Gerald B. Wells

SPHINGOLIPIDS ARE POTENTIAL HEAT STRESS SIGNALS IN SACCHAROMYCES

The ability of organisms to quickly respond to stresses requires the activation of many intracellular signal transduction pathways. The sphingolipid intermediate ceramide is thought to be particularly important for activating and coordinating signaling pathways during mammalian stress responses. Here we present the first evidence that ceramide and other sphingolipid intermediates are signaling molecules in the Saccharomyces cerevisiaeheat stress response. Our data show a 2–3-fold transient increase in the concentration of C18-dihydrosphingosine and C18-phytosphingosine, more than a 100-fold transient increase in C20-dihydrosphingosine and C20-phytosphingosine, and a more stable 2-fold increase in ceramide containing C18-phytosphingosine and a 5-fold increase in ceramide containing C20-phytosphingosine following heat stress. Treatment of cells with dihydrosphingosine activates transcription of the TPS2 gene encoding a subunit of trehalose synthase and causes trehalose, a known thermoprotectant, to accumulate. Dihydrosphingosine induces expression of aSTRE-LacZ reporter gene, showing that the global stress response element, STRE, found in many yeast promoter sequences can be activated by sphingolipid signals. TheTPS2 promoter contains four STREs that may mediate dihydrosphingosine responsiveness. Using genetic and other approaches it should be possible to identify sphingolipid signaling pathways in S. cerevisiae and quantify the importance of each during heat stress.

Synthesis of Mannose-(inositol-P)2-ceramide, the Major Sphingolipid in Saccharomyces cerevisiae, Requires the IPT1 (YDR072c) Gene

Knowledge of the Saccharomyces cerevisiaegenes and proteins necessary for sphingolipid biosynthesis is far from complete. Such information should expedite studies of pathway regulation and sphingolipid functions. Using the Aur1 protein sequence, recently identified as necessary for synthesis of the sphingolipid inositol-P-ceramide (IPC), we show that a homolog (open reading frameYDR072c), termed Ipt1 (inositolphosphotransferase1) is necessary for synthesis of mannose-(inositol-P)2-ceramide (M(IP)2C), the most abundant and complex sphingolipid in S. cerevisiae. This conclusion is based upon analysis of an ipt1-deletion strain, which fails to accumulate M(IP)2C and instead accumulates increased amounts of the precursor mannose-inositol-P-ceramide. The mutant also fails to incorporate radioactive precursors into M(IP)2C, and membranes prepared from it do not incorporate [3H-inositol]phosphatidylinositol into M(IP)2C, indicating a lack of M(IP)2C synthase activity (putatively phosphatidylinositol:mannose-inositol-P-ceramide phosphoinositol transferase). M(IP)2C synthase activity is inhibited in the micromolar range by aureobasidin A, but drug sensitivity is over 1000-fold lower than reported for IPC synthase activity. An ipt1-deletion mutant has no severe phenotypic effects but is slightly more resistant to growth inhibition by calcium ions. Identification of the IPT1 gene should be helpful in determining the function of the M(IP)2C sphingolipid and in determining the catalytic mechanism of IPC and M(IP)2C synthases.

Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids.

Sphingolipids comprise a large, widespread family of complex eucaryotic-membrane constituents of poorly defined function. The yeast Saccharomyces cerevisiae is particularly suited for studies of sphingolipid function because it contains a small number of sphingolipids and is amenable to molecular genetic analysis. Moreover, it is the only eucaryote in which mutants blocked in sphingolipid biosynthesis have been isolated. Beginning with a nonreverting sphingolipid-defective strain that requires the addition of the long-chain-base component of sphingolipids to the culture medium for growth, we isolated two strains carrying secondary, suppressor mutations that permit survival in the absence of exogenous long-chain base. Remarkably, the suppressor strains made little if any sphingolipid. A study of how the suppressor gene products compensate for the lack of sphingolipids may reveal the function(s) of these membrane lipids in yeast cells.

Rapid separation of acetylated oligosaccharides by reverse-phase high-pressure liquid chromatography.

Abstract Peracetylated saccharides were separated by chromatography on a reverse-phase support, eluting with mixtures of acetonitrile-water. Gradient elution for 2.5 h gave significant separations of all linear glucose oligomers containing up to 35 sugar residues. With isocratic elution retention was exponentially related to molecular mass and only slightly affected by linkage or anomeric configuration. The presence of glucosamine in various saccharides markedly reduced their retention.

Separation of dolichylpyrophosphoryloligosaccharides by liquid chromatography.

Abstract Fourteen dolichylpyrophosphoryloligosaccharides, precursors of the asparagine-linked oligosaccharides of glycoproteins, have been separated by liquid chromatography on silica gel. The dolichylpyrophosphoryl- N -acetylglucosamine and the dolichylpyrophosphoryl-( N -acetylglucosamine) 2 -(mannose) 9 (glucose) 2,3 thus resolved were shown to retain their activity as substrates in enzyme catalyzed reactions. The chromatography procedure for the first time makes available many of these single intermediates for further study.

[9] Resolution of dolichylpyrophosphoryl oligosaccharides by high-pressure liquid chromatography

Publisher Summary Current information indicates that asparagine-linked oligosaccharides of glycoproteins arise from a precursor whose probable composition is dolichol-P2-(N-acetylglucosamine) 2 (mannose) 9 (glucose) 3 . This compound is formed by the stepwise addition of saccharide units to dolichol phosphate, presumably yielding 14 components. A procedure is presented in this chapter that rapidly resolves these 14 dolichylpyrophosphoryl oligosaccharides by liquid chromatography on silica gel. Liquid chromatography of dolichol-linked oligosaccharides is carried out on six 0.32 × 100 cm columns linked in series, packed with silicic acid and maintained at 58 °. A 0.32 × 3.5 cm precolumn with the same packing can be used. Separation of the dolichylpyrophosphoryl oligosaccharides is carried out with a nonlinear gradient formed with solvent A, CHCl 3 -CH 3 OH-conc. NH 3 and solvent B, CHCl 3 -CH 3 OH conc. NH 3 -H 2 O. Both solvents contain 0.6 g of ammonium chloride per liter. The gradient is run for 120 min from 0 to 100% solvent B, followed by pumping solvent B for 75 min. The solvent composition during the gradient at t minutes is given by: % solvent B = 100(t/120).