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.

Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides.

All 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 lag1Delta lac1Delta mutants lacking acyl-CoA-dependent ceramide synthesis, which require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2Delta.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, 2Delta.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, W303lag1Delta lac1Delta ypc1Delta ydc1Delta 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.

Yeast sphingolipids do not need to contain very long chain fatty acids.

Synthesis of VLCFAs (very long chain fatty acids) and biosynthesis of DHS (dihydrosphingosine) both are of vital importance for Saccharomyces cerevisiae. The bulk of VLCFAs and DHS are used for ceramide synthesis by the Lag1p (longevity-assurance gene 1)/Lac1p (longevity-assurance gene cognate 1)/Lip1p (Lag1p/Lac1p interacting protein) ceramide synthase. LAG1 and LAC1 are redundant but LIP1 is essential. Here we show that 4Delta (lag1Deltalac1Deltaypc1Deltaydc1Delta) cells devoid of all known endogenous ceramide synthesis pathways are unviable but can be rescued by the expression of Lass5, a mouse LAG1 homologue. Ceramide synthase activity of 4Delta.Lass5 cells only utilizes C16 and C18 fatty acids and does not require the help of Lip1p, an essential cofactor of Lag1p/Lac1p. HPLC-electrospray ionization-MS/MS analysis demonstrated that in IPCs (inositolphosphorylceramides) of 4Delta.Lass5, the very long chain fatty acids (C26 and C24) account for <1% instead of the normal >97%. Notwithstanding, IPCs incorporated into glycosylphosphatidylinositol anchors of 4Delta.Lass5 show normal mobility on TLC and the ceramide- and raft-dependent traffic of Gas1p (glycophospholipid-anchored surface protein) from endoplasmic reticulum to Golgi remains almost normal. Moreover, the biosynthesis of C24:0 fatty acids remains essential. Thus, C(24:0) and dihydrosphingosine are both necessary for survival of yeast cells even if they utilize C16 and C18 fatty acids for sphingolipid biosynthesis.