Wild type and mutant Czf1p interact with Efg1p in a yeast two-hybrid assay.
S. cerevisiae strains carrying a lacZ reporter plasmid, a lexA DNA-binding domain bait fusion (indicated below columns) and a Gal4 activation domain prey fusion (black bars, Gal4AD-Efg1; white bars, Gal4AD-Slk19) were grown in synthetic complete medium lacking uracil, leucine and histidine (CM-ULH) and β-galactosidase activity was determined using o-nitrophenyl β-D-galactopyranoside (ONPG) as the substrate. Analysis was performed in triplicate and mean and standard deviation are shown. Differences in activity of Czf1-LexA fusions with Gal4AD-Efg1p and the same fusion with Gal4AD-Slk19 were statistically significant (p<5×10−4 for all Czf1-LexA fusions; two tailed t test). (Panel B) S. cerevisiae strains were grown in CM-ULH and immunoblot analysis was performed on crude extracts using an anti-LexA antibody to visualize Czf1-lexA protein fusions. Actin was detected as the loading control. Samples from two independent cultures of a given strain are shown. Lanes 1 and 2, strain with lacZ reporter and Gal4AD-Efg1 fusions only; lanes 3 and 4, strains carrying lacZ reporter, Gal4AD-Efg1 and CZF1-lexA fusion; lanes 5 and 6, strains carrying lacZ reporter, Gal4AD-Efg1 and czf1K322A-lexA fusion; lanes 7 and 8, strains carrying lacZ reporter, Gal4AD-Efg1 and czf1R321A-lexA fusion. A cross-reacting band, marked with *, is apparent above the Czf1-LexA fusion.