Figure 1. Pho8 activity and protein accumulation requires zinc.

(A) Wild type and pep4 mutant cells bearing the vector (pRS316-GAL1-LEU2) or pGAL1-PHO8 were assayed for ALP activity under both low Zn and high Zn conditions. Cells were grown in LZM plus 1 µM ZnCl₂ (−Zn, filled bars) or 300 µM ZnCl₂ (+Zn, open bars). (B) Wild type cells bearing the pGAL1-PHO8 plasmid were grown overnight in LZM supplemented with the indicated concentration of zinc and then assayed for ALP activity. For panels A and B, each value is the mean of three replicates and the error bars represent ± 1 S.D. (C) Immunoblot analysis of protein samples prepared from cells bearing the vector or pGAL1-PHO8. Wild type cells transformed with pGAL1-PHO8 were grown in LZM supplemented with a range of zinc concentrations. Previous studies showed that cell viability is unaffected by these growth conditions (MacDiarmid et al., 2003). Designations of Pho8 forms are p, precursor; m1, mature form 1; m2, mature form 2. Pgk1 was used as a loading control. (D) S1 nuclease protection assays were performed with pho8 mutant cells or wild type cells bearing pGAL1-PHO8 grown in LZM with three representative zinc concentrations. ³²P labeled DNA oligonucleotides that detect PHO8 and CMD1 mRNA were used as probes.