Visualizing the complex functions and mechanisms of the anaphase promoting complex/cyclosome (APC/C)

Abstract

The anaphase promoting complex or cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that orchestrates cell cycle progression by mediating the degradation of important cell cycle regulators. During the two decades since its discovery, much has been learnt concerning its role in recognizing and ubiquitinating specific proteins in a cell-cycle-dependent manner, the mechanisms governing substrate specificity, the catalytic process of assembling polyubiquitin chains on its target proteins, and its regulation by phosphorylation and the spindle assembly checkpoint. The past few years have witnessed significant progress in understanding the quantitative mechanisms underlying these varied APC/C functions. This review integrates the overall functions and properties of the APC/C with mechanistic insights gained from recent cryo-electron microscopy (cryo-EM) studies of reconstituted human APC/C complexes.

Keywords: APC/C; cell cycle; cryo-EM; spindle assembly checkpoint.

Figure 1.

Overall structure of the human APC/C^Cdh1.Emi1 complex. (a) and (b) Two orthogonal views of the APC/C. Large APC/C subunits are represented as cartoons, whereas small APC/C subunits (Apc12, Apc13, Apc15, Apc16), the IR tails of Cdh1 and Apc10, the Cdh1 NTD and the Emi1 inhibitor are shown as space filling representations. The TPR and platform sub-structures are labelled. The two subunits of the canonical homo-dimeric TPR subunits (Apc3, Apc6, Apc7 and Apc8) and Apc12 are labelled with the suffix ‘A’ and ‘B’. Apc2^CTD and Apc11^RING form the catalytic module, Cdh1 and Apc10 generate the substrate recognition module. PDB 4UI9, from Chang et al. [80].

Figure 2.

The IR-tail and C-box binding sites of Apc3 and Apc8 respectively, are homologous. (a) Cdh1 IR-tail binding site. (b) Apc10 IR-tail binding site. (c) C-box binding site on Apc8B. The Ile and Arg side chains of the IR tail of both Cdh1 and Apc10 interact with a site on Apc3 that is homologous to the binding sites for Arg(47) and Ile(49) of the Cdh1 C box on Apc8B. The C box (DR[F/Y]IPxR) forms additional contacts to Apc8B as shown. PDB 4UI9, from Chang et al. [80].

Figure 3.

Coactivators interact with Apc1 and Apc3 and create a D-box co-receptor with Apc10. (a) Overview of the APC/C with the Cdh1 coactivator subunit. Based on the APC/C^Cdh1.Emi1 coordinates (PDB 4UI9) [80] with the KEN box and ABBA motif modelled on the S. cerevisiae Cdh1–Acm1 complex (PDB: 4BH6) [71]. Except for the D box, Emi1 coordinates are not shown. (b) Close-up view of the D-box co-receptor formed from Cdh1 and Apc10. (c) Cdk1-dependent phosphorylation of the NTD of Cdh1 blocks its binding to the APC/C. Red spheres indicate sites of inhibitory phosphorylation.

Figure 4.

Substrate recognition is mediated by coactivators and Apc10. (a) D-box receptor on Cdh1, (b) D-box co-receptor (Cdh1 and Apc10), (c) KEN-box receptor on Cdh1, (d) ABBA-motif interactions with Cdh1. Coordinates in (a,c,d) are based on the S. cerevisiae Cdh1–Acm1 complex (PDB: 4BH6) [71]. (b) Based on APC/C^Cdh1.Emi1 complex (PDB 4UI9) [80].

Figure 5.

Degron consensus sequences. (a) Sequence motif of D box derived from 68 APC/C substrates [71]. Sequence motif determined using multiple expectation maximization for motif elicitation (MEME) [116]. (b) Sequence motif of KEN box derived from 46 APC/C substrates [71]. (c) Alignment of consensus D box degron with non-canonical D box degrons. (d) Consensus KEN box. Adapted from [71].

Figure 6.

APC/C ubiquitination reaction. (a) Apo APC/C. In the absence of coactivator the catalytic module adopts a ‘down’ inactive conformation. UbcH10 binding to Apc11^RING is blocked by Apc5, and Apc5 prevents the correct location of Apc2^WHB required to engage UbcH10. EM density for Apc11^RING is weak indicating RING domain flexibility. PDB 5G05 from Zhang et al. [60]. (b) Complex of APC/C^{Cdh1.substrate} with a UbcH10 ∼ ubiquitin conjugate. Apc2^WHB becomes ordered and engages UbcH10. dUb: modelled donor ubiquitin conjugated to UbcH10. The C-terminus of dUb is indicated with a red sphere. PDB 5A31, from Chang et al. [80]. PDB for Apc2^WHB 4YII Chang et al. [111]. (c) APC/C^{Cdh1.substrate}-Ube2S∼Ub complex. Ube2S is partially built. aUb: acceptor ubiquitin bound to the Apc11^RING exosite. PDB 5L9T, from Brown et al. [112]. The figure is based on previous work [60,80,111,112].

Figure 7.

Schematic of ubiquitination reaction catalysed by the APC/C. (a) In the apo state, the downward position of the catalytic module would cause a clash between Apc5 and both UbcH10 and Apc2^WHB (as in the APC/C^{Cdh1.substrate}–UbcH10 ∼ ubiquitin complex). (b) Binding of coactivator shifts the catalytic module (Apc2 and Apc11) to an upward position. Apc2^CTD together with Apc2^WHB and Apc11^RING are highly flexible. Target lysines on the APC/C substrate are shown as ‘K’. (c) UbcH10-catalysed monoubiquitination. dU: UbcH10-conjugated donor ubiquitin. Apc2^WHB rigidifies by binding to UbcH10, Apc11^RING is less flexible. (d) UbcH10-catalysed multiubiquitination. The substrate-conjugated ubiquitin (U) engages the ubiquitin-binding exosite of Apc11^RING. (e) Ube2S-catalysed polyubiquitination. The distal acceptor ubiquitin (aU) of the polyubiquitin chain engages the ubiquitin-binding exosite of Apc11^RING positioning Lys 11 adjacent to the catalytic site of Ube2S. dU: donor ubiquitin conjugated to Ube2S. Dashed lines around Apc11^RING and Apc2^WHB denote conformational flexibility. Based on schemes from Brown et al. [112] and Chang & Barford [162].

Figure 8.

Overall structure of the phosphorylated APC/C^{Cdc20.substrate} complex. (a) and (b) Two orthogonal views of the APC/C^{Cdc20.substrate}. The substrate is the high affinity budding yeast substrate Hsl1 (residues 667 to 872 containing a D box and KEN box). EM density for Apc11^RING is weak indicating RING domain flexibility. PDB 5G04, from Zhang et al. [60].

Figure 9.

Control of APC/C^Cdc20 by phosphorylation. (a) In the unphosphorylated state an auto-inhibitory segment (AI; dark green) within the Apc1^300s loop of Apc1^WD40 mimics the Cdc20 C-box motif and binds to the C-box binding site, blocking Cdc20 association. The AI segment is located on the same face of the APC/C as the hyperphosphorylated Apc3 loop. (b) Zoomed view of the AI segment of Apc1^WD40 associated with the C-box binding site of Apc8B. (c) Superposition of the AI segment with the Cdc20 C box (purple) shows that a conserved Arg residue anchors both the C box and the AI segment to the C-box binding site. Sites of mitotic phosphorylation present within the AI segment that activate APC/C^Cdc20 are depicted as red spheres. From Zhang et al. [60].

Figure 10.

Schematic of control by phosphorylation. In the unphosphorylated state an auto-inhibitory (AI) segment of Apc1^WD40 mimics the Cdc20 C-box motif and binds to the C-box binding site, blocking Cdc20 association. Initial Cdk-dependent phosphorylation of a kinase recruitment loop in Apc3 recruits Cdk-cyclin-Cks to the APC/C to facilitate intramolecular phosphorylation of the AI segment (when transiently displaced from the C-box binding site). The phosphorylated AI segment is stably displaced from the C-box binding site, permitting Cdc20 association to generate APC/C^Cdc20. This scheme indicates the relay mechanism by which initial phosphorylation of exposed consensus Cdk1 sites on Apc3 allow recruitment of Cks-Cdk-cyclin to the APC/C to promote intramolecular phosphorylation of Apc1. From Zhang et al. [60].

Figure 11.

APC/C regulation by the MCC. (a) Atomic structure of APC/C^MCC. Ordered regions of BubR1 C-terminal to the TPR domain are shown in space-filling representation, as is the NTD and IR tail of Cdc20^MCC. (b) Schematic of BubR1 showing positions of D-box (D1, D2), KEN-box (K1, K2) and ABBA motifs (A1–A3) and schematic of Cdc20. (c) Details of the MCC-Cdc20^APC/C module with BubR1 forming extensive interactions with Cdc20^MCC and Cdc20^APC/C. (d) Schematic of interactions formed by Cdc20^MCC and Cdc20^APC/C with BubR1 and APC/C subunits. PDB 5LCW, from Alfieri et al. [92].

Figure 12.

APC/C^MCC adopts open and closed states that allows for reciprocal control by the MCC. (a) In APC/C^MCC-closed, the MCC inhibits both substrate (for example, securin and cyclin B) and UbcH10 recognition. Upper panel: overall APC/C^MCC structure. Lower panel: shows how binding of the MCC in APC/C^MCC-closed causes an upward movement of Apc5^NTD and Apc4^HBD (compare with b) and concomitant disordering of the N-terminal helix of Apc15 (Apc15^NTH). (b) In APC/C^MCC-open, the catalytic module is exposed, Apc5^NTD rotates, Apc4^HBD translates down by 10 Å, and Apc15^NTH is ordered. Movements of Apc4^HBD, Apc5^NTD and Apc11^RING domain are indicated with arrows. (c) In the APC/C^MCC-UbcH10 complex, APC/C^MCC adopts the open conformation with Apc15^NTH ordered and UbcH10 docking to its canonical position on Apc11^RING and Apc2^WHB. The C-terminal tail of Cdc20^MCC engages the catalytic site of UbcH10 for auto-ubiquitination of Lys485 and Lys490. In APC/C^MCC-closed, the C-terminal IR tail of Cdc20^MCC engages the C-box binding site of Apc8A. From Alfieri et al. [92].

Figure 13.

Reciprocal regulation of the APC/C and MCC at the SAC. Cartoon illustrating reciprocal regulation of APC/C and MCC by APC/C^MCC. MCC binding to APC/C^Cdc20 generates APC/C^MCC that blocks D-box- and KEN-box-dependent substrate binding. APC/C^MCC interconverts between APC/C^MCC-closed and APC/C^MCC-open, with APC/C^MCC-closed predominating. In APC/C^MCC-open the UbcH10 binding site is exposed. UbcH10 binding to APC/C^MCC-open catalyses Cdc20^MCC auto-ubiquitination. Ube2S elongates Ub-conjugates initiated by UbcH10. Cdc20^MCC ubiquitination promotes disassembly of APC/C^MCC to generate APC/C^Cdc20. APC/C^MCC disassembly may also be mediated by BubR1 ubiquitination. p31^comet and TRIP13 participate in MCC disassembly. During an active SAC, sustained assembly of MCC regenerates APC/C^MCC. (U: ubiquitin, dU: donor ubiquitin). The position of Bub3 within APC/C^MCC is unknown. Adapted from Alfieri et al. [92].