Protein degradation during the cell cycle is controlled by the opposing activities of ubiquitin ligases and deubiquitinating enzymes (DUBs). Although the functions of ubiquitin ligases in the cell cycle have been studied extensively, the roles of DUBs in this process are less well understood. Here, we used an overexpression screen to examine the specificities of each of the 21 DUBs in budding yeast for 37 cell cycle-regulated proteins. We find that DUBs up-regulate specific subsets of proteins, with five DUBs regulating the greatest number of targets. Overexpression of Ubp10 had the largest effect, stabilizing 15 targets and delaying cells in mitosis. Importantly, UBP10 deletion decreased the stability of the cell cycle regulator Dbf4, delayed the G1/S transition, and slowed proliferation. Remarkably, deletion of UBP10 together with deletion of four additional DUBs restored proliferation to near-wild-type levels. Among this group, deletion of the proteasome-associated DUB Ubp6 alone reversed the G1/S delay and restored the stability of Ubp10 targets in ubp10Δ cells. Similarly, deletion of UBP14, another DUB that promotes proteasomal activity, rescued the proliferation defect in ubp10Δ cells. Our results suggest that DUBs function through a complex genetic network in which their activities are coordinated to facilitate accurate cell cycle progression.

Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates Cks1-dependent phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.

A network of transcription factors (TFs) coordinates transcription with cell cycle events in eukaryotes. Most TFs in the network are phosphorylated by cyclin-dependent kinase (CDK), which limits their activities during the cell cycle. Here, we investigate the physiological consequences of disrupting CDK regulation of the paralogous repressors Yhp1 and Yox1 in yeast. Blocking Yhp1/Yox1 phosphorylation increases their levels and decreases expression of essential cell cycle regulatory genes which, unexpectedly, increases cellular fitness in optimal growth conditions. Using synthetic genetic interaction screens, we find that Yhp1/Yox1 mutations improve the fitness of mutants with mitotic defects, including condensin mutants. Blocking Yhp1/Yox1 phosphorylation simultaneously accelerates the G1/S transition and delays mitotic exit, without decreasing proliferation rate. This mitotic delay partially reverses the chromosome segregation defect of condensin mutants, potentially explaining their increased fitness when combined with Yhp1/Yox1 phosphomutants. These findings reveal how altering expression of cell cycle genes leads to a redistribution of cell cycle timing and confers a fitness advantage to cells.

Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates processive phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.