Figure 2.

sjl1Δ sjl2^ts sjl3Δ cells display growth defects and generate increased levels of PtdIns(4,5)P₂ at the restrictive temperature. (A) sjl1Δ sjl2Δ sjl3Δ cells expressing either SJL2 (YCS157) or sjl2^ts (YCS176) from LEU2 CEN plasmids were grown in YPD at the permissive temperature. Tenfold serial dilutions of cells were spotted onto YPD and grown for 2 d at 26°C or 38°C. (B and C) Cells were preincubated at either 26°C or 38°C for 10 min and labeled with myo-[2-³H]inositol for 50 min at the permissive or nonpermissive temperatures. Lipids were then deacylated from cellular membranes, and glycero-phosphoinositols were extracted and analyzed by HPLC. (B) Quantitative comparisons of glycero-phosphoinositols generated by sjl1Δ SJL2 sjl3Δ cells (YCS157; open columns) and sjl1Δ sjl2^ts sjl3Δ cells (YCS176; black columns) at 26°C (left) and 38°C (right) are shown. These data represent the means ± SD of three independent experiments. (C) Typical HPLC elution profiles are shown for sjl1Δ SJL2 sjl3Δ cells (YCS157; left) and sjl1Δ sjl2^ts sjl3Δ cells (YCS176; right) at 38°C. Peaks corresponding to glycero-Ins(3)P, glycero-Ins(4)P, glycero-Ins(3,5)P2, and glycero-Ins(4,5)P₂ are indicated.