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  • The majority of APC C subunits form a giant scaffold

    2019-10-09

    The majority of APC/C subunits form a giant scaffold, which was originally named the arc lamp, based on its shape when viewing APC/C from one side (Figure 1 and Box 1) [24]. The scaffold consists of two modules: the TPR lobe resembles the curved post and lamp, and the platform resembles the cox inhibitor supporting the arched lamp-post. Although this visual analogy lacked functional relevance, it did reveal the organization that enables concentrating the two functional modules – the substrate recognition module (either CDC20 or CDH1 coactivator and in many cases also the core subunit APC10) and a catalytic module (i.e., the cullin–RING catalytic core consisting of the cullin subunit APC2 and its associated RING partner APC11) – within the central cavity through their respective interactions inside the TPR lobe and platform ([24]; reviewed in [15]). The termini of the substrate recognition and catalytic modules are anchored to opposite sides of the scaffold so that they face each other. Within the substrate recognition module, the coactivators CDC20 and CDH1 have three domains: intrinsically disordered N- and C-terminal regions containing so-called C boxes and IR tails, respectively, and a central β propeller that binds to D-box, KEN-box, and ABBA-motif sequences found in substrates and APC/C regulators (reviewed in 15, 44). The APC/C core subunit APC10 has two cox inhibitor domains, an N-terminal jellyroll that along with a coactivator cobinds to D-box sequences, and a C-terminal IR-tail 39, 41, 45. The IR tails from a coactivator and APC10 engage the TPR lobe through grooves at the C termini of the two APC3 protomers, while the coactivator C box docks in a homologous groove in one APC8 21, 36, 46, 47. This arrangement flexibly projects the β propeller of the coactivator toward APC10 and the APC2–APC11 catalytic module, with its position determined by its binding partners. Across the scaffold, the platform anchors the N terminus of the elongated cullin structure of APC2 [21]. This connects to the flexible cullin–RING catalytic core consisting of the C-terminal region of APC2 and the associated APC11 11, 12, 21. The flexibility and positions of the catalytic core are controlled by the orientation of the platform, and by proteins interacting directly with APC2–APC11 to regulate ubiquitylation 21, 24, 47, 48, 49, 50, 51, 52.
    Visualizing APC/C Interactions and Functions around the Cell Cycle Throughout the cell cycle, APC/C undergoes a series of transformations between assemblies for which cryo-EM data revealed that functions are dictated in part by conformations of the coactivator and the cullin–RING catalytic core. By the end of interphase, APC/C is hypophosphorylated, coactivator free, and inactive, in part due to autoinhibition, whereby intramolecular interactions block access of CDC20 and restrain the cullin–RING catalytic core 21, 24, 53, 54, 55 (Figure 2A, Conformation I). In prophase, phosphorylation conformationally activates APC/C binding to the coactivator CDC20, which in turn conformationally activates the catalytic core (see Video S1 in the Supplemental information online) 21, 24, 53, 54, 55. APC/CCDC20 is, in principle, competent to recruit substrates for ubiquitylation 21, 24 (Figure 2A, Conformation IIA). However, the timing of substrate binding is regulated by the mitotic checkpoint complex (MCC), which serves as a brake during the spindle assembly checkpoint when APC/C has been activated by CDC20 but cells are not yet prepared for division 48, 51 (Figure 2B, Conformation III). When all chromosomes are properly bioriented on the mitotic spindle, both MCC and the catalytic core are reoriented (Figure 2B, Conformation IV) so MCC can itself be ubiquitylated as a prelude to liberating APC/C for substrate ubiquitylation that triggers anaphase 48, 51, 56, 57 (Figure 2B, Conformation V). In anaphase, APC/CCDC20 recruits substrates, while the APC2–APC11 cullin–RING catalytic core engages, positions, and activates transient E2∼Ub intermediates (e.g., UBE2C∼Ub, where ‘∼’ refers to thioester bond) from which Ub is transferred to a coactivator-bound substrate (Figure 2B, Conformation VI) or to a substrate-linked Ub molecule during polyubiquitylation (Figure 2B, conformation VII) 47, 49, 50, 53. Following anaphase, CDC20 itself undergoes APC/C-mediated ubiquitylation and subsequent degradation, but is replaced by the homologous but distinct coactivator CDH1 that recruits other substrates for ubiquitylation (Figure 2A, Conformation IIB) 47, 49, 51. After substrates are degraded, then EMI1 inhibits APC/CCDH147, 58, 59, 60 (Figure 2B, Conformation VIII) to enable accumulation of G1 cyclins and CDK-dependent inactivation of CDH1.