Figure 4

SAC1 expression is independent of inositol levels and ER stress. (A) Analysis of cell growth in sac1 Δ and opi1 Δ mutants. Cells were grown at 30°C, plated in 5-fold serial dilutions starting with a density of 10⁷ cells/ml on rich growth medium (YPD) or on inositol-free medium and incubated for 3 days. (B) SAC1 promoter activity in opi1 Δ mutants. Cells were transformed with a CEN-based plasmid containing the SAC1(-500/-1)-GFP fusion construct and grown to early log phase at 30°C. Cell extracts were analyzed by SDS-PAGE and immunoblotting. Relative GFP expression levels were quantified. Data are from at least three independent experiments (+/-SE). (C) Influence of inositol on SAC1 promoter activity. sac1 Δ cells transformed with a CEN-based plasmid containing the SAC1(-500/-1)-GFP fusion construct were grown in media containing a range of inositol concentrations. Relative GFP expression levels were quantified as above. Data are from at least three independent experiments (+/-SE). (D) Influence of ER stress on SAC1 promoter activity. Wild-type and sac1 Δ cells transformed with a CEN-based plasmid containing the SAC1(-500/-1)-GFP fusion construct were cultivated in media with or without 7 mM DTT. Cell extracts were analyzed by SDS-PAGE and immunoblotting using anti-GFP, anti-glucose-6-phosphate dehydrogenase (Zwf1p), and anti-Kar2p antibodies. Relative GFP expression levels were quantified. Data are from at least three independent experiments (+/-SE).