Abstract
A role for sphingolipids in the yeast heat stress response has been suggested by the isolation of suppressors of mutants lacking these lipids, which are unable to grow at elevated temperatures. The current study examines the possible role of sphingolipids in the heat adaptation of yeast cells as monitored by growth and viability studies. The suppressor of long chain base auxotrophy (SLC, strain 7R4) showed a heat-sensitive phenotype that was corrected by transformation with serine palmitoyltransferase. Thus, the deficiency in sphingolipids and not the suppressor mutation was the cause of the heat-sensitive phenotype of the SLC strain 7R4. The ability of sphingolipids to rescue the heat-sensitive phenotype was examined, and two endogenous yeast sphingoid backbones, phytosphingosine and dihydrosphingosine, were found to be most potent in this effect. Next, the effect of heat stress on the levels of the three major classes of sphingolipids was determined. The inositol phosphoceramides showed no change over a 1.5-h time course. However, the four detected species of sphingoid bases increased after 15 min of heat stress from 1.4- to 10.8-fold. The largest increases were seen in two sphingoid bases, C20 phytosphingosine and C20 dihydrosphingosine, which increased 6.4- and 10.8-fold over baseline, respectively. At 60 min of heat stress two species of yeast ceramide increased by 9.2- and 10.6-fold over baseline. The increase seen in the ceramides was partially decreased by Fumonisin B1, a ceramide synthase inhibitor. Therefore, heat stress induces accumulation of sphingoid bases and of ceramides, probably through de novo synthesis. Taken together, these results demonstrate that sphingolipids are involved in the yeast heat stress adaptation.
Highlights
Saccharomyces cerevisiae has been shown to respond to a transfer of 25–37 or 39 °C with the physiology defined as a heat stress response [1, 2], which appears to involve two phases
The novel inositol glycerolipids contain: 1) the 26 carbon fatty acid that is ordinarily exclusively found in the yeast sphingolipids; and 2) the same head groups that are found in the inositol phosphoceramides, attached to a glycerol backbone [8, 9]
The current study examines the role of sphingolipids in the heat adaptation of yeast cells
Summary
Drosphingosine, C2 phytoceramide, C2 and C6 ceramides, and C2 and C6 dihydroceramides were synthesized as described previously [11, 12]. The LCB1 gene in the plasmid was cut at its unique SmaI site at base 263 and EcoRI site at base 936 to obtain a 573-base pair fragment with which to transform the yeast strain 7R4. Plate Growth Studies—Yeast strains were streaked and grown for 2 to 4 days at 37 °C. Lipid Extraction—Yeast sphingolipids were extracted from liquid cultures via the method of Bligh and Dyer [14]. Yeast lipid extracts were prepared and run on TLC plates as described [17]. The resultant TLC plates were exposed to film to obtain a permanent image. An aliquot for the measurement of organic phosphate was taken before the lipids were run on TLC plates. The resultant phosphate value was used to normalize the values obtained via Image Quant
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