Ime2 activation is uncoupled from meiotic progression. Measurement of Ime2 kinase activity during meiosis in wild type and cell cycle mutants. Ime2-3V5 was immunoprecipitated from lysate collected at the indicated time points. On-bead Ime2-3V5 was incubated with histone H1 and γ-32P ATP. Ime2 kinase activity toward histone H1 was determined by autoradiography, and Ime2-3V5 abundance in the reaction was determined by immunoblotting. Ime2 kinase activity is plotted with the background from a no-tag control (A15055) subtracted from each value. Values are scaled to the maximum background-subtracted value from the experiment. In addition, mitochondrial detachment frequency was scored using the Cit1-GFP mitochondrial marker and meiotic staging determined by tubulin immunofluorescence (n = 100 cells per time point per condition for each analysis). (A) pGAL-NDT80 GAL4.ER cells (UB10554) were induced to sporulate, then after 5 h treated with 1 µM β-estradiol (+Ndt80) or ethanol vehicle control (−Ndt80). (B) Sporulating pGAL-NDT80 GAL4.ER cdc5-mn (UB18612) and pGAL-NDT80 GAL4.ER cdc20-mn (UB18614) cells were treated with 1 µM β-estradiol at 5 h. pGAL-NDT80 GAL4.ER cdc28-as1 (UB18845) cells were simultaneously treated with 1 µM β-estradiol and 1 µM 1-NM-PP1 at 5 h.
