Figure 4.

Processing of Pho8 depends on the growth phase and not zinc. A, schematic of Pho8-GFP, highlighting the positions of the transmembrane domain (TM), cleavage site, C-terminal propeptide, and GFP. B, WT cells bearing the empty vector and WT and MGF317 cells expressing Pho8-GFP were grown for 16 h overnight in ZL-EMM supplemented with 0 (−Zn) or 100 μm (+Zn) zinc, and protein extracts were prepared for immunoblot analysis. Immunoblots were incubated with anti-GFP antibodies and anti-Act1 antibodies as a loading control. The positions of the molecular mass markers in kilodaltons are shown on the left. C, alkaline phosphatase activity (ALP) was assayed in the indicated strains following growth for 16 h in ZL-EMM supplemented with 0 or 100 μm zinc. Each is the mean of three independent repeats, with the error bars representing standard deviations. D and E, pho8Δ and pho2Δ pho8Δ cells expressing Pho8-GFP were grown overnight in ZL-EMM supplemented with 0 or 100 μm zinc and harvested at an A₆₀₀ of ∼4.0, ∼6.0, and ∼8.0. Protein extract preparation and immunoblot analysis were performed as described above (D), or cell lysates were assayed for alkaline phosphatase activity as described above (E).