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.
Highlights
Proper progression through the eukaryotic cell cycle is achieved in large part through coordinated regulation of protein degradation mediated by E32 ubiquitin ligases
Mnd2 Is Hyperphosphorylated in Vivo during Mitosis—Recent mass spectrometric analyses have shown that at least five of 11 different human anaphase-promoting complex (APC) subunits are phosphorylated on a total of 43 different Ser or Thr residues, many of which are differentially phosphorylated in a cell cycle-dependent manner [45]
Only three of 13 APC subunits in yeast have been shown to be phosphorylated in vivo [25, 26], and specific sites have been inferred from canonical cyclin-dependent kinase consensus sequences, there has not been a focused proteomics scale analysis of APC subunits far
Summary
MATa ade can100 his leu112 trp ura MATa cdc bar1⌬::URA3. mnd2⌬::KanMX4 trp1::pRS404-mnd2(S/T-A)-TRP1 mnd2⌬::KanMX4 trp1::pRS404-MND2-TRP1. The sporulation defect of the alanine mutant strain can be explained by the relatively low levels of mnd2-(S/T-A) found in APC co-precipitations compared with the aspartic acid mutant and wild type Mnd isoforms in vivo. In vitro binding between the APC and each Mnd isoform was found to be almost identical, and treatment of the APC with phosphatase did not eliminate Mnd co-precipitation, suggesting that phosphorylation may be necessary to maintain the stability of Mnd before entry into meiosis. Taken together, these data support a role for mitotic phosphorylation in the regulation of Mnd as an APCAma inhibitor in early meiosis
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