Fig. 1. Synthesis of sphingolipids in yeast from endogenous and exogenous precursors. DHS and PHS can be internalized by cells and phosphorylated by the LCB4 and LCB5 gene products to give DHS-1P and PHS-1P. The phosphorylated sphingoid bases can be dephosphorylated by the LCB3 and YSR3 gene products. LCB1 encodes a subunit of serine palmitoyltransferase. The SUR2 gene product converts DHS into PHS. Synthesis of ceramides from DHS or PHS and C26 fatty acyl-CoA is inhibited by australifungin. Ceramides are used to synthesize the sphingolipids IPC (inositolphosphorylceramide), MIPC (mannosylated inositolphosphorylceramide) and M(IP)₂C (mannosylated di-inositolphosphorylceramide).

Fig. 2. Conditional sphingolipid synthesis and endocytosis in lcb1-100 cells. (A) Wild-type (RH1800, wt) or lcb1 mutant (RH3809) cells were grown overnight to A₆₀₀ = 0.4, preshifted to the appropriate temperature for 15 min in the absence or presence of DHS, and labeled with [³H]myoinositol at 24 or 37°C in the absence or presence of DHS. Incorporation of [³H]myoinositol into the total lipid fraction was quantified and equal c.p.m. were applied to TLC plates, or treated with mild base to identify sphingolipids and applied to TLC plates. Incorporation of radioactivity into sphingolipids was quantified using a phosphorimager [sum of IPC + MIPC + M(IP)₂C] and the relative amount of sphingolipid synthesis determined after setting the amount in wild-type cells at 24°C to 100%. PI, phosphatidylinositol; IPC; MIPC; LYSO-PI, lysophosphatidylinositol; M(IP)₂C; X, unidentified lipid. The products were analyzed by TLC using solvent system I. (B) Yeast cells were grown overnight in SDYE. Internalization of [³⁵S]α-factor by RH3809 cells was measured at 24 or at 37°C in SDYE in the presence or absence of DHS or PHS. (C) Yeast were grown overnight in YPUAD. Internalization of [³⁵S]α-factor by RH3809 and RH1597 cells was measured in SDYE at 37°C in the presence or absence of DHS.

Fig. 3. Incorporation of [³H]DHS into sphingolipids and DHS-1P. The lcb1-100 (RH3809) or triple mutants (RH3859, lcb1/3/ ysr3 and RH4355, lcb1/4/5) were labeled with [³H]DHS at 37°C. Lipids were analyzed by TLC using solvent system II. Images of portions of individual lanes are shown and were scanned to quantify the incorporation. The incorporation of [³H]DHS into DHS-1P or sphingolipids in the lcb1-100 mutant was set to 100%. The positions of PI, IPC, DHS-1P, M(IP)₂C, as well as two unknown products, Y and Z, are marked.

Fig. 4. Suppression of the α-factor internalization defect by DHS in mutants in sphingoid base metabolism. Strains RH4355 (lcb1/4/5), RH3859 (lcb1/3/ysr3) and RH4363 (lcb1/sur2) were assayed for [³⁵S]α-factor internalization at 24 or at 37°C with or without exogenous DHS or PHS.

Fig. 5. Australifungin inhibits sphingolipid synthesis, but not endocytosis. (A) RH3809 cells were labeled with [³H]DHS at 37°C in the presence or absence of 1 or 2 mg/l australifungin and lipids were analyzed by TLC using solvent system II. Positions of PI, IPC, DHS-1P, M(IP)₂C, and two unknown compounds, Y and Z, are marked. (B) Internalization of [³⁵S]α-factor was measured in RH3809 cells in the presence of DHS and the presence or absence of australifungin at 37°C.

Fig. 6. Phosphorylation of the α-factor receptor. RH1800 (wild-type), RH3809 (lcb1) or RH3162 (ste2Δ) cells were incubated in the presence or absence of α-factor at 37°C. Total proteins were extracted and the α-factor receptor revealed by western blotting. N, normal phosphorylated α-factor receptor seen in the absence of pheromone; P, mobility of hyperphosphorylated form of the α-factor receptor after pheromone treatment; *, non-specific band seen in ste2Δ mutant.

Fig. 7. LY accumulation requires sphingoid base. RH1800 (wt), RH3809 (lcb1) and RH1597 (sla2) cells were incubated with LY at 37°C in the presence or absence of PHS. Cells were washed and visualized by fluorescence optics (left panels) or Nomarski optics (right panels).

Fig. 8. Actin staining in wild-type and mutant cells. RH1800 (wt) or RH3809 (lcb1) cells were incubated at 24 or at 37°C in the presence or absence of DHS or PHS, then fixed, and filamentous actin was visualized using TRITC–phalloidin (left panels) or Nomarski optics (right panels).