Figure 5

SAC1 expression responds to changes in PI(4)P levels. (A) PI(4)P levels in sac1 Δ and sac1 Δ PI 4-kinase double mutants. Yeast cells were grown at 33°C and labeled with [³H]myo-inositol. Phosphoinositides were extracted, deacylated and quantified by HPLC. Data are from three independent experiments (+/-SE).(B) SAC1 promoter activity in sac1 Δ and sac1 Δ PI 4-kinase double mutants. Yeast cells were transformed with a CEN-based plasmid containing the SAC1(-500/-1)-GFP fusion construct and grown to early log phase at 33°C. Cell extractswere analyzed by SDS-PAGE and immunoblotting. Relative GFP expression levels were quantified. Data are from at least three independent experiments (+/-SE). (C) Correlation of increased Sac1 protein levels and PI(4)P phosphatase deficiency. Wild-type and sac1 Δ yeast expressing either a myc-tagged wild-type Sac1p or phosphatase-deficient mutant myc-Sac1-22p from the SAC1(-500/-1) promoter were grown to early log phase at 30°C. Cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-GFP and anti-glucose-6-phosphate dehydrogenase (Zwf1p) antibodies.