Pho4 is regulated by the cyclin-dependent kinase Pho85 and responds to phosphate deprivation by translocating to nuclei. Pho4 is regulated by the cyclin-dependent kinase Pho85 and responds to phosphate deprivation by translocating to nuclei. (A) YPD-grown cells were incubated for 3 h in MM plus 1 mM KH2PO4 (MM Pi+) and dimethyl sulfoxide (DMSO) (negative control) or MM Pi+ with purvalanol A (50 μM). MM without KH2PO4 supplemented with DMSO served as a positive control (Pi−). Cell-associated APase activity, which served as a readout for Pho4 activation, was measured using the chromogenic substrate pNPP. Results represent the means ± SDs (n = 3 biological replicates). (B) Cells expressing Pho4-mCherry were incubated for 2 h in MM-KCl (Pi−) or MM-KH2PO4 (Pi+) and viewed using a DeltaVision fluorescence microscope. Nuclei were visualized with Hoechst stain (5 min, 25 µg/ml stain in the growth medium). DIC, differential interference contrast. Sophie Lev et al. mSphere 2017; doi:10.1128/mSphere.00381-16