FIGURE 4.

Blockage of the S1P metabolic pathway in tsc13Δ cells is recovered by expression of mammalian TER. A, ABY83 (tsc13Δ cells bearing the pTW6 plasmid encoding TSC13 (pTSC13) and URA3; ABY83/pTW6) were transfected with the pAKNF313 (HIS3 vector, 1), pAB119 (3xFLAG-CERS5 (pCERS5), 2) or pAB134 (3xFLAG-TSC13 (pTSC13), 3) plasmids. Cells were grown on a plate of SC medium lacking uracil and histidine (SC-His-Ura) or SC-His containing 5-FOA at 30 °C for 2 days. B–E, BY4741 (wild type)/pAB119 (3xFLAG-CERS5) cells and ABY80 (tsc13Δ cells/pAB119) cells were grown in SC-His medium and labeled with 0.07 μCi of [11,12-³H]SPH (B and E), 0.07 μCi of [4,5-³H]DHS (C), or 0.07 μCi of [9,10-³H]palmitic acid (D) at 30 °C for 2 h. B–D, lipids were extracted, separated by normal-phase TLC with chloroform, methanol, 4.2 n ammonia (9:7:2, v/v), and detected by autoradiography. CER, ceramide; DHS1P, DHS 1-phosphate; IPC, inositol phosphorylceramide; MIPC, mannosylinositol phosphorylceramide; M(IP)₂C, mannosyldiinositol phosphorylceramide. E, lipids were extracted and subjected to trans-methylation. The generated FAMEs were isolated by hexane/methanol phase separation, treated with or without KMnO₄/KIO₄, and separated by reverse-phase TLC with chloroform/methanol/water (5:15:1, v/v). F, double bond cleavage reactions in E for palmitic acid methyl ester (C16:0 FAME), trans-2-hexadecenoic acid methyl ester (trans-2-C16:1 FAME), and palmitoleic acid (cis-9-hexadecenoic acid) methyl ester (cis-9-C16:1 FAME). KMnO₄/KIO₄ treatment causes a double bond cleavage and generation of two carboxylic acids. Note that two tritium atoms in palmitoleic acid methyl ester are removed during the oxidation reactions. Dots represent tritium atoms. Me, methyl group. G, BY4741 (wild type)/pAB119 (pCERS5)/pAK158 (vector), ABY80 (tsc13Δ/pCERS5)/pAK158, and ABY80 (tsc13Δ/pCERS5)/pAB128 (2xHA-TER) cells grown in SC-His-Leu medium were labeled with 0.07 μCi of [11,12-³H]SPH for 2 h. Lipids were extracted, separated by normal-phase TLC with chloroform, methanol, 4.2 n ammonia (9:7:2, v/v), and detected by autoradiography.