Regulation of the anaphase-promoting complex by phosphorylation

Regulation of the eukaryotic cell cycle is accomplished in large part by well-timed and targeted destruction of proteins that inhibit or activate passage through cell cycle transitions. Ubiquitin-mediated proteolysis is well established as the primary mechanism through which this occurs, and the anaphase-promoting complex (APC), an E3 ubiquitin ligase, is a vital regulatory component of this system. The APC is a multi-subunit complex required for cell cycle transitions that include progression through anaphase, exit from mitosis, as well as many events throughout meiosis. Two APC subunits, Apc2 and Apc11, are necessary for catalytic transfer of ubiquitin to target substrates, while the remaining subunits participate in a variety of regulatory mechanisms that coordinate APC activity with other cell cycle events. The primary mechanisms of APC regulation include co-activator protein association and phosphorylation of specific subunits in the complex. In budding yeast, two proteins, Mnd2 and Swm1, were identified by mass spectrometry after co-purification with the APC subunit, Cdc27. Here, I show that the APC co-purifies with epitope-tagged Mnd2 or Swm1 and that both proteins are present on the APC at stoichiometric levels throughout the mitotic cell cycle. Mnd2 that co-purifies with the APC is phosphorylated in a cell cycle dependent manner. Using a variety of mass spectrometry techniques, I found that phosphorylation occurs on a least 8 serine or threonine residues within the Mnd2 primary structure. Yeast strains that harbor alanine phosphorylation site mutations in Mnd2 progress normally through pre-meiotic S phase, but are unable to progress efficiently through the first nuclear division of meiosis. In contrast, yeast harboring aspartic acid phosphorylation site mutations display a partial recovery of the wild type meiosis phenotype. Alanine phosphorylation site mutants also displayed low levels of the APCAma1 meiotic substrate, Clb5. Taken together, these results demonstrate that Mnd2 is a stoichiometric component of the APC during mitosis and that Mnd2 phosphorylation is necessary for APC-mediated progression beyond the first meiotic nuclear division