A large fraction of the proteome displays cell cycle-dependent expression, which is important for cells to accurately grow and divide. Cyclical protein expression requires protein degradation via the ubiquitin proteasome system (UPS), and several ubiquitin ligases (E3) have established roles in this regulation. Less is understood about the roles of deubiquitinating enzymes (DUB), which antagonize E3 activity. A few DUBs have been shown to interact with and deubiquitinate cell cycle-regulatory E3s and their protein substrates, suggesting DUBs play key roles in cell cycle control. However, in vitro studies and characterization of individual DUB deletion strains in yeast suggest that these enzymes are highly redundant, making it difficult to identify their in vivo substrates and therefore fully understand their functions in the cell. To determine if DUBs play a role in the cell cycle, I performed a screen to identify specific DUB targets in vivo and then explored how these interactions contribute to cell cycle control. I conducted an in vivo overexpression screen to identify specific substrates of DUBs from a sample of UPS-regulated proteins and I determined that DUBs regulate different subsets of targets, confirming they display specificity in vivo. Five DUBs regulated the largest number of substrates, with Ubp10 stabilizing 40% of the proteins tested. Deletion of Ubp10 delayed the G1-S transition and reduced expression of Dbf4, a regulatory subunit of Cdc7 kinase, demonstrating Ubp10 is important for progression into S-phase. We hypothesized that compound deletion strains of these five DUBs would be deficient in key cellular processes because they regulated the largest number of cell cycle proteins from our screen. I performed genetic analysis to determine if redundancies exist between these DUBs. Our results indicate that most individual and combination deletion strains do not have impaired proliferation, with the exception of cells lacking UBP10. However, I observed negative interactions in some combinations when cells were challenged by different stressors. This implies the DUB network may activate redundant pathways only upon certain environmental conditions. While deletion of UBP10 impaired proliferation under standard growth conditions, I discovered that deletion of the proteasome-regulatory DUBs Ubp6 or Ubp14 rescues the cell cycle defect inubp10∆ cells. This suggests in the absence of Ubp10 substrates such as Dbf4 are rapidly degraded by the proteasome, but deletion of proteasome-associated DUBs restores cell cycle progression. Our work demonstrates that in unperturbed cells DUBs display specificity for their substrates in vivo and that a coordination of DUB activities promotes cell cycle progression.

Cyclin-dependent kinase (Cdk1) orchestrates progression through the cell cycle by coordinating the activities of cell-cycle regulators. Although phosphatases that oppose Cdk1 are likely to be necessary to establish dynamic phosphorylation, specific phosphatases that target most Cdk1 substrates have not been identified. In budding yeast, the transcription factor Hcm1 activates expression of genes that regulate chromosome segregation and is critical for maintaining genome stability. Previously we found that Hcm1 activity and degradation are stimulated by Cdk1 phosphorylation of distinct clusters of sites. Here we show that, upon exposure to environmental stress, the phosphatase calcineurin inhibits Hcm1 by specifically removing activating phosphorylations and that this regulation is important for cells to delay proliferation when they encounter stress. Our work identifies a mechanism by which proliferative signals from Cdk1 are removed in response to stress and suggests that Hcm1 functions as a rheostat that integrates stimulatory and inhibitory signals to control cell proliferation.

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.