Sphingoid long-chain base kinase Lcb4 catalyzes the production of the bioactive lipid molecules the longchain base 1-phosphates. Although Lcb4 has no apparent transmembrane-spanning domain, it is tightly associated with the membrane. Here, we demonstrate that Lcb4 is modified by palmitoylation. This modification was greatly reduced in mutants for AKR1, which was recently identified as encoding a protein acyltransferase. In vitro experiments revealed that Akr1 indeed acts as a protein acyltransferase for Lcb4. Studies using site-directed mutagenesis indicated that Cys-43 and Cys-46 are palmitoylated. The loss of palmitoylation on Lcb4 caused several effects, including mislocalization of the protein to the cytosol, reduced phosphorylation, and loss of downregulation during the stationary phase. Although Akr2 is highly homologous to Akr1, the deletion of AKR2 did not result in any remarkable phenotypes. However, overproduction of Akr2 resulted in reduced amounts of Lcb4. We demonstrated that Akr2 is an unstable protein and is degraded in the vacuole. Akr2 exhibits high affinity for Lcb4, and in Akr2-overproducing cells this interaction caused unusual delivery of Lcb4 to the vacuole and degradation.

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 ⌬hem14 and ⌬hmg1 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 (⌬erg2, ⌬erg3, and ⌬erg6). Finally, plasma membrane localization of Lcb4p was found to be reduced in ⌬erg6 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.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) 2 with a fatty acid. The major mammalian LCB is sphingosine, whereas in the yeast Saccharomyces cerevisiae, dihydrosphingosine (DHS) and phytosphingosine (PHS) serve as LCBs. LCBs are bioactive lipid molecules involved in apoptosis, endocytosis, and G 1 cell cycle arrest (1-3). 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. Interestingly, LCBPs possess completely different biological functions compared with the original LCBs.In mammalian cells, S1P is known to elicit various cellular responses via the cell-surface SIP/Edg (endothelial differentiation gene) receptors, such as proliferation, motility, differentiation, and immunity (4 -7). In addition to its role as an extracellular signal mediator, S1P also functions intracellularly. Intracellular S1P has been proposed to be involved in Ca 2ϩ mobilization, cell proliferation, G 1 /S cell cycle transition, and inhibition of apoptosis (4 -7), although its intracellular target molecules and signaling pathways remain unclear.Because yeast cells do not possess a cell-surface receptor for LCBP/ S1P, their LCBPs function only intracellularly (8 -11). Yeast LCBPs and mammalian intracellular S1P share some effects. For instance, both influence Ca 2ϩ mob...