FIGURE 7.

A higher proportion of newly synthesized PI is used to support inositol-containing sphingolipid synthesis in the dga1Δlro1Δare1Δare1Δ mutant. Cells were grown and labeled as described in Fig. 6 except that the cells were pulse-labeled for 30 min instead of 20 min to allow sufficient time for ³²P label to enter through PI and be transferred to the inositol-containing sphingolipids. The total lipids were extracted and deacylated. The nondeacylated fraction contained the inositol-containing sphingolipids. Panel A, absolute ³²P incorporation into IPC, MIPC, and M(IP)₂C is shown. The inset shows the ³²P incorporated into the total of inositol containing sphingolipids plus PI. The rate of ³²P incorporation into PI was measured from lipid extracts before the deacylation step that were prepared from pulse-labeled parallel cultures as describe under “Experimental Procedures.” Panel B, data from the panel A inset is plotted as a percentage of total ³²P label derived from PI (i.e. PI + SL) to illustrate the synthesis of each lipid relative to PI + SL. SL, total inositol-containing sphingolipids. ODU, optical density units.