No abstract

UPC2 and ECM22 belong to a Zn(2)-Cys(6) family of fungal transcription factors and have been implicated in the regulation of sterol synthesis in Saccharomyces cerevisiae and Candida albicans. Previous reports suggest that double deletion of these genes in S. cerevisiae is lethal depending on the genetic background of the strain. In this investigation we demonstrate that lethality of upc2D ecm22D in the S288c genetic background is attributable to a mutation in the HAP1 transcription factor. In addition we demonstrate that strains containing upc2D ecm22D are also inviable when carrying deletions of ERG6 and ERG28 but not when carrying deletions of ERG3, ERG4, or ERG5. It has previously been demonstrated that UPC2 and ECM22 regulate S. cerevisiae ERG2 and ERG3 and that the erg2D upc2D ecm22D triple mutant is also synthetically lethal. We used transposon mutagenesis to isolate viable suppressors of hap1D, erg2D, erg6D, and erg28D in the upc2D ecm22D genetic background. Mutations in two genes (YND1 and GDA1) encoding apyrases were found to suppress the synthetic lethality of three of these triple mutants but not erg2D upc2D ecm22D. We show that deletion of YND1, like deletion of GDA1, alters the sphingolipid profiles, suggesting that changes in sphingolipids compensate for lethality produced by changes in sterol composition and abundance.

KEG1/YFR042w of Saccharomyces cerevisiae is an essential gene that encodes a 200-amino acid polypeptide with four predicted transmembrane domains. The green fluorescent proteinor Myc 6 -tagged Keg1 protein showed the typical characteristics of an integral membrane protein and was found in the endoplasmic reticulum by fluorescence imaging. Immunoprecipitation from the Triton X-100-solubilized cell lysate revealed that Keg1 binds to Kre6, which has been known to participate in ␤-1,6-glucan synthesis. To analyze the essential function of Keg1 in more detail, we constructed temperature-sensitive mutant alleles by error-prone polymerase chain reaction. The keg1-1 mutant cells showed a common phenotype with ⌬kre6 mutant including hypersensitivity to Calcofluor white, reduced sensitivity to the K1 killer toxin, and reduced content of ␤-1,6-glucan in the cell wall. These results suggest that Keg1 and Kre6 have a cooperative role in ␤-1,6-glucan synthesis in S. cerevisiae.The fungal cell wall is an essential extracellular network composed of polysaccharides and proteins that contributes to protect the cell from internal turgor pressure and external harmful agents. It also contributes in the selective uptake and excretion of various materials and works in communication with the environment. The yeast Saccharomyces cerevisiae cell wall is composed of polysaccharides (ϳ85%) and proteins (ϳ15%) and represents ϳ30% of the cell dry weight (1). Polysaccharides form a basal fibril network with branches and covalent linkage among the components. The proteins are heavily glycosylated with N-and O-linked saccharides mostly composed of mannose. Many of these mannoproteins are covalently linked to polysaccharide networks through glycosylphosphatidylinositol-anchor remnant or alkali-labile linkage (1, 2). Some proteins are physically trapped in the mesh of networks or are chemically linked with other proteins by a disulfide bond. Polysaccharides are long fibrillar and tensile ␤-1,3-glucans of ϳ1500 glucose units/chain, amorphous and flexible ␤-1,6-glucans of ϳ350 glucose units/chain, and linear microfibril chitins of ␤-1,4-linked N-acetylglucosamine (1-2%). ␤-1,3-Glucan and chitin are synthesized by specific polymerases embedded in the plasma membrane using cytoplasmic UDP-sugars as the substrates. The localization and activity of polymerases are controlled by the actin cytoskeleton and secretory system according to the environmental stimuli and cell cycle regulatory system. Conversely, ␤-1,6-glucan synthesis is more complex, and the gene products whose mutations have defects in the ␤-1,6-glucan content are localized throughout the secretory pathway and at the cell surface (3). KRE5 and CNE1, which encode UDP-glucose:glycoprotein glucosyltransferase-and calnexin-related proteins, CWH41 and ROT2, which encode glucosidases I and II, respectively, fungus-specific genes BIG1, ROT1, KRE9 and its homologue KNH1, KRE6, and SKN1, which encode type II membrane protein with sequence and structural similarity to family 16 glycoside hydrolases, a...

No abstract

No abstract