Insights Into Substrate Recognition by the Anaphase Promoting Complex (APC)
Mitotic exit depends on the proper degradation of numerous cell cycle-regulated proteins, which is executed by an E3 ubiquitin ligase called the anaphase promoting complex (APC). APC adds polyubiquitin chains to numerous substrates, which leads to their subsequent degradation in late mitosis and G1 phase. The selective and timely recognition and degradation of APC substrates is essential for proper cell cycle progression and maintenance of genome stability. However, a key question is how APC specifically recognizes such a diversity of substrates. The activation of APC requires binding to one of its co-activators, Cdh1 or Cdc20. The co-activators directly facilitate APC enzymatic activity, but also contribute to the highly selective recognition of substrates via binding to degrons, such as the destruction box (D-box), KEN-box, and ABBA motif. However, not all substrates contain these degrons. Moreover, D- and KEN-box sequences are found in many proteins that are clearly not APC substrates, indicating that additional factors must contribute to recognition of these degrons. APC activity is tightly regulated by pseudosubstrate inhibitors, which bind tightly to the degron receptor sites on the APC co-activators to competitively block substrate binding. Pseudosubstrate inhibitors have been identified throughout the eukaryotes, including in yeast, plant, and metazoan species. In budding yeast, Cdh1 is regulated by a pseudosubstrate inhibitor called Acm1, which uses both D-box and KEN-box motifs and a unique sequence called the ABBA motif to stably bind and inhibit Cdh1. Although high- resolution structure of the Cdh1-Acm1 complex has been solved, an important question still remains unanswered: what distinguishes Acm1 from true substrates to allow it to evade ubiquitination and degradation? The answer will help us better understand the requirements for APC substrate recognition and specificity. Moreover, since APC is considered an attractive cancer chemotherapeutic target, knowledge of the inhibitory mechanism of Acm1, a natural APC inhibitor, can guide development of selective APC inhibitors in the future. Here I found that the D-box receptor on Cdh1 is required for normal proteolysis of most, possibly all, APCCdh1 substrates, including many lacking consensus D-box sequences. In contrast, the KEN-box and ABBA motif receptors are only required for proteolysis of a subset of Cdh1 substrates. Moreover, Acm1 was shown to inhibit the in vivo proteolysis of diverse APCCdh1 substrates in budding yeast, including those without canonical D- and/or KEN boxes. Therefore, at least part of the substrate recognition mechanisms appears to be shared by all APCCdh1 substrates. Moreover, I found that Acm1 can be converted into an APC substrate in vivo solely by mutation of its D-box degron. I identified a new component of the D-box that is responsible for Acm1 acting as an inhibitor. Replacing this sequence just C-terminal to the D-box core consensus in Acm1 with the corresponding sequence from Hsl1, a true substrate, is sufficient to convert Acm1 into an APCCdh1 substrate. Conversely, several APC Cdh1 substrates can be stabilized by replacing this D-box extension (DBE) with the corresponding one from Acm1, and these mutant APCCdh1 substrates also acquire weak inhibitory function. These results together suggest that the DBE in Acm1 has unique properties that lead to potent APC inhibition, likely through high affinity binding coupled with perturbation of APC’s enzymatic activity. APCCdh1 substrates can also be stabilized by mutating the residues of their DBEs to just alanine. This indicates that the DBE is generally important for substrate processing by APCCdh1. In summary, my study reveal the critical role of D-box, which includes both RxxL core sequence and the extension, in substrate recognition by APCCdh1.
Hall, Purdue University.
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