Abstract
Sphingoid long-chain base 1-phosphates (LCBPs) are widely conserved, bioactive lipid molecules. In yeast, LCBPs are known to be involved in several cellular responses such as heat shock resistance and Ca(2+) mobilization, although their target molecules and signaling pathways remain unclear. To identify genes involved in LCBP signaling and in regulation of LCBP synthesis, we performed transposon mutagenesis in cells lacking the LCBP lyase DPL1 and LCBP phosphatase LCB3 genes and screened for phytosphingosine-resistant clones. Further isolation and identification revealed eight genes (PBP1, HEM14, UFD4, HMG1, TPS1, KES1, WHI2, and ERG5), in addition to the previously characterized LCB4 and PDR5 genes, that are involved in phytosphingosine resistance. Of these eight, four are ergosterol-related genes (HEM14, HMG1, KES1, and ERG5). We also demonstrated that protein expression of the long-chain base kinase Lcb4p was reduced in Deltahem14 and Deltahmg1 cells, likely as a consequence of decreased activity of the heme-dependent transcription factor Hap1p. In addition, phosphorylation of Lcb4p was decreased in all the ergosterol-related mutants isolated and other ergosterol mutants constructed (Deltaerg2, Deltaerg3, and Deltaerg6). Finally, plasma membrane localization of Lcb4p was found to be reduced in Deltaerg6 cells. These results suggest that changes in sterol composition affect the phosphorylation of Lcb4p because of the altered localization. The other genes isolated (PBP1, UFD4, TPS1, and WHI2) may be involved in LCBP signaling.
Highlights
Sphingolipids are major membrane components of eukaryotic cells
Further isolation and identification revealed eight genes (PBP1, HEM14, UFD4, HMG1, TPS1, KES1, WHI2, and ERG5), in addition to the previously characterized LCB4 and PDR5 genes, that are involved in phytosphingosine resistance
Exogenous PHS is readily imported into yeast cells and converted to PHS 1-phosphate (PHS1P) by Lcb4p
Summary
Sphingolipids are major membrane components of eukaryotic cells. Ceramide, the backbone of sphingolipids, is formed by the amide linkage of a sphingoid long-chain base (LCB) with a fatty acid. The phosphorylation of LCBs at C-1 results in the production of long-chain base 1-phosphates (LCBPs), sphingosine 1-phosphate (S1P) in mammals and PHS 1-phosphate (PHS1P) and DHS 1-phosphate in yeast. Intracellular S1P has been proposed to be involved in Ca2ϩ mobilization, cell proliferation, G1/S cell cycle transition, and inhibition of apoptosis (4 –7), its intracellular target molecules and signaling pathways remain unclear. LCBPs confer protection from environmental stresses, as in the inhibition of apoptosis in mammalian cells and heat resistance in yeast. Such shared functions imply evolutionarily conserved target molecules and signaling pathways for LCBPs. The enzymes responsible for both the production and degradation of LCBPs are, completely conserved from yeast to mammals. We recently found that the stability of yeast Lcb4p is regulated through its phosphorylation by the cyclin-dependent protein kinase Pho85p via a ubiquitin-dependent pathway [17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
