Chemogenetic E-MAP in Saccharomyces cerevisiae for Identification of Membrane Transporters Operating Lipid Flip Flop.

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.

Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated.

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 endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found 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 under this condition. We established hydroxylated C26 fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only 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 revealed that vesicle-mediated transport between Golgi apparatus, 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-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals 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, the 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.

Enzyme-coupled assays for flip-flop of acyl-Coenzyme A in liposomes.

Acyl-Coenzyme A is made in the cytosol. Certain enzymes using acyl-CoA seem to operate in the lumen of the ER but no corresponding flippases for acyl-CoA or an activated acyl have been described. In order to test the ability of purified candidate flippases to operate the transport of acyl-CoA through lipid bilayers in vitro we developed three enzyme-coupled assays using large unilamellar vesicles (LUVs) obtained by detergent removal. The first assay uses liposomes encapsulating a water-soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate (G3P). It measures formation of [(3)H]lyso-phosphatidic acid inside liposomes after [(3)H]palmitoyl-CoA has been added from outside. Two other tests use empty liposomes containing [(3)H]palmitoyl-CoA in the inner membrane leaflet, to which either soluble acyl-CoA:glycerol-3-phosphate acyl transferase plus glycerol-3-phosphate or alkaline phosphatase are added from outside. Here one can follow the appearance of [(3)H]lyso-phosphatidic acid or of dephosphorylated [(3)H]acyl-CoA, respectively, both being made outside the liposomes. Although the liposomes may retain small amounts of detergent, all these tests show that palmitoyl-CoA crosses the lipid bilayer only very slowly and that the lipid composition of liposomes barely affects the flip-flop rate. Thus, palmitoyl-CoA cannot cross the membrane spontaneously implying that in vivo some transport mechanism is required.

Cdc1 removes the ethanolamine phosphate of the first mannose of GPI anchors and thereby facilitates the integration of GPI proteins into the yeast cell wall.

Temperature-sensitive cdc1(ts) mutants are reported to stop the cell cycle upon a shift to 30°C in early G2, that is, as small budded cells having completed DNA replication but unable to duplicate the spindle pole body. A recent report showed that PGAP5, a human homologue of CDC1, acts as a phosphodiesterase removing an ethanolamine phosphate (EtN-P) from mannose 2 of the glycosylphosphatidylinositol (GPI) anchor, thus permitting efficient endoplasmic reticulum (ER)-to-Golgi transport of GPI proteins. We find that the essential CDC1 gene can be deleted in mcd4∆ cells, which do not attach EtN-P to mannose 1 of the GPI anchor, suggesting that Cdc1 removes the EtN-P added by Mcd4. Cdc1-314(ts) mutants do not accumulate GPI proteins in the ER but have a partial secretion block later in the secretory pathway. Growth tests and the genetic interaction profile of cdc1-314(ts) pinpoint a distinct cell wall defect. Osmotic support restores GPI protein secretion and actin polarization but not growth. Cell walls of cdc1-314(ts) mutants contain large amounts of GPI proteins that are easily released by ß-glucanases and not attached to cell wall ß1,6-glucans and that retain their original GPI anchor lipid. This suggests that the presumed transglycosidases Dfg5 and Dcw1 of cdc1-314(ts) transfer GPI proteins to cell wall ß1,6-glucans inefficiently.

Characterization of yeast mutants lacking alkaline ceramidases YPC1 and YDC1.

Humans and yeast possess alkaline ceramidases located in the early secretory pathway. Single deletions of the highly homologous yeast alkaline ceramidases YPC1 and YDC1 have very little genetic interactions or phenotypes. Here, we performed chemical-genetic screens to find deletions/conditions that would alter the growth of ypc1∆ydc1∆ double mutants. These screens were essentially negative, demonstrating that ceramidase activity is not required for cell growth even under genetic stresses. A previously reported protein targeting defect of ypc1∆ could not be reproduced and reported abnormalities in sphingolipid biosynthesis detected by metabolic labeling do not alter the mass spectrometric lipid profile of ypc1∆ydc1∆ cells. Ceramides of ypc1∆ydc1∆ remained normal even in presence of aureobasidin A, an inhibitor of inositolphosphorylceramide synthase. Moreover, in caloric restriction conditions Ypc1p reduces chronological life span. A novel finding is that, when working backwards as a ceramide synthase in vivo, Ypc1p prefers C24 and C26 fatty acids as substrates, whereas it prefers C16:0, when solubilized in detergent and working in vitro. Therefore, its physiological activity may not only concern the minor ceramides containing C14 and C16. Intriguingly, so far the sole discernable benefit of conserving YPC1 for yeast resides with its ability to convey relative resistance toward H2O2.

Topology of the microsomal glycerol-3-phosphate acyltransferase Gpt2p/Gat1p of Saccharomyces cerevisiae.

All glycerophospholipids are made from phosphatidic acid, which, according to the traditional view, is generated at the cytosolic surface of the ER. In yeast, phosphatidic acid is synthesized de novo by two acyl-CoA-dependent acylation reactions. The first is catalysed by one of the two homologous glycerol-3-phosphate acyltransferases Gpt2p/Gat1p and Sct1p/Gat2p, the second by one of the two 1-acyl-sn-glycerol-3-phosphate acyltransferases Slc1p and Ale1p/Slc4p. To study the biogenesis and topology of Gpt2p we observed the location of dual topology reporters inserted after various transmembrane helices. Moreover, using microsomes, we probed the accessibility of natural and substituted cysteine residues to a membrane impermeant alkylating agent and tested the protease sensitivity of various epitope tags inserted into Gpt2p. Finally, we assayed the sensitivity of the acyltransferase activity to membrane impermeant agents targeting lysine residues. By all these criteria we find that the most conserved motifs of Gpt2p and its functionally relevant lysines are oriented towards the ER lumen. Thus, the first step in biosynthesis of phosphatidic acid in yeast seems to occur in the ER lumen and substrates may have to cross the ER membrane.