The hydrolysis of ceramides in yeast is catalysed by the alkaline ceramidases Ypc1p and Ydc1p, two highly homologous membrane proteins localized to the ER (endoplasmic reticulum). As observed with many enzymes, Ypc1p can also catalyse the reverse reaction, i.e. condense a non-esterified fatty acid with PHS (phytosphingosine) or DHS (dihydrosphingosine) and thus synthesize ceramides. When incubating microsomes with [(3)H]palmitate and PHS, we not only obtained the ceramide PHS-[(3)H]C(16:0), but also a more hydrophobic compound, which was transformed into PHS-[(3)H]C(16:0) upon mild base treatment. The biosynthesis of a lipid with similar characteristics could also be observed in living cells labelled with [(14)C]serine. Its biosynthesis was dependent on the diacylglycerol acyltransfereases Lro1p and Dga1p, suggesting that it consists of an acylceramide. The synthesis of acylceramide could also be monitored using fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazole)-ceramides as an acceptor substrate for microsomal assays. The Lro1p-dependent transfer of oleic acid on to NBD-ceramide was confirmed by high-resolution Fourier transform and tandem MS. Immunopurified Lro1p was equally able to acylate NBD-ceramide. Lro1p acylates NBD-ceramide by attaching a fatty acid to the hydroxy group on the first carbon atom of the long-chain base. Acylceramides are mobilized when cells are diluted into fresh medium in the presence of cerulenin, an inhibitor of fatty acid biosynthesis.

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.

b 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 C 26 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. Y east sphingolipids have been recognized to be essential for cell growth and survival ever since the initial demonstration by Bob Dickson and colleagues that LCB1 and LCB2, coding for two components of serine palmitoyltransferase, are essential genes (1) (Fig. 1). In 1997 the same group also demonstrated that the essential AUR1 gene encodes the enzyme making inositolphosphorylceramides (IPCs), implying that IPCs are essential for cell growth (2); in contrast, the synthesis of mannosyl-IPCs (MIPCs) and inositol-phospho-MIPCs [M(IP) 2 Cs] was found to be dispensable (3-5). The essentiality of IPCs, however, has been questioned, since cells lacking all ceramide synthases are highly resistant to the inhibitor aureobasidin A (AbA), a very specific and potent inhibitor of Aur1, which rapidly induces cell death in wild-type (WT) yeast cells (6-9). Nevertheless, several lines of evidence suggest that IPCs are essential for growth. This had been already suggested by early studies showing that the lethality of the lcb1⌬ mutation (Fig. 1), eliminating all sphingolipid biosynthesis, is suppressed in cells harboring the SLC1-1 gain-of-function mutation, which allows making phosphatidylinositol (PI) with the very-long-chain fatty acid (VLCFA) C 26:0 in the sn-2 position. Structurally, thi...