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While most yeast enzymes for the biosynthesis of glycerophospholipids, sphingolipids and ergosterol are known, genes for several postulated transporters allowing the flopping of biosynthetic intermediates and newly made lipids from the cytosolic to the lumenal side of the membrane are still not identified. An E-MAP measuring the growth of 142'108 double mutants generated by systematically crossing 543 hypomorphic or deletion alleles in genes encoding multispan membrane proteins, both on media with or without an inhibitor of fatty acid synthesis, was generated. Flc proteins, represented by 4 homologous genes encoding presumed FAD or calcium transporters of the ER, have a severe depression of sphingolipid biosynthesis and elevated detergent sensitivity of the ER. FLC1, FLC2 and FLC3 are redundant in granting a common function, which remains essential even when the severe cell wall defect of flc mutants is compensated by osmotic support. Biochemical characterization of some other genetic interactions shows that Cst26 is the enzyme mainly responsible for the introduction of saturated very long chain fatty acids into phosphatidylinositol and that the GPI lipid remodelase Cwh43, responsible for introducing ceramides into GPI anchors having a C26:0 fatty acid in sn-2 of the glycerol moiety can also use lyso-GPI protein anchors and various base resistant lipids as substrates. Furthermore, we observe that adjacent deletions in several chromosomal regions show strong negative genetic interactions with a single gene on another chromosome suggesting the presence of undeclared suppressor mutations in certain chromosomal regions that need to be identified in order to yield meaningful E-map data., Author Summary All living cells define their boundaries by lipid-containing membranes, which are impermeable to ions and water-soluble metabolic intermediates, and thus allow maintaining constant conditions inside the cells and stopping metabolic intermediates from diffusing away. Membranes are formed by amphiphilic lipids that have a hydrophilic and a hydrophobic component. Such lipids form flat double-layered sheets (bilayers) wherein the hydrophilic components of the constituent lipids are directed towards the aqueous surroundings, the hydrophobic ones populate the center of the bilayer. Membranes grow when enzymes resident in the bilayer synthesize new amphiphilic lipids. These enzymes have their active site on one side of the membrane and insert the newly made lipids in only one of the two layers. To ensure symmetric growth of membranes, cells need flippases catalyzing the transfer of lipids from one into the other layer. To identify unknown flippases we performed a chemogenetic interaction screen able to bring to light functions of unknown proteins through their genetic interaction with genes of known function. The data point to Flc proteins as potential lipid flippases of the endoplasmic reticulum, reveal novel lipid modifying activities of Cst26 and Cwh43 and suggest that undeclared suppressor mutations in certain chromosomal regions can generate false genetic interactions.

Inositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by Aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes ER stress, an increase in cytosolic calcium, reactive oxygen production and mitochondrial damage leading to apoptosis. We find that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidase YPC1 rescues cells in this condition. We establish hydroxylated C26 fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis only occurs when YPC1 is overexpressed. In contrast, overexpression of YPC1 has no beneficial effect when Aur1 is acutely repressed by AbA. A high throughput genetic screen reveals that vesicle mediated transport between Golgi, endosomes and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and Quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidant N-acetyl-cysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals (ROS) induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway but osmotic support does not improve the viability of wild type cells on AbA. Altogether, data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance.

In yeast, the inositolphosphorylceramides mostly contain C26:0 fatty acids. Inositolphosphorylceramides were considered to be important for viability because the inositolphosphorylceramide synthase AUR1 is essential. However, lcb1Δ cells, unable to make sphingoid bases and inositolphosphorylceramides, are viable if they harbor SLC1-1, a gain of function mutation in the 1-acyl-glycerol-3-phosphate acyltransferase SLC1. SLC1-1 allows the incorporation of C26:0 fatty acids into phosphatidylinositol (PI), thus generating PI″, an abnormal, C26-containing PI, presumably acting as surrogate for inositolphosphorylceramide. Here we show that the lethality of the simultaneous deletion of the known ceramide synthases LAG1/LAC1/LIP1 and YPC1/YDC1 can be rescued by the expression of SLC1-1 or the overexpression of AUR1. Moreover, lag1Δ lac1Δ ypc1Δ ydc1Δ (4Δ) quadruple mutants have been reported to be viable in certain genetic backgrounds but to still make some abnormal uncharacterized inositol-containing sphingolipids. Indeed, we find that 4Δ quadruple mutants make substantial amounts of unphysiological inositolphosphorylphytosphingosines but that they also still make small amounts of normal inositolphosphorylceramides. Moreover, 4Δ strains incorporate exogenously added sphingoid bases into inositolphosphorylceramides, indicating that these cells still possess an unknown pathway allowing the synthesis of ceramides. 4Δ cells also still add quite normal amounts of ceramides to glycosylphosphatidylinositol anchors. Synthesis of inositolphosphorylceramides and inositolphosphorylphytosphingosines is operated by Aur1p and is essential for growth of all 4Δ cells unless they contain SLC1-1. PI″, however, is made without the help of Aur1p. Furthermore, mannosylation of PI″ is required for the survival of sphingolipid-deficient strains, which depend on SLC1-1. In contrast to lcb1Δ SLC1-1, 4Δ SLC1-1 cells grow at 37 °C but remain thermosensitive at 44 °C.

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors the thus modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction, which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells having been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide based anchors. Such microsomes, when incubated with [³H]DHS, generate radiolabeled, ceramide-containing anchor lipids of the same structure as made by intact cells. Microsomes from cwh43Δ or mcd4Δ mutants, unable to make ceramide-based anchors in vivo, do not incorporate [³H]DHS into anchors in vitro. Moreover, gup1Δ microsomes incorporate [³H]DHS into the same abnormal anchor lipids as gup1Δ cells synthesize in vivo. Thus, the in vitro assay of ceramide incorporation into GPI anchors faithfully reproduces the events that occur in mutant cells. Incorporation of [³H]DHS into GPI proteins is observed with microsomes alone, but the reaction is stimulated by cytosol or bovine serum albumin, ATP plus coenzyme A or C26:0-Coenzyme A, particularly if microsomes are depleted of acyl-CoA. Thus, [³H]DHS cannot be incorporated into proteins in the absence of acyl-CoA.