Regulation of the sphingoid long-chain base kinase Lcb4p by ergosterol and heme: studies in phytosphingosine-resistant mutants.
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.
Sano
Takamitsu
T
Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan.
Kihara
Akio
A
Kurotsu
Fumiko
F
Iwaki
Soichiro
S
Igarashi
Yasuyuki
Y
D015415:Biomarkers; D002462:Cell Membrane / Q000378:metabolism; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins / Q000378:metabolism; D004875:Ergosterol / Q000494:pharmacology*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006418:Heme / Q000494:pharmacology*; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D010744:Phosphoric Monoester Hydrolases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors
D015415:Biomarkers; D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; C058452:HAP1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D006418:Heme; C113962:LCB4 protein, S cerevisiae; D017853:Phosphotransferases (Alcohol Group Acceptor); C110252:LCB3 protein, S cerevisiae; D010744:Phosphoric Monoester Hydrolases; C012491:phytosphingosine; D013110:Sphingosine; D004875:Ergosterol