%) and [3H]oleic acid (99%) into the sn-2 position of PtdCho, and the preferential incorporation of these fatty acids into the sn-2 position of PtdEtn (70 and 90%, respectively, for palmitic and oleic acid) are compatible with the postulate that phospholipase A2-mediated deacylation followed by reacylation of the lysophospholipids is involved in the generation of phospholipid species in yeast.