Figure 6.

Mouse Ssc1 restores glycosphingolipid levels in the Δsur4 yeast mutant. The Δsur4 yeast mutant cells were transformed either with the empty expression vector (Δsur4 alone), or with plasmids expressing FEN1, SUR4, Cig30, Ssc2, or Ssc1. Along with these transformants, the wild-type cells (EMA3) and the sur4Δfen1 double mutant transformed with Ssc1 were metabolically labeled with [³H]serine for 6 h, [³H]sphinganine, or [³H]inositol for 1.5 h. Total lipid extracts were prepared and separated by thin-layer chromatography. Before chromatography, [³H]serine-labeled lipids shown here were subjected to mild alkaline hydrolysis with methylamine. The plates were treated with EN³HANCE (New England Nuclear Life Science Products) and exposed to X-ray films for several weeks. The bands were identified by their relative mobility compared with radioactive standards ([³H]sphinganine) and glycerolipid compounds were determined in parallel experiments on the basis of their sensitivity to methylamine (not shown). CER, Ceramide; GPC, glycerophosphorylcholine; GPE, glycerophosphorylethanolamine; GPS, glycerophosphorylserine; IPC, inositolphosphorylceramide; MIPC, mannose inositolphosphorylceramide; M(IP)₂C, mannose diinositolphosphorylceramide; SPH, sphinganine. Similar results were obtained in a duplicate experiment.