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  • The best characterized APC C

    2019-07-10

    The best-characterized APC/C substrates are recruited via various linear motifs (e.g., D boxes, KEN boxes, and ABBA motifs) that serve as degrons, binding to distinct regions of the β-propeller domain of a coactivator (Figure 1, Figure 3B) (reviewed in [44]). In addition, D boxes also simultaneously bind the APC/C core subunit APC10, thereby fastening the coactivator propeller adjacent to APC10 (Figures 2 A and 3 B) 39, 41, 45, 47. The positions of potential target lysines within substrates are thus determined by their locations relative to degrons bound to an APC/C–coactivator complex, and the location of the coactivator propeller determined by the presence or absence of a D box. The E2 UBE2C (also known as UBCH10) is recruited to APC/C by a specialized mechanism that places the active site proximal to substrates (Figure 4B). One side of the UBE2C∼Ub intermediate engages the RING domain of APC11 through canonical RING-E2∼Ub interactions 47, 49. However, as in other cullin–RING ligases [12], the RING domain of APC11 is loosely tethered to the cullin-binding region by a flexible linker that rotates (Figure 4). How then, does the RING-bound UBE2C∼Ub intermediate achieve a position with the active site facing substrates? Unexpectedly, the flexibly tethered cullin element at the C terminus of APC2 – the WHB subdomain – binds the so-called E2 backside of UBE2C distal from the active site (Figure 2B) [49]. Thus, the two flexibly tethered domains of APC11 and APC2 together grasp opposite sides of UBE2C, acting like a clamp to direct the catalytic center toward substrates [49]. This structural arrangement provides a potential rationale for why many APC/C substrates are ubiquitylated in intrinsically disordered regions: flexible polypeptides bound to a coactivator can access the relatively proximal but immobilized UBE2C active site 47, 49, 50, 69. Indeed, the disordered N-terminal domain of Solithromycin B can receive enough individual Ubs from UBE2C for proteasomal targeting even without generation of poly-Ub chains [29]. The confined space between the active site of UBE2C and a coactivator-bound degron may also explain why UBE2C preferentially modifies substrates with individual Ubs and short chains rather than long poly-Ub chains [70].
    Ubiquitin Capture by the Mobile APC11 RING Domain Contributes to Processive Substrate Ubiquitylation Although the rules of Ub-mediated proteolysis are only beginning to emerge, the rate and order in which different APC/C substrates are degraded during the cell cycle correlates with processivity of their ubiquitylation. Highly processive substrates receive enough Ubs in a single binding event to enable proteasome binding, and are apparently degraded earlier during the cell cycle than nonprocessive substrates that must cycle on and off APC/C numerous times to receive enough Ubs for proteasomal targeting [71]. Degradation of less-processive substrates can be further slowed by competition with other substrates preventing their rebinding to APC/C, and deubiquitylation removing the few initially linked Ubs [72]. Processivity is determined in part by the rate of dissociation of a substrate from APC/C, which depends on the affinity and arrangement of degron sequences. In addition, a substrate evolves during ubiquitylation, which affects processivity as revealed by recent single-molecule experiments. In a process called processive affinity amplification, ubiquitylation increases the duration of a substrate on APC/C and propensity for further ubiquitylation [73]. Processive affinity amplification is determined in part by mobile elements within the APC/C catalytic core, whereby a substrate-linked Ub binds an unprecedented Ub-binding site that is distinct from the UBE2C–Ub binding site on the RING domain of APC11 [74]. The RING domain simultaneously captures a Ub linked to a substrate and places UBE2C to ubiquitylate another site on a substrate [50]. Accordingly, mutationally eliminating the secondary RING–ubiquitin interaction decreases processivity in vitro, reducing the number of Ubs received by a given substrate molecule, while increasing the number of substrates receiving at least one Ub [50], although other Ub-binding sites within APC/C that contribute to processive affinity amplification or other functions remain to be described.