Sphingoid Base Is Required for Translation Initiation during Heat Stress inSaccharomyces cerevisiae

Abstract

Sphingolipids are required for many cellular functions including response to heat shock. We analyzed the yeast lcb1-100 mutant, which is conditionally impaired in the first step of sphingolipid biosynthesis and shows a strong decrease in heat shock protein synthesis and viability. Transcription and nuclear export of heat shock protein mRNAs is not affected. However, lcb1-100 cells exhibited a strong decrease in protein synthesis caused by a defect in translation initiation under heat stress conditions. The essential lipid is sphingoid base, not ceramide or sphingoid base phosphates. Deletion of the eIF4E-binding protein Eap1p in lcb-100 cells restored translation of heat shock proteins and increased viability. The translation defect during heat stress in lcb1-100 was due at least partially to a reduced function of the sphingoid base-activated PKH1/2 protein kinases. In addition, depletion of the translation initiation factor eIF4G was observed in lcb1-100 cells and ubiquitin overexpression allowed partial recovery of translation after heat stress. Taken together, we have shown a requirement for sphingoid bases during the recovery from heat shock and suggest that this reflects a direct lipid-dependent signal to the cap-dependent translation initiation apparatus. INTRODUCTIONEukaryotic cells have developed several mechanisms to respond to rapid increases in temperature. On heat stress, cells reduce the rate of synthesis of proteins that were expressed before the heat shock and change their transcription profile dramatically to produce mainly heat shock responsive proteins (Gasch et al., 2000;Murray et al., 2004). In the budding yeast Saccharomyces cerevisiae changes in transcription upon heat stress are fairly well understood, involving control by two transcription factors, the heat shock factor Hsf1p and Msn2p/4p. Hsf1p binds to heat shock elements (HSEs) found in the promoter region of many heat shock protein genes (Wu, 1995). Genes that do not contain HSEs, but whose transcription is induced by heat and other stress signals, including osmotic shock, DNA damage, and oxidative stress, contain stress response elements (STREs) in their promoters. On these stresses, Msn2/4p shuttles from the cytosol to the nucleus and activates transcription through binding the STREs (Schmitt and McEntee, 1996;Gorner et al., 1998). After transcription, the corresponding mRNAs are exported from the nucleus and are translated (Stutz and Rosbash, 1998). Proteins encoded by heat stress responsive genes are responsible for the synthesis of the thermoprotectant trehalose (Singer and Lindquist, 1998), for the folding of proteins and for the degradation of unfolded and aggregated proteins (Imai et al., 2003;Riezman, 2004).In addition to the induction of heat shock proteins, yeast cells induce the de novo synthesis of free sphingoid bases, followed by ceramides and sphingolipids (Jenkins, 2003). The first steps in the biosynthesis of sphingolipids in animal cells and in yeast are similar, but differ in production of complex sphingolipids. ...

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