Mechanisms of sphingolipid functions during heat stress in "Saccharomyces cerevisiae"

Abstract

Sphingolipids are important membrane constituents in all eukaryotic cells. Ongoing sphingolipid synthesis is required for a variety of cellular processes including response to heat shock. During heat stress, de novo sphingolipid synthesis is upregulated and mutants defective in the biosynthesis of sphingolipids show a significantly greater loss of viability. To dissect the mechanisms of sphingolipid functions during heat stress we made use of the Saccharomyces cerevisiae lcb1-100 mutant. This mutant is conditionally impaired in serine palmitoyltransferase activity, the first committed step in sphingolipid biosynthesis. lcb1-100 cells are highly sensitive to heat shock and die at elevated temperatures. In this study we could show that the increased sensitivity to heat stress in lcb1-100 cells correlated with a lack of induction of the major heat shock proteins at high temperature. This defect could be restored by the overexpression of ubiquitin, a situation that increased turnover of proteins and prevented accumulation of protein aggregates in lcb1-100 cells. These data showed that the essential function of heat shock protein induction is the removal of misfolded or aggregated proteins. Furthermore it suggested that heat stressed cells do not die because of the loss of protein activity due to their denaturation, but because of the inherent toxicity of the denatured and/or aggregated proteins. In addition we tried to explain the lack of induction of heat shock proteins in lcb1-100 cells. We could show that transcription and nuclear export of heat shock protein mRNA was not affected in these cells. Under heat stress conditions, lcb1-100 cells exhibit a strong decrease in protein synthesis and polysome analyses demonstrated a defect in translation initiation. Furthermore we demonstrate that efficient translation under these conditions relies upon the synthesis of sphingoid base. Deletion of the eIF4E binding protein Eap1p partially restores translation initiation and the synthesis of heat shock proteins in lcb-100 cells. Thus, sphingoid bases signal to the cap-dependent translation initiation apparatus to enhance heat shock protein synthesis. In addition, ubiquitin overexpression in the lcb1-100 mutant allowed recovery of translation, but not at the initial phase where heat shock proteins are made. Therefore the recovery process seems to be dependent upon the function of the heat shock proteins that are made during the initial phase of heat stress. Altogether we have uncovered a new function for sphingoid bases and provide an explanation for the sphingoid base synthesis requirement for survival during heat stress.