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Updated: Jul 15, 2026
Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae
Published on: October 11, 2022
Mitotic phosphorylation of the anaphase-promoting complex inhibitory subunit Mnd2 is necessary for efficient
Matthew P Torres1, Christoph H Borchers
1Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Abstract:
The yeast anaphase-promoting complex (APC) subunit Mnd2 is necessary for maintaining sister chromatid cohesion in prophase I of meiosis by inhibiting premature ubiquitination and subsequent degradation of substrates by the APC(Ama1) ubiquitin ligase. In a proteomics screen for post-translational modifications on the APC, we discovered that Mnd2 is phosphorylated during mitosis in a cell cycle-dependent manner. We identified and characterized the sites of mitotic Mnd2 phosphorylation during the cell cycle. Collective mutation of Mnd2 phosphorylation sites to alanine had no effect on vegetative growth but a striking effect (>85% reduction) on the percentage of tetrad-forming cells compared with the wild type strain. Similar to the MND2 deletion strain, cells harboring the alanine mutant that did not form spores arrested after premeiotic S phase with a single undivided nucleus and low levels of the APC(Ama1) meiotic substrate, Clb5, relative to wild type cells. In contrast, collective mutation of Mnd2 phosphorylation sites to aspartic acid resulted in partial suppression of the sporulation defect. No differences were observed in the binding between each Mnd2 isoform and the APC in vitro. However, in vivo, we observed a gradient in the abundance of APC-associated Mnd2 in each strain that was proportional to the observed differences in sporulation and Clb5 levels. Taken together, these data suggest that mitotic phosphorylation of Mnd2 is necessary for APC-mediated progression beyond the first meiotic nuclear division.
Insights
Mitotic phosphorylation of Mnd2 is crucial for yeast meiosis. This modification ensures proper cell division by regulating the anaphase-promoting complex (APC), preventing meiotic progression defects.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- The anaphase-promoting complex (APC) is a critical ubiquitin ligase regulating cell cycle progression.
- Mnd2 is an APC subunit essential for maintaining sister chromatid cohesion during meiotic prophase I.
- Mnd2 prevents premature degradation of APC substrates by the APC(Ama1) ubiquitin ligase.
Purpose of the Study:
- To investigate the role of Mnd2 phosphorylation in regulating APC function during meiosis.
- To identify and characterize the cell cycle-dependent phosphorylation sites on Mnd2 during mitosis.
Main Methods:
- Proteomics screen to identify post-translational modifications on APC subunits.
- Site-directed mutagenesis to create Mnd2 phosphorylation site mutants (alanine and aspartic acid).
- Analysis of meiotic progression, sporulation efficiency, and substrate levels (Clb5) in wild-type and mutant yeast strains.
- In vitro and in vivo binding assays to assess Mnd2-APC interactions.
Main Results:
- Mitotic Mnd2 phosphorylation was identified and characterized.
- Mutating phosphorylation sites to alanine severely impaired sporulation (>85% reduction) and caused cell cycle arrest.
- Alanine mutants showed reduced levels of the meiotic substrate Clb5, similar to Mnd2 deletion strains.
- Mutation to aspartic acid partially rescued the sporulation defect.
- In vivo APC-associated Mnd2 abundance correlated with sporulation efficiency and Clb5 levels.
Conclusions:
- Mitotic phosphorylation of Mnd2 is essential for proper APC function during meiosis.
- Phosphorylation regulates Mnd2's interaction with or stability within the APC complex in vivo.
- This modification is critical for progression through the first meiotic nuclear division.
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