Pho8 stability is not affected by zinc status.
A, schematic of Pho8-HA and Pho8-HA ΔC, highlighting the positions of the transmembrane domain (TM), 3×HA epitope, cleavage site, and C-terminal propeptide (blue box). B, alkaline phosphatase (ALP) activity was assayed in the indicated strains, which were grown in ZL-EMM supplemented with 0 or 100 μm zinc to an A600 of ∼5. Activity is the mean of three independent repeats, with the error bars representing standard deviations. C, the indicated strains were grown in ZL-EMM supplemented with 0 or 100 μm zinc to an A600 of ∼5, and protein extracts were prepared for immunoblot analysis. Immunoblots were incubated with anti-HA antibodies and anti-Act1 antibodies as a loading control. D, pho8D pho2D cells bearing the empty vector, Pho8-HA, or the indicated Pho8-HA zinc-binding mutants were grown overnight in ZL-EMM supplemented with 0 (−Zn) or 100 μm zinc (+Zn) to an A600 of ∼5. Protein extract preparation and immunoblot analysis were performed as described above. Bottom panel, the mean ratio of mature to unprocessed Pho8-HA proteins from three independent repeats, with error bars representing standard deviations. *, p < 0.05; ns, not significant.