Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1.

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APC(Cdh1) inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APC(Cdh1) inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APC(Cdh1) whereas lysine removal from the APC substrate Hsl1 converted it into a potent APC(Cdh1) inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APC(Cdh1).

Transcriptional and post-transcriptional regulation of Cdc20 during the spindle assembly checkpoint in S. cerevisiae.

The anaphase-promoting complex (APC) is a ubiquitin ligase responsible for promoting the degradation of many cell cycle regulators. One of the activators and substrate-binding proteins for the APC is Cdc20. It has been shown previously that Cdc20 can promote its own degradation by the APC in normal cycling cells mainly through a cis-degradation mode (i.e. via an intramolecular mechanism). However, how Cdc20 is degraded during the spindle assembly checkpoint (SAC) is still not fully clear. In this study, we used a dual-Cdc20 system to investigate this issue and found that the cis-degradation mode is also the major pathway responsible for Cdc20 degradation during the SAC. In addition, we found that there is an inverse relationship between APCCdc20 activity and the transcriptional activity of the CDC20 promoter, which likely occurs through feedback regulation by APCCdc20 substrates, such as the cyclins Clb2 and Clb5. These findings contribute to our understanding of how the inhibition of APCCdc20 activity and enhanced Cdc20 degradation are required for proper spindle checkpoint arrest.

The Ubp15 deubiquitinase promotes timely entry into S phase in Saccharomyces cerevisiae.

The anaphase-promoting complex in partnership with its activator, Cdh1, is an E3 ubiquitin ligase responsible for targeting cell cycle proteins during G1 phase. In the budding yeast Saccharomyces cerevisiae, Cdh1 associates with the deubiquitinating enzyme Ubp15, but the significance of this interaction is unclear. To better understand the physiological role(s) of Ubp15, we examined cell cycle phenotypes of cells lacking Ubp15. We found that ubp15∆ cells exhibited delayed progression from G1 into S phase and increased sensitivity to the DNA synthesis inhibitor hydroxyurea. Both phenotypes of ubp15∆ cells were rescued by additional copies of the S-phase cyclin gene CLB5. Clb5 is an unstable protein targeted for proteasome-mediated degradation by several pathways. We found that during G1 phase, the APC(Cdh1)-mediated degradation of Clb5 was accelerated in ubp15∆ cells. Ubp15 interacted with Clb5 independent of Cdh1 and deubiquitinated Clb5 in a reconstituted system. Thus deubiquitination by Ubp15 counteracts APC activity toward cyclin Clb5 to allow Clb5 accumulation and a timely entry into S phase.

Identification of anaphase promoting complex substrates in S. cerevisiae.

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/C(Cdh1), we developed two complementary approaches to identify additional APC/C(Cdh1) 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/C(Cdh1)-mediated degradation of these proteins may help to coordinate re-entry into the cell cycle following environmental stresses.

Securin' M-phase entry.

The anaphase-promoting complex (APC) mediates the ubiquitination and degradation of key M-phase regulators, including cyclins and the anaphase inhibitor securin. Intriguingly, securin can also inhibit the degradation of cyclin B. This competition between substrates permits the accumulation of enough cyclin to drive entry into M phase.

Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation.

The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.

Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint.

Inappropriate attachment/tension between chromosomal kinetochores and the kinetochore microtubules activates the spindle assembly checkpoint, which delays anaphase by blocking the ubiquitin-mediated degradation of securin/Pds1p by APCCdc20. The checkpoint proteins Mad2 and Mad3/BubR1 bind to Cdc20, although how they inhibit APCCdc20 is unclear. We investigated the roles of two evolutionarily conserved KEN boxes and a D box within Mad3/BubR1. Although such motifs usually mediate APC-substrate recognition and ubiquitination, they have no apparent role in Mad3p turnover in Saccharomyces cerevisiae. Instead, these motifs are important for Mad3p function in the checkpoint and for binding to Cdc20p. We show that the Mad3p D box and KEN boxes function together to mediate Cdc20p-Mad3p interaction and that Mad3p and an anaphase-promoting complex (APC) substrate, Hsl1p, compete for Cdc20p binding in a D-box- and KEN-box-dependent manner. In vivo, we observed an increased binding of Cdc20p to Mad3p and decreased binding to Hsl1p upon checkpoint activation. Furthermore, we demonstrate that Mad2p stimulates the association between Mad3p and Cdc20p and that this stimulated binding requires KEN box 1 within Mad3p. These findings implicate Mad3p as a pseudosubstrate inhibitor of APCCdc20, competing with APC substrates for Cdc20p binding. We present a model aimed at unifying previous analyses of checkpoint function by focusing on the Mad3-Cdc20 interaction.

Assembly of an APC-Cdh1-substrate complex is stimulated by engagement of a destruction box.

The anaphase-promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators. Cdh1p is an APC coactivator that directly binds APC substrates. A genetic screen in budding yeast identified residues within Cdh1p critical for its function. Cdh1p proteins containing mutations within the "C box" or the "IR" motif could bind substrate, but not the APC, whereas mutants that only bound the APC were not identified, suggesting an ordered assembly of the ternary APC-Cdh1p-substrate complex. Supporting this hypothesis, we found that substrate binding to wild-type Cdh1p enhanced its association with the APC in yeast cells. We used peptide competition assays to demonstrate that Cdh1p interacts directly with the D box and the KEN box, two motifs within APC substrates known to be required for APC-mediated degradation. Moreover, an intact D box domain within a substrate was required to stimulate the association between the Cdh1p-substrate complex and the APC.