Date Published: September 26, 2012
Publisher: Public Library of Science
Author(s): Denis Ostapenko, Janet L. Burton, Mark J. Solomon, Michael Polymenis. http://doi.org/10.1371/journal.pone.0045895
The Anaphase-Promoting Complex/Cyclosome (APC/C) is an essential ubiquitin ligase that targets numerous proteins for proteasome-mediated degradation in mitosis and G1. To gain further insight into cellular pathways controlled by APC/CCdh1, we developed two complementary approaches to identify additional APC/CCdh1 substrates in budding yeast. First, we analyzed the stabilities of proteins that were expressed at the same time in the cell cycle as known APC/C substrates. Second, we screened for proteins capable of interacting with the Cdh1 substrate-binding protein in a yeast two-hybrid system. Here we characterize five potential APC/C substrates identified using these approaches: the transcription factors Tos4 and Pdr3; the mRNA processing factor Fir1; the spindle checkpoint protein kinase Mps1; and a protein of unknown function, Ybr138C. Analysis of the degradation motifs within these proteins revealed that the carboxyl-terminal KEN box and D-boxes of Tos4 are important for its interaction with Cdh1, whereas the N-terminal domain of Ybr138C is required for its instability. Functionally, we found that a stabilized form of Mps1 delayed cell division upon mild spindle disruption, and that elevated levels of Ybr138C reduced cell fitness. Interestingly, both Tos4 and Pdr3 have been implicated in the DNA damage response, whereas Mps1 regulates the spindle assembly checkpoint. Thus, the APC/CCdh1-mediated degradation of these proteins may help to coordinate re-entry into the cell cycle following environmental stresses.
The orderly progression of cell cycle events requires the coordinated synthesis and degradation of multiple regulatory proteins. Degradation of these proteins occurs via the ubiquitin-dependent proteasome pathway, which entails sequential enzymatic reactions carried out by an E1 (ubiquitin activating enzyme), an E2 (ubiquitin conjugating enzyme), and an E3 (ubiquitin ligase). The resulting formation of poly-ubiquitin chains targets proteins for degradation by the 26S proteasome . Two major classes of RING-type E3s that play critical roles targeting predominantly non-overlapping sets of proteins for degradation during cell cycle progression are the Anaphase-Promoting Complex/Cyclosome (APC/C) and the SCF complex –.
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.