Figure 2.

Phospholipid profiles of wild-type and sac1 mutant yeast strains. (A) Yeast strains were pulse radiolabeled for 20 min with [³²P]orthophosphate at 26°C in inositol- and choline-supplemented medium. Radiolabeled phospholipids were extracted using an acidic extraction solvent suitable for recovery of phosphoinositides (see MATERIALS AND METHODS) and resolved by two-dimensional paper chromatography. Phospholipid species are numbered. Relevant phospholipids are identified as follows: 1, PtdIns-4-P; 4, PtdIns; 5, PtdSer; 6, PtdCho; 7, PtdEtn; 8, PtdOH. The spot 1 lipid also labels with [3H]inositol, and its assignment as PtdIns-4-P is justified below. The sac1-22 strain exhibited a dramatic elevation in both PtdIns-4-P and PtdCho and a significant reduction in PtdSer, relative to wild-type yeast. These aberrant levels of PtdCho, PtdIns-4-P, and PtdSer were typical of Δsac1 strains as well. Phospholipids extracted from the equivalent number of wild-type and sac1-22 cells were chromatographically resolved in this experiment. (B) Deacylation of [³H]inositol-labeled lipids from sac1 strains reveals a dramatic increase in a soluble species that coelutes with the glycerophosphoinositol 4-P standard (i.e. the deacylated product of PtdIns-4-P). Total cellular lipids prepared from equal cell equivalents of [³H]inositol-labeled wild-type (CTY182) or isogenic Δsac1 strain (CTY244) were deacylated, and resulting glycerophosphoinositols were analyzed by HPLC. The radioactive profile from the region corresponding to glycerophosphoinositol monophosphates is shown for CTY182 (dotted line, lower panel) and CTY244 (solid line, upper panel). The elution position of glycerophosphoinositol-4-P was identified by an internal ³²P standard included in HPLC analysis of deacylated fractions prepared from CTY244 (open circles, upper panel). In other experiments, radiolabeled glycerophosphoinositol, glycerophosphoinositol 4-P, and glycerophosphoinositol 4,5-P₂ were used as standards to further verify assignments. (C) Inositol polyphosphate 1-phosphatase digestion of soluble head groups derived by deglyceration of deacylated inositol lipids prepared from the Δsac1 strain CTY244. Reactants of control (upper panel) or inositol polyphosphate-1-phosphatase-treated (lower panel) deglycerated samples were separated by anion-exchange HPLC. Peaks corresponding to Ins-1-P, Ins-4-P, Ins-1,3-P₂, and Ins-1,4-P₂ eluted at 17, 18, 33, and 35 min, respectively.