Figure 7. Pho8 zinc metallation is impaired in a zrc1 cot1 mutant.
(A) Immunoblot analysis of lysates from wild type and zrc1 cot1 cells bearing pGAL1-PHO8 plasmid and grown in LZM supplemented with the indicated ZnCl2 concentrations. (B) Immunoblot analysis of lysates from wild type or zrc1 cot1 cells bearing either pGAL1-PHO8 plasmid or the vector after overnight induction with different amounts of β-estradiol to equalize expression levels. The low zinc medium for both wild type and zrc1 cot1 strains contained 3 µM zinc and 1 µM β-estradiol. For zinc-replete conditions (300 µM zinc), the wild type cells were induced with 0.01 µM β-estradiol and the zrc1 cot1 cells were induced with 1 µM β-estradiol. (C) Wild type and zrc1 cot1 cells bearing pGAL1-PHO8 were grown as described for panel B and then assayed for ALP activity. The activity values were then normalized to Pho8 (m1 and m2) protein levels quantified from panel B. Zinc-replete cells were also treated with 50 µM pyrithione, a zinc ionophore, for 20 minutes prior to harvesting (+Zn +Pyr, hatched bars). Each value is the mean of three replicates and the error bars represent ± 1 S.D.