We have developed two complementary approaches for the identification of APC/C substrates. The first approach identified candidates encoded by genes whose cell-cycle expression paralleled that of known APC/C substrates and the second approach used a yeast two-hybrid library to identify proteins that interacted with the APC/C activator Cdh1. Using these approaches we have identified five potential APC/CCdh1 substrates, providing insight into additional cell cycle processes regulated by the APC/C. All of these proteins were unstable in G1 and were either stabilized or partially stabilized by APC/CCdh1 mutations. As additional support, several of these proteins were destabilized following expression of the constitutively active Cdh1-m11 protein and interacted with Cdh1 in vitro. Of the proteins we examined, we would classify Fir1, Mps1 and Ybr138C as proven APC/C substrates, Tos4 as a likely substrate, and Pdr3 as a potential substrate requiring further study. With the exception of Mps1, the functions of the proteins identified in this study are relatively unknown. Given that the majority of characterized APC/C substrates function in cell cycle regulation, we anticipate that most of the proteins we identified will also have roles in cell division. We summarize what is known about the functions of these proteins below.
Fir1 has been implicated in mRNA processing. It interacts with RNA cleavage/polyadenylation factors and its deletion results in the shortening of polyA tails 
. Fir1 interacts with SUMO, a small ubiquitin-like modifier, and contains a SUMO-interacting motif (SIM) 
. Although we found that Fir1 interacts with Cdh1 and that it is an APC/CCdh1
substrate, mutations within eight putative D-boxes did not stabilize Fir1. Understanding how APC/C-mediated degradation of Fir1 impacts the cell cycle will need to await the identification of a stabilized form of the protein.
We found that Ybr138C expression is cell cycle regulated, that it is an APC/CCdh1
substrate in G1, and that its elevated expression delays cell growth. Together, these findings suggest that the degradation of Ybr138C is important for normal cell cycle progression. Although we were unable to stabilize Ybr138C through mutations within three potential D-boxes, we found that deletion of the N-terminal region, which lacks any obvious degradation motifs, increased protein stability. A similar situation exists with Cik1, a kinesin-associated protein that is expressed as two isoforms with the longer, mitotic form of Cik1 being targeted for ubiquitination by APC/CCdh1
through a unique N-terminal region 
. Although both Cik1 and Ybr138C contain several D-box motifs, their instability depended upon an unidentified motif(s) within their first 80 amino acids. Interestingly, the interaction of the N-terminal portion of Ybr138C with Cdh1 in the yeast two-hybrid system required Cdh1 residues important for D-box interaction suggesting that this new motif(s) could interact with Cdh1 in a similar manner despite the absence of obvious D-box motifs. More detailed analyses will be required to identify these non-canonical degradation motifs.
Mps1 is a multifunctional protein kinase required for activation and maintenance of the spindle assembly checkpoint (SAC) and for proper duplication of the spindle pole bodies 
. While this work was in progress, Mps1 was reported to be an APC/CCdc20
substrate whose overexpression in anaphase could reactivate the spindle checkpoint 
. The Ufd2 ubiquitin ligase has also been implicated in Mps1 degradation 
. We observed that the bulk of endogenous Mps1 was degraded in G1, though we cannot exclude the possibility that some kinetochore-bound Mps1 might be degraded in mitosis. Given that Mps1 overexpression can activate the spindle checkpoint 
, we were surprised that expression of a stabilized form of Mps1 (Mps1-3mdb) resulted in neither toxicity nor a cell cycle delay, despite its overexpression by several-fold. Interestingly, we found that in the presence of a low concentration of nocodazole, cells expressing Mps1-3mdb delayed progression through mitosis and had reduced cell fitness compared to wild-type cells. Thus, Mps1 degradation is nonessential under normal growth conditions but important for the proper exit from the spindle assembly checkpoint.
Screening of a yeast two-hybrid library for Cdh1-interacting proteins identified two transcription factors, Tos4 and Pdr3, both of which were found to be unstable and whose degradation was at least partially dependent upon APC/CCdh1. For Tos4, we identified four carboxyl-terminal D-boxes and a KEN box that were important for both interaction with Cdh1 and ubiquitination by APC/CCdh1
in vitro. We have not yet investigated what motifs are important for Pdr3 degradation. However, it is interesting to note that the Pdr3 protein isolated in the yeast two-hybrid screen consisted of amino acids 411–976, which contains all five putative D-boxes within Pdr3.
At present, we do not know how APC/CCdh1
-mediated degradation of either Tos4 or Pdr3 influences cell cycle progression. A recent study demonstrated that Tos4 levels increase dramatically upon DNA replication stress in a Rad53-dependent manner 
. Interestingly, Pdr3 has also been implicated in the DNA damage response, independent of its role in the pleiotropic drug response pathway. Pdr3, but not the related transcription factor Pdr1, co-activates the transcription of the DNA damage inducible genes MAG1
through binding a bi-directional promoter element located between these two genes 
. In pdr3Δ
cells, these genes were not induced upon exposure to DNA damaging agents. Since both Tos4 and Pdr3 have been linked to the DNA damage response, it is tempting to speculate that their turnover may be important for resuming cell division after DNA repair. Since we found that APC/C inactivation leads to only partial stabilization of Tos4 and Pdr3, it is likely that other E3s may also regulate their stability, as has been demonstrated for other APC/C substrates 
. Therefore, considerable effort will be required to design stabilized mutant versions of these proteins in order to investigate how their degradation might influence cell cycle progression after DNA damage.
Various systematic screens for APCCdh1
substrates have been performed and different substrate proteins have been uncovered in each of these screens. Likewise, in the present study we have identified APC/CCdh1
substrates that were not previously found using other systematic approaches. This outcome may have to do with differences in how the screens were conducted. For example our first approach analyzed endogenous low-abundance proteins rather than screening libraries of fluorescently-tagged proteins, which favored identification of more abundant substrates 
. In addition, we screened a yeast two-hybrid library to identify proteins that specifically interacted with Cdh1 in a D-box dependent manner rather than looking at protein instability as a first step in the screening process. Initial screening for Cdh1 binding may be useful for uncovering proteins that are targeted for ubiquitination by more than one ubiquitin ligase or that are recognized by APC/CCdh1
under different physiological conditions, such as during environmental stresses. For instance, the APC/C targets heat shock factor 2 and the DNA damage checkpoint protein Rad17 in response to protein-damaging and genotoxic stresses, respectively 
. Similarly, the APC/C may target Tos4 and Pdr3 for degradation following similar stresses and Mps1 for degradation after exposure to the spindle disrupting agent nocodazole.
Even though CDH1
is non-essential, cdh1Δ
cells grow slowly and exhibit various morphological abnormalities 
. Upon superficial analysis, stabilization of none of the Cdh1 substrates identified in this study caused major cell cycle delays. However, overexpression of Mps1, Pdr3, or Tos4 is toxic for cell growth, and overexpressed Fir1 leads to accumulation of sumoylated proteins 
. Our co-culture experiments revealed that stabilization of Mps1 and elevated expression of Ybr138C reduced cell fitness in the presence and absence of nocodazole, respectively. Similarly, stabilization of two other APC/CCdh1
substrates, Nrm1 and Yhp1, also resulted in modest reductions in cell fitness 
. The combined effects of stabilizing many such “minor” Cdh1 substrates likely contributes to the stronger phenotype exhibited by cdh1Δ
cells and to the coordination of cell cycle transitions with other cellular events.