Ume6p regulation during meiosis. Panel A. Vegetative steady state levels of T7-epitope tagged wild-type strain RSY1079 (T7-Ume6p) and destruction box 1 mutant (RSY1180, Ume6p^db1Δ) proteins were determined Western blot analysis. The parental control (RSY335) lacks a T7-tagged UME6 allele. Molecular weight standards (kDa) are given on the left of the panel. Panel B. The indicated strains UME6 (RSY335), T7-UME6 (RSY1079), ume6Δ (RSY853) and T7-dbox1Δ (RSY1080) were transformed with the spo13-URA3 reporter plasmid (pMS49). Three independent transformants were patched on medium lacking uracil (-uracil) or containing the uracil analog 5-FOA. Plates were incubated two days and photographed. Panel C. A meiotic timecourse was conducted with strain RSY1079 and timepoints taken prior (0 hr) and following the shift to sporulation medium (SPM). T7-Ume6p levels were determined by Western blot analysis. Northern blot analysis of SPO13 mRNA was conducted from the same timepoints. The execution of meiotic S phase (meiS), the meiotic divisions (MI, MII) and spore wall formation were determined by FACS analysis, fluorescent microscopy of DAPI stained cells and light microscopy, respectively. Tub1p and ENO1 mRNA levels served as loading controls for the Western and Northern blot analyses, respectively.

Cdc20p is required for Ume6p destruction. Panel A. The cdc20-1 strain RSY809 was transferred to SPM at the permissive temperature (23°) then shifted to the restrictive temperature (34.5°) four hrs later (arrow). T7-Ume6p levels were determined as before. Wild type (RSY335) and cdc20-1 mutant strain (RSY809) harboring CLB1-3HA plasmid pKC426 were treated as just described and Clb1p levels followed by Western blot analysis. Tub1p levels served as loading controls. Panel B. In vitro ubiquitylation assays were conducted with constant Cdc20p concentrations (2.5 μl) for the times indicated (left panel). Increased amounts of Cdc20p (2.5 and 5 μl) were added to the reaction (90 min) with Ume6p or Ume6p^db1Δ as substrate (right panel). The arrow heads indicate unmodified substrate, brackets, ubiquitylated species. The right panel was overexposed compared to the left panel to determine if weak ubiquitylation activity was observed with the destruction box mutant substrate.

Cdc20p is required for normal meiotic gene expression. Wild-type (RSY335) and mutant cdc20-1 (RSY809) strains were induced to enter meiosis at 23°C. After 4 hours in SPM, the cultures were shifted to the restrictive temperature (34.5°C). Total RNA was prepared from samples taken at the times indicated (hrs) following shift to SPM and the transcript levels of the genes indicated were analyzed by Northern blot analysis. ENO1 serves as a loading control. MI and MII indicate the approximate times of meiosis I and meiosis II in the wild-type culture as determined by DAPI analysis.

Ume6p destruction is required for normal EMG transcription and meiotic nuclear divisions. Panel A. Schematic of Ume6p indicating location of destruction box motifs and zinc cluster domain (Zn). Sequence alignment of Ume6p destruction box motifs and starting amino acid residue corresponding to the arginine is presented. The consensus sequence is given with X = any amino acid. Panel B. Wild type (RSY1079) and UME6^db1Δ (RSY1080) mutant strains were induced to enter meiosis and timepoints taken as indicated (hrs). T7-Ume6p and T7-Ume6p^db1Δ levels were followed by Western blot analysis. SPO13 mRNA levels were monitored by Northern blot analysis. ENO1 mRNA served as the loading control for the Northern analysis. Panel C. The timepoints described in Panel B were examined for the execution of meiosis I and meiosis II by DAPI analysis. The percentage of the population that had undergone one or both divisions in the wild type (shaded bars) or UME6^db1Δ strain (open bars) is depicted.

Ume6p is a substrate of APC/C^Cdc20 during G2-M stage of the cell cycle. Panel A. Haploid strain RSY1069 (T7-UME6, top panel) or RSY1070 (T7-UME6^db1Δ) was treated with hydroxyurea (HU) for 2 hrs to arrest the cells in S phase. The cells were washed and placed in fresh medium and timepoints taken as indicated. Ume6p and Ume6p^db1Δ levels were determined by Western blot analysis for every timepoint. Tib1p served as a loading control. Panel B. The execution of anaphase was determined in the samples described in Panel A by monitoring the presence of separated nuclei by DAPI staining and fluorescent microscopy.

Ume6p levels are regulated by the RAS pathway. Panel A. Wild type (RSY1079) and UME6^db1Δ (RSY1080) strains were grown in dextrose (D) or acetate (Ac) medium (two independent cultures) to mid-log phase. Ume6p or Ume6p^db1Δ levels (arrow) were monitored by Western blot analysis. Tub1p served as a loading control. Panel B. T7-Ume6p levels were monitored in the cdc20-1 mutant strain (RSY809) grown to mid-log in acetate medium at permissive (23°), or following two hrs at restrictive (37°), temperature. Panel C. Ume6p levels (arrows) were determined in log phase cultures with the indicated genotypes grown at permissive temperature (23°) and following two hrs at the restrictive (37°) temperature. V = vector control. Panel D. Extracts prepared from log phase RSY1079 cultures in dextrose (Dex), acetate (Ac) or after transfer to sporulation medium (SPM) for 4hr were immunoprecipitated with the indicated antibodies or no antibody (∅). The immunoprecipitates were subjected to Western blot analysis probing for the presence of Cdc20p-13xmyc. Cdc20p-13xmyc migration and expression were verified in the 4 hr sporulation timepoint (right lane). No antibody lane controls for non-specific association of Cdc20p-13xmyc to protein A beads. Molecular weight markers (kDa) are provided on the left. Panel E. Ume6p or Ume6p^db1Δ levels were followed in a cdc20-1 strain (RSY809) harboring wild type (WT), S52A, or S88A CDC20 expression plasmids (see Experimental procedures for details). Error bars indicate standard deviation from the mean.

Ume6p destruction requires meiotic induction and Ime1p association. Panel A. T7-Ume6p levels were monitored by Western blot analysis in a MATa/MATa diploid (RSY303) following transfer to SPM (in hrs). Panel B. T7-Ume6p levels were monitored by Western blot analysis in an ime1Δ diploid (RSY1128) as just described in Panel A. Panel C. T7-Ume6p levels were monitored by Western blot analysis in wild type (RSY1079) and UME6^T99N (RSY1395) diploid strains as described in Panel A. Panel D. Model for two-step destruction of Ume6p. The presence of dextrose and nitrogen (left panel) stimulates PKA which in turn phosphorylates Cdc20p. This modification prevents Cdc20p-Ume6p interaction. Growth on a non-fermentable carbon source (acetate) and high nitrogen (+N2) reduces PKA activity (grey arrows) reducing Cdc20p phosphorylation leading to partial degradation of Ume6p (Step 1). Step 2 (right panel) requires meiotic entry and Ime1p induction. A proposed ternary complex between Ime1p, Ume6p and Cdc20p completes Ume6p degradation.