Figure 4
SsPAQR1 yeast-based assay. The agonist of SsPAQR1 was identified using a yeast-based assay as described in Methods. S. cerevisiae BY4742 was transformed with YEp353 (FET3-lacZ) containing a fragment of the FET3 promoter fused to lacZ driven by a minimal CYC1 promoter and with pYES2CT w/wo the sspaqr1 gene insert. S. cerevisiae were grown in LIM-Fe medium containing 2% galactose and FET3 activity is measured using the FET3-lacZ construct as a reporter. Black bars show FET3-lacZ activity in yeast treated with the solvent only (H2O or ethanol) and gray bars show activity in yeast treated with different possible agonist; thaumatin, adiponectin or progesterone. FET3-lacZ activity was measured as the β-galactosidase activity expressed as the percentage of the untreated vector control. Panel (A) shows that SsPAQR1 does not repress FET3-lacZ when over-expressed in yeast by using the GAL1 promoter. Panel (B) shows β-galactosidase activity in cells expressing SsPAQR1 in the presence of 1 mM progesterone, panel (C) shows β-galactosidase activity in cells expressing SsPAQR1 in the presence of 50 μM thaumatin and panel (D) shows β-galactosidase activity in cells expressing SsPAQR1 in the presence of 0.1 μM adiponectin.