1. Meiosis-Specific Destruction of the Ume6p Repressor by the Cdc20-Directed APC/C 
Michael J. Mallory, Katrina F. Cooper, Randy Strich 
Molecular Cell 
Volume 27, Issue 6, Pages (September 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 Ume6p Regulation during Meiosis
(A) Vegetative steady-state levels of T7 epitope-tagged wild-type strain RSY1079 (T7-Ume6p) and destruction box 1 mutant (RSY1180, Ume6pdb1Δ) proteins were determined by 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.
(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 (−ura) or containing the uracil analog 5-FOA. Plates were incubated 2 days and photographed.
(C) A meiotic time course was conducted with strain RSY1079, and time points were 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 time points. 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2 Cdc20p Is Required for Ume6p Destruction
(A) The cdc20-1 strain RSY809 was transferred to SPM at the permissive temperature (23°C), then shifted to the restrictive temperature (34.5°C) 4 hr later (arrow). T7-Ume6p levels were determined as before. Wild-type (RSY335) and cdc20-1 mutant (RSY809) strains harboring CLB1-3HA plasmid pKC426 were treated as just described, and Clb1p levels were followed by western blot analysis. Tub1p levels served as loading controls.
(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 Ume6pdb1Δ as substrate (right panel). The arrowheads indicate unmodified substrate, and brackets indicate ubiquitylated species. The right panel was overexposed compared to the left panel to determine whether weak ubiquitylation activity was observed with the D box mutant substrate.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 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 hr in SPM, the cultures were shifted to the restrictive temperature (34.5°C). Total RNA was prepared from samples taken at the times indicated (hr) 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Ume6p Destruction Is Required for Normal EMG Transcription and Meiotic Nuclear Divisions
(A) Schematic of Ume6p indicating location of D box motifs and zinc cluster domain (Zn). Sequence alignment of Ume6p D box motifs and starting amino acid residue corresponding to the arginine is presented. The consensus sequence is given with X = any amino acid.
(B) Wild-type (RSY1079) and UME6db1Δ (RSY1080) mutant strains were induced to enter meiosis, and time points were taken as indicated (hr). T7-Ume6p and T7-Ume6pdb1Δ 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.
(C) The time points described in (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 UME6db1Δ strain (open bars) is depicted.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Ume6p Is a Substrate of APC/CCdc20 during G2-M Stage of the Cell Cycle
(A) Haploid strain RSY1069 (T7-UME6, top panel) or RSY1070 (T7-UME6db1Δ) was treated with hydroxyurea (HU) for 2 hr to arrest the cells in S phase. The cells were washed and placed in fresh medium, and time points were taken as indicated. Ume6p and Ume6pdb1Δ levels were determined by western blot analysis for every time point. Tub1p served as a loading control.
(B) The execution of anaphase was determined in the samples described in (A) by monitoring the presence of separated nuclei by DAPI staining and fluorescent microscopy.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6 Ume6p Levels Are Regulated by the RAS Pathway
(A) Wild-type (RSY1079) and UME6db1Δ (RSY1080) strains were grown in dextrose (D) or acetate (Ac) medium (two independent cultures) to mid-log phase. Ume6p or Ume6pdb1Δ levels (arrow) were monitored by western blot analysis. Tub1p served as a loading control.
(B) T7-Ume6p levels were monitored in the cdc20-1 mutant strain (RSY809) grown to mid-log in acetate medium at permissive (23°C) temperature or following 2 hr at restrictive (37°C) temperature.
(C) Ume6p levels (arrows) were determined in log phase cultures with the indicated genotypes grown at permissive temperature (23°C) and following 2 hr at the restrictive (37°C) temperature. V, vector control.
(D) Extracts prepared from log phase RSY1079 cultures in dextrose (Dex), in acetate (Ac), or after transfer to sporulation medium (SPM) for 4 hr were immunoprecipitated with the indicated antibodies or no antibody (∅). The immunoprecipitates were subjected to western blot analysis probing for the presence of Cdc20p-13×myc. Cdc20p-13×myc migration and expression were verified in the 4 hr sporulation time point (right lane). No antibody lane controls for nonspecific association of Cdc20p-13×myc to protein A beads. Molecular weight markers (kDa) are provided on the left.
(E) Ume6p or Ume6pdb1Δ 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
Ume6p Destruction Requires Meiotic Induction and Ime1p Association
(A) T7-Ume6p levels were monitored by western blot analysis in a MATa/MATa diploid (RSY303) following transfer to SPM (in hr).
(B) T7-Ume6p levels were monitored by western blot analysis in an ime1Δ diploid (RSY1128) as just described in (A).
(C) T7-Ume6p levels were monitored by western blot analysis in wild-type (RSY1079) and UME6T99N (RSY1395) diploid strains as described in (A).
(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 nonfermentable carbon source (acetate) and high nitrogen (+N2) reduces PKA activity (gray 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions