Figure 2.
Synthesis rate of inositol-containing sphingolipids is altered in lag1Δlac1Δ cells. (A) [3H]Inositol labeling. Cells of wild-type (lane 1), lag1Δlac1Δ (lane 2), lag1Δlac1Δ expressing LAG1 (lane 3), lag1Δlac1Δ expressing LAC1 (lane 4), and lag1Δlac1Δ expressing LAG1 and LAC1 (lane 5) were pulse labeled with [3H]inositol for 20 min followed by a chase of 60 min. Further energy-dependent metabolism was stopped by adding NaN3/NaF, and the labeled lipids were extracted and analyzed on HPTLC plates, resolved in solvent A, and subjected to fluorography. (B) [3H]DHS labeling. Cells of wild-type (lane 1) and lag1Δlac1Δ (lane 2) were pulse labeled with [3H]DHS for 2 h, and the labeled lipids were extracted and analyzed on HPTLC plates, resolved in solvent B, and subjected to fluorography. CER, ceramide; DHS, dihydrosphingosine; DHS-1P, DHS-1-phosphate; IPC, inositolphosphorylceramide; MIPC, mannosylinositolphosphorylceramide; M(IP)2C, mannosyldiinositolphosphorylceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PHS, phytosphingosine; PI, phosphatidylinositol; unidentified lipids are marked with an asterisk.