Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1⌬ cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1⌬ and slc4⌬ cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1⌬ and slc4⌬ cells incubated with [ 14 C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1⌬ and slc4⌬ cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg 2؉ -dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1⌬ lac1⌬ double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1⌬ lac1⌬ yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1⌬ lac1⌬ yeast mutants.The only structural sphingolipids of Saccharomyces cerevisiae are the inositol phosphorylceramides (IPCs), 1 mannosylIPCs (MIPCs), and inositol phosphoryl-MIPC, which together represent a significant fraction of membrane lipids, especially in the plasma membrane (1-3). Recent progress has resulted in the identification of yeast genes involved in all enzymatic steps that are required for their biosynthesis (4). The intermediates in sphingolipid synthesis, dihydrosphingosine (DHS), phytosphingosine (PHS), and their 1-phosphorylated derivatives, as well as free ceramides, have been proposed to act as signal transduction molecules governing heat stress responses, endocytosis, glycosyl phosphatidylinositol protein transport, ubiquitin-dependent degradation of membrane channels, and progression through G 1 (for review, see Ref. 4).A key role in the sphingolipid pathway is played by ceramide synthase, as it not only catalyzes an essential biosynthetic reaction, but also influences the levels of long chain bases and ceramides, which have signaling function. The simultaneous deletion of LAG1 and its close homologue LAC1 eliminates all detectable acyl-CoA-dependent ceramide biosynthesis in yeast microsomes (5, 6). Moreover, lag1⌬ lac1⌬ cells have a drastically reduced amount of normal ceramides and IPCs, but exhibit a marked accumulation of free DHS and a compensatory increase of C26:0 fatty acids, which seem to be used for making a new form of phosphatidylinositol (PI) that we call PIЈ (5, 6). Whereas the single deletion of LAG1 or LAC1 had no abnormal growth phenotype, the concomitant deletion of LAG1 and LAC1 caused osmotic fragility, calcofluor white hypersensitivity, and a significant decrease of the growth rate in the genetic background of W303 cells, and the same double mutation was lethal in the background of YPK9 cells (5,7,8). Lethality of YPK9 lag1⌬lac1⌬ (herein named YPK9.2⌬) can be overcome by overexpression of LAG1 homologues from man (LA...

SummaryAll mature Saccharomyces cerevisiae sphingolipids comprise inositolphosphorylceramides containing C26:0 or C24:0 fatty acids and either phytosphingosine or dihydrosphingosine. Here we analysed the lipid profile of lag1D lac1D mutants lacking acyl-CoAdependent ceramide synthesis, which require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2D.YDC1, make sphingolipids containing exclusively dihydrosphingosine and an abnormally wide spectrum of fatty acids with between 18 and 26 carbon atoms. Like wild-type cells, 2D.YDC1 cells stop growing when exposed to Aureobasidin A (AbA), an inhibitor of the inositolphosphorylceramide synthase AUR1, yet their ceramide levels remain very low. This finding argues against a current hypothesis saying that yeast cells do not require inositolphosphorylceramides and die in the presence of AbA only because ceramides build up to toxic concentrations. Moreover, W303lag1D lac1D ypc1D ydc1D cells, reported to be AbA resistant, stop growing on AbA after a certain number of cell divisions, most likely because AbA blocks the biosynthesis of anomalous inositolphosphorylsphingosides. Thus, data argue that inositolphosphorylceramides of yeast, the equivalent of mammalian sphingomyelins, are essential for growth. Data also clearly confirm that wild-type strains, when exposed to AbA, immediately stop growing because of ceramide intoxication, long before inositolphosphorylceramide levels become subcritical.

The essential CDC1 gene of yeast encodes a Mn2+-dependent lipid phosphatase of the endoplasmic reticulum. Hypomorphic alleles affect Ca2+ signaling, actin polarization, Golgi inheritance, and cell cycle progression. Cdc1 removes an ethanolamine phosphate from the glycosylphosphatidylinositol (GPI) anchor and thereby facilitates integration of GPI proteins into the yeast cell wall.

Background:The active sites of bacterial and mammalian 1-acyl-sn-glycerol-3-phosphate acyltransferases are assumed to reside in the cytosol. Results: The topology of yeast 1-acyl-sn-glycerol-3-phosphate acyltransferases has been determined. Their most conserved residues and motifs are located in the ER lumen. Conclusion: Biosynthesis of phosphatidic acid in yeast may occur on the lumenal side of the ER. Significance: The data invite additional, complementary experimentation.