title: Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated.
abstract: 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.
authors.affiliation: Division of Biochemistry, Department of Biology, University of Fribourg, Fribourg, Switzerland.
authors.forename: Natalia S
authors.initials: NS
authors.lastname: Voynova
authors.affiliation: Division of Biochemistry, Department of Biology, University of Fribourg, Fribourg, Switzerland.
authors.forename: Carole
authors.initials: C
authors.lastname: Roubaty
authors.affiliation: Division of Biochemistry, Department of Biology, University of Fribourg, Fribourg, Switzerland.
authors.forename: Hector M
authors.initials: HM
authors.lastname: Vazquez
authors.affiliation: Division of Biochemistry, Department of Biology, University of Fribourg, Fribourg, Switzerland.
authors.forename: Shamroop K
authors.initials: SK
authors.lastname: Mallela
authors.affiliation: Department of Biochemistry and Molecular Biology, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark.
authors.forename: Christer S
authors.initials: CS
authors.lastname: Ejsing
authors.affiliation: Division of Biochemistry, Department of Biology, University of Fribourg, Fribourg, Switzerland andreas.conzelmann@unifr.ch.
authors.forename: Andreas
authors.initials: A
authors.lastname: Conzelmann
mesh_terms: D000483:Alleles; D001692:Biological Transport / Q000187:drug effects; D053898:Biosynthetic Pathways / Q000187:drug effects; D002518:Ceramides / Q000378:metabolism; D047630:Depsipeptides / Q000494:pharmacology; D004318:Doxycycline / Q000494:pharmacology; D004843:Epistasis, Genetic / Q000187:drug effects; D017353:Gene Deletion; D063990:Gene Ontology; D005820:Genetic Testing; D006028:Glycosphingolipids / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006868:Hydrolysis; D066292:Lipid Droplets / Q000187:drug effects / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011796:Quinacrine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013107:Sphingolipids / Q000096:biosynthesis; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism*
chemical_list: D002518:Ceramides; D047630:Depsipeptides; D006028:Glycosphingolipids; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; C477937:inositolphosphorylceramide; C071398:aureobasidin A; D006602:Hexosyltransferases; C088714:phosphatidylinositol-ceramide phosphoinositol transferase; D011796:Quinacrine; D004318:Doxycycline