br Inhibiting APC C during Interphase and

Inhibiting APC/C during Interphase and prior to Anaphase Because ubiquitylation by APC/C triggers cell division, it is essential that APC/C is restrained until cells are prepared for its substrates to be degraded. In addition to regulation by phosphorylation, an additional layer of control comes from cellular inhibitors also restricting ubiquitylation until needed. This is best understood for APC/CCDH1 inhibition by early mitotic inhibitor (EMI)1 in interphase [58], and APC/CCDC20 inhibition by the MCC prior to azd6244 76, 77, 78, 79, 80. EMI1 and MCC may also play roles in localizing APC/C to specific subcellular structures or substrates [14], although the structural basis for this regulation remains unknown. EMI1 and MCC hijack the mobile substrate recognition and catalytic modules by binding multiple sites on APC/CCDH1 and APC/CCDC20, respectively (Box 1). However, they achieve these functions through completely different routes. In addition, MCC does not block UBE2S-dependent polyubiquitylation and even binds APC/C in distinct configurations that differentially modulate ubiquitylation, while the available structure of a single EMI1 molecule bound to APC/C shows inhibition of all ubiquitylation. EMI1 regulates the coupling of mitosis and DNA replication [81]. Briefly, after cells divide, APC/CCDH1 activity must be restrained. Although this is ultimately achieved by CDH1 phosphorylation, the required CDK activity is too low in G1 due to APC/CCDH1-dependent degradation of cyclins. Thus, accumulation of cyclins during G1 depends on EMI1 inhibiting APC/CCDH1. The best-characterized portion of the 50-kDa EMI1 protein is its 16-kDa C-terminal domain, which consists of four inhibitory elements: a D box, linker, zinc binding region (ZBR), and C-terminal tail 47, 58, 59, 60. Each individual EMI1 element only weakly interacts with APC/CCDH1, but together they synergistically bind numerous APC/CCDH1 domains to avidly inhibit (Figure 4D and Box 1) 47, 58, 59, 60. The EMI1 D box binds simultaneously to CDH1 and APC10 to block substrate access 47, 58, 59, 60. The linker and ZBR together act like a wedge to simultaneously capture the UBE2C-binding surface of the APC11 RING domain and elements of APC2 and APC1 47, 59. This has several effects including seizing the RING domain away from a catalytic conformation, while also walling off a portion of the central cavity. Finally, the C-terminal tail of EMI1 shares sequence homology with the C-terminal tail of UBE2S, docks in the same groove between APC4 and APC2, and prevents UBE2S binding 47, 59, 60. Although EMI1 locks the APC/C structure in a rigid conformation, the intrinsic flexibility of the APC11 RING domain enables its initial capture by EMI1. It is also critical that APC/CCDC20 is inhibited by MCC during the spindle assembly checkpoint, prior to correct chromosome alignment on the mitotic spindle (Box 1, reviewed in 13, 34). As proposed in [13] and demonstrated in [82], human MCC is a heterotetrameric complex consisting of its own molecule of CDC20, along with three other proteins associated with regulating the spindle assembly checkpoint (MAD2, BUBR1, and BUB3) (reviewed in [34]). The APC-bound CDC20 is denoted as CDC20A (i.e., in APC/CCDC20), and that in MCC as CDC20M. To date, BUB3 has not been visualized in cryo-EM maps despite its presence in the complexes, leaving open the structure and role of the BUB3 subunit. Nonetheless, the core of MCC – the CDC20M–MAD2–BUBR1 subcomplex, which corresponds to the entire MCC in some organisms, and is sufficient to recapitulate many biochemical properties of full human MCC in vitro (reviewed in [34]) – has been visualized bound to APC/CCDC20 by cryo-EM (Figures 2 B and 4 E–G) 38, 48, 51. APC/CCDC20–MCC is conformationally dynamic, with different architectures specifying distinct activities. The different configurations are achieved in part by tilting and rotation of the CDC20A propeller domain, and by variation in conformation of the APC2–APC11 cullin–RING subcomplex.