Cdc5-mediated Ulp2 phosphorylation controls the timing of polySUMOylation during the cell cycle

SUMOylation is a posttranslational modification, and polySUMOylation can further trigger protein ubiquitination and relocalization to facilitate cell cycle progression. Previous studies show cell cycle–dependent polySUMOylation in budding yeast, and depletion of SUMO protease Ulp2 causes premature polySUMOylation. Furthermore, Ulp2 undergoes phosphorylation in a manner dependent on mitotic kinase Cdc5. In this study, we report that Cdc5 is necessary for protein polySUMOylation and artificially tethering Cdc5 to Ulp2 is sufficient to trigger polySUMOylation. We further identified serine 734 as the primary phosphorylation site on Ulp2, which is regulated by Cdc5 kinase and Rts1-associated PP2A phosphatase. Notably, phosphodeficient ulp2^S734A mutant suppressed the polySUMOylation induced by CDC5 overexpression or RTS1 deletion. Finally, we found that Ulp2 phosphorylation at serine 734 compromised its binding to SUMO chains. Collectively, these results demonstrate that Cdc5-dependent phosphorylation of SUMO protease Ulp2 reduces its SUMO chain affinity and induces protein polySUMOylation, but PP2A^Rts1 counteracts this to prevent premature polySUMOylation.