Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit

The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin ligase that is responsible for the metaphase to anaphase transition and the exit from mitosis. One of the subunits of the APC that is required for its ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology domain that has been identified in a number of diverse putative E3 ubiquitin ligases. Here, we present the crystal structure of Saccharomyces cerevisiae Doc1/Apc10 at 2.2A resolution. The Doc1 homology domain forms a beta-sandwich structure that is related in architecture to the galactose-binding domain of galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1. Residues that are invariant amongst Doc1/Apc10 sequences, including a temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the molecule, whose counterpart in galactose oxidase, the coagulation factor C2 domains and XRCC1, mediate bio-molecular interactions. This finding suggests the identification of the functionally important and conserved region of Doc1/Apc10 and, since invariant residues of Doc1/Apc10 colocalise with conserved residues of other Doc1 homology domains, we propose that the Doc1 homology domains perform common ubiquitination functions in the APC and other E3 ubiquitin ligases.     

Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27

The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.     

Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets specific cell cycle-related proteins for degradation, regulating progression from metaphase to anaphase and exit from mitosis. The APC is regulated by binding of the coactivator proteins Cdc20p and Cdh1p, and by phosphorylation. We have developed a purification strategy that allowed us to purify the budding yeast APC to near homogeneity and identify two novel APC-associated proteins, Swm1p and Mnd2p. Using an in vitro ubiquitylation system and a native gel binding assay, we have characterized the properties of wild-type and mutant APC. We show that both the D and KEN boxes contribute to substrate recognition and that coactivator is required for substrate binding. APC lacking Apc9p or Doc1p/Apc10 have impaired E3 ligase activities. However, whereas Apc9p is required for structural stability and the incorporation of Cdc27p into the APC complex, Doc1p/Apc10 plays a specific role in substrate recognition by APC-coactivator complexes. These results imply that Doc1p/Apc10 may play a role to regulate the binding of specific substrates, similar to that of the coactivators.     

The Doc1 subunit is a processivity factor for the anaphase-promoting complex

Ubiquitin-mediated proteolysis of securin and mitotic cyclins is essential for exit from mitosis. The final step in ubiquitination of these and other proteins is catalysed by the anaphase-promoting complex (APC), a multi-subunit ubiquitin-protein ligase (E3). Little is known about the molecular reaction resulting in APC-dependent substrate ubiquitination or the role of individual APC subunits in the reaction. Using a well-defined in vitro system, we show that highly purified APC from Saccharomyces cerevisiae ubiquitinates a model cyclin substrate in a processive manner. Analysis of mutant APC lacking the Doc1/Apc10 subunit (APC(doc1 Delta)) indicates that Doc1 is required for processivity. The specific molecular defect in APC(doc1 Delta) is identified by a large increase in apparent K(M) for the cyclin substrate relative to the wild-type enzyme. This suggests that Doc1 stimulates processivity by limiting substrate dissociation. Addition of recombinant Doc1 to APC(doc1 Delta) fully restores enzyme function. Doc1-related domains are found in mechanistically distinct ubiquitin-ligase enzymes and may generally stimulate ubiquitination by contributing to substrate-enzyme affinity.     

The APC subunit Doc1 promotes recognition of the substrate destruction box

Background: Accurate chromosome segregation during mitosis requires the coordinated destruction of the mitotic regulators securin and cyclins. The anaphase-promoting complex (APC) is a multisubunit ubiquitin-protein ligase that catalyzes the polyubiquitination of these and other proteins and thereby promotes their destruction. How the APC recognizes its substrates is not well understood. In mitosis, the APC activator Cdc20 binds to the APC and is thought to recruit substrates by interacting with a conserved target protein motif called the destruction box. A related protein, called Cdh1, performs a similar function during G1. Recent evidence, however, suggests that the core APC subunit Doc1 also contributes to substrate recognition. Results: To better understand the mechanism by which Doc1 promotes substrate binding to the APC, we generated a series of point mutations in Doc1 and analyzed their effects on the processivity of substrate ubiquitination. Mutations that reduce Doc1 function fall into two classes that define spatially and functionally distinct regions of the protein. One region, which includes the carboxy terminus, anchors Doc1 to the APC but does not influence substrate recognition. The other region, located on the opposite face of Doc1, is required for Doc1 to enhance substrate binding to the APC. Importantly, stimulation of binding by Doc1 also requires that the substrate contain an intact destruction box. Cells carrying DOC1 mutations that eliminate substrate recognition delay in mitosis with high levels of APC substrates. Conclusions: Doc1 contributes to recognition of the substrate destruction box by the APC. This function of Doc1 is necessary for efficient substrate proteolysis in vivo.     

The Apc5 subunit of the anaphase-promoting complex/cyclosome interacts with poly(A) binding protein and represses internal ribosome entry site-mediated translation

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G(1). We used a yeast three-hybrid screen to identify proteins that interact with the internal ribosome entry site (IRES) of platelet-derived growth factor 2 mRNA. Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain. We found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element. PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect. In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction. In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation. Expression of Apc5 in differentiated cells abolished IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex.     

Apc10 and Ste9/Srw1, two regulators of the APC-cyclosome, as well as the CDK inhibitor Rum1 are required for G1 cell-cycle arrest in fission yeast.

Many eukaryotic cells arrest the cell cycle at G1 phase upon nutrient deprivation. In fission yeast, during nitrogen starvation, cells divide twice and arrest at G1. We have isolated a novel type of sterile mutant, which undergoes one additional S phase upon starvation and, as a result, arrests at G2. Three loci (apc10, ste9/srw1 and rum1) were identified. The apc10 mutants, previously unidentified, show, in addition to sterility, temperature-sensitive growth with defects in chromosome segregation. apc10(+) is essential for viability, encodes a conserved protein (a homologue of budding yeast Apc10/Doc1) and is required for ubiquitination and degradation of mitotic B-type cyclins. Apc10 does not co-sediment with the 20S APC-cyclosome, a ubiquitin ligase for B-type cyclins, and in the apc10 mutant the 20S complex is intact, suggesting that it is a novel regulator for this complex. A subpopulation of Apc10 does co-immunoprecipitate with the anaphase-promoting complex (APC). A second gene, ste9(+)/srw1(+), encodes a member of the fizzy-related family, also regulators of the APC. Finally, Rum1 is a cyclin-dependent kinase (CDK) inhibitor which exists only in G1. The results suggest that dual downregulation of CDK, one via the APC and the other via the CDK inhibitor, is a universal mechanism that is used to arrest cell cycle progression at G1.     

Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex

The anaphase-promoting complex (APC), or cyclosome, is a cell cycle-regulated ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle. The APC is composed of at least 11 subunits; no structure has been determined for any of these subunits. The subunit APC10/DOC1, a one-domain protein consisting of 185 amino acids, has a conserved core (residues 22-161) that is homologous to domains found in several other putative ubiquitin ligases and, therefore, may play a role in ubiquitination reactions. Here we report the crystal structure of human APC10 at 1.6 A resolution. The core of the protein is formed by a beta-sandwich that adopts a jellyroll fold. Unexpectedly, this structure is highly similar to ligand-binding domains of several bacterial and eukaryotic proteins, such as galactose oxidase and coagulation factor Va, raising the possibility that APC10 may function by binding a yet unidentified ligand. We further provide biochemical evidence that the C-terminus of APC10 binds to CDC27/APC3, an APC subunit that contains multiple tetratrico peptide repeats.     

The APC11 RING-H2 finger mediates E2-dependent ubiquitination

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that the Saccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.     

Dynamic regulation of mitotic ubiquitin ligase APC/C by coordinated Plx1 kinase and PP2A phosphatase action on a flexible Apc1 loop

The anaphase‐promoting complex/cyclosome (APC/C), a multi‐subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin‐dependent kinase 1 (Cdk1) promotes Cdc20 co‐activator loading in mitosis to form active APC/C‐Cdc20. However, detailed phospho‐regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo‐like kinase (Plx1) and PP2A‐B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1‐loop⁵⁰⁰) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1‐loop⁵⁰⁰ in a phosphorylation‐dependent manner and promotes the formation of APC/C‐Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A‐B56 is recruited to the Apc1‐loop⁵⁰⁰ and differentially promotes dissociation of Plx1 and PP2A‐B56 through dephosphorylation of Plx1‐binding sites. Stable Plx1 binding, which prevents PP2A‐B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1‐loop⁵⁰⁰ is controlled by distant Apc3‐loop phosphorylation. Our study suggests that phosphorylation‐dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.     

Crystal Structure of the N-terminal Domain of Anaphase-promoting Complex Subunit 7

Anaphase-promoting complex or cyclosome (APC/C) is an unusual E3 ubiquitin ligase and an essential protein that controls mitotic progression. APC/C includes at least 13 subunits, but no structure has been determined for any tetratricopeptide repeat (TPR)-containing subunit (Apc3 and -6-8) in the TPR subcomplex of APC/C. Apc7 is a TPR-containing subunit that exists only in vertebrate APC/C. Here we report the crystal structure of quad mutant of nApc7 (N-terminal fragment, residues 1-147) of human Apc7 at a resolution of 2.5 Å. The structure of nApc7 adopts a TPR-like motif and has a unique dimerization interface, although the protein does not contain the conserved TPR sequence. Based on the structure of nApc7, in addition to previous experimental findings, we proposed a putative homodimeric structure for full-length Apc7. This model suggests that TPR-containing subunits self-associate and bind to adaptors and substrates via an IR peptide in TPR-containing subunits of APC/C.Anaphase-promoting complex/cyclosome (APC/C)² is an E3 ubiquitin ligase that controls mitotic progression (1). APC/C is an ∼1.7-MDa protein complex that is composed of at least 13 subunits, and it contains a cullin homolog (Apc2), a ring-H2 finger domain (Apc11), and a tetratricopeptide repeat (TPR)-containing subunit (TPR subunit; Apc3 and -6-8) (2). Most TPR subunits are essential and evolutionarily conserved in eukaryotes (3).APC/C requires two adaptors that contain a C-terminal WD40 domain, Cdc20 and Cdh1, to recruit and select various substrates at different stages of the cell cycle. Moreover, both adaptors and specific APC/C subunits contribute to substrate recognition (4).APC/C specifically ubiquitinates cell cycle regulatory proteins that contain destruction (D) or KEN box motifs (5-7), which target them for destruction by the 26 S proteosome (8). During the cell cycle, APC/C mediates the metaphase-anaphase transition by ubiquitinating and degrading securin, a separase inhibitor, which participates in the degradation of chromatic cohesion complexes and ubiquitinates B-type cyclin, thereby accelerating transition from the late mitotic phase to G₁ (9). In addition to its primary role in cell cycle regulation, APC/C participates in postmitotic processes, such as regulation of synaptic size and axon growth (10, 11).To assess the mechanism that underlies cell cycle regulation by APC/C and the various roles of its subunits, we need to understand how APC/C is organized into higher order structures and the manner in which the subunits assemble. Although little is known regarding the crystal structures of APC/C components, three-dimensional models of APC/C have recently been obtained by cryo-negative staining EM in human, Xenopus laevis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe (12-15). Several studies have indicated that APC/C assumes an asymmetric triangular shape that is composed of an outer shell and a cavity that extends through its center (12, 14). Furthermore, APC/C includes a catalytic subcomplex (Doc1/Apc10, Apc11, and Apc2), a structural complex (Apc1, Apc4, and Apc5), and a TPR subcomplex (TPR-containing subunits and nonessential subunits) (16).A TPR unit consists of a 34-residue repeat motif that adopts a helix-turn-helix conformation, which is associated with protein-protein interactions (17). Multiple copies of TPR-containing subunits are organized into the TPR subcomplex within APC/C, and this subcomplex is functionally important for the recruitment of adaptors and substrates (18). In fact, adaptors (Cdc20 and Cdh1) and Doc1/Apc10 bind to the C-terminal domain of the TPR-containing subunits Apc3 and Apc7 via the IR peptide tail sequence (7, 16, 19). It is unknown, however, how TPR-containing subunits form homo- and heterosubunit complexes, although studies have demonstrated that TPR-containing subunits self-associate in vivo and in vitro (15) and that they interact with other TPR-containing subunits (20).Apc7 is found only in vertebrate APC/C and is estimated to contain 9-15 TPR motifs, similar to other TPR-containing subunits (9). Apc7 is considered to be a molecular descendant of the same ancestral protein that gave rise to Apc3. Furthermore, the N-terminal domain of Apc7 has been reported to contain cell cycle-regulated phosphorylation sites (21), and the C-terminal TPR domain of Apc7 interacts with Cdh1 and Cdc20 (19). In Drosophila APC/C, the homolog of vertebrate Apc7 participates in synergistic genetic interactions with other TPR-containing subunits (22).The function of Apc7 within vertebrate APC/C, however, is poorly understood. Moreover, although the C-terminal regions of Apc3 and Apc7 include a tandem of nine TPR motifs, the N-terminal domains of human Apc3 and Apc7 share little homology with the canonical TPR sequence. Therefore, the N-terminal domain of human Apc7 is expected to have a significant function in vertebrate APC/C.In this study, we determined the crystal structure of the N-terminal fragment of human Apc7 (residues 1-147, denoted nApc7), and the homodimeric self-association of nApc7 structure led us to insights into mechanisms of vertebrate APC/C.     

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.     

Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex

The anaphase-promoting complex (APC) or cyclosome is a multi-subunit ubiquitin ligase that controls progression through mitosis and the G1-phase of the cell cycle. The APC ubiquitinates regulatory proteins such as securin and cyclin B and thereby targets them for destruction by the 26S proteasome. Activation of the APC depends on the activator proteins Cdc20 and Cdh1, which are thought to recruit substrates to the APC. In vitro, APC's RING finger subunit Apc11 alone can also function as a ubiquitin ligase. Here, we review different methods that have been used to measure the ubiquitination activity of the APC in vitro and to analyze APC-mediated degradation reactions either in vitro or in vivo. We describe procedures to isolate the APC from human cells or from Xenopus eggs, to activate purified APC with recombinant Cdc20 or Cdh1 and to measure the ubiquitination activity of the resulting APC(Cdc20) and APC(Cdh1) complexes. We also describe procedures to analyze the ubiquitination activity associated with recombinant Apc11.     

In vivo characterization of the nonessential budding yeast anaphase-promoting complex/cyclosome components Swm1p, Mnd2p and Apc9p

We have examined the in vivo requirement of two recently identified nonessential components of the budding yeast anaphase-promoting complex, Swm1p and Mnd2p, as well as that of the previously identified subunit Apc9p. swm1Delta mutants exhibit synthetic lethality or conditional synthetic lethality with other APC/C subunits and regulators, whereas mnd2Delta mutants are less sensitive to perturbation of the APC/C. swm1Delta mutants, but not mnd2Delta mutants, exhibit defects in APC/C substrate turnover, both during the mitotic cell cycle and in alpha-factor-arrested cells. In contrast, apc9Delta mutants exhibit only minor defects in substrate degradation in alpha-factor-arrested cells. In cycling cells, degradation of Clb2p, but not Pds1p or Clb5p, is delayed in apc9Delta. Our findings suggest that Swm1p is required for full catalytic activity of the APC/C, whereas the requirement of Mnd2p for APC/C function appears to be negligible under standard laboratory conditions. Furthermore, the role of Apc9p in APC/C-dependent ubiquitination may be limited to the proteolysis of a select number of substrates.     

Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex

Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tags (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.     

Subunits and substrates of the anaphase-promoting complex

The initiation of anaphase and exit from mitosis depend on a ubiquitination complex called the anaphase-promoting complex (APC) or cyclosome. The APC is composed of more than 10 constitutive subunits and associates with additional regulatory factors in mitosis and during the G1 phase of the cell cycle. At the metaphase-anaphase transition the APC ubiquitinates proteins such as Pds1 in budding yeast and Cut2 in fission yeast whose subsequent degradation by the 26S proteasome is essential for the initiation of sister chromatid separation. Later in anaphase and telophase the APC promotes the inactivation of the mitotic cyclin-dependent protein kinase 1 by ubiquitinating its activating subunit cyclin B. The APC also mediates the ubiquitin-dependent proteolysis of several other mitotic regulators, including other protein kinases, APC activators, spindle-associated proteins, and inhibitors of DNA replication.     

Depletion of anaphase-promoting complex or cyclosome (APC/C) subunit homolog APC1 or CDC27 of Trypanosoma brucei arrests the procyclic form in metaphase but the bloodstream form in anaphase

The anaphase-promoting complex or cyclosome (APC/C) is a multiprotein subunit E3 ubiquitin ligase complex that controls segregation of chromosomes and exit from mitosis in eukaryotes. It triggers elimination of key cell cycle regulators such as securin and mitotic cyclins during mitosis by polyubiquitinating them for proteasome degradation. Seven core subunit homologs of APC/C (APC1, APC2, APC11, CDC16, CDC23, CDC27, and DOC1) were identified in the Trypanosoma brucei genome data base. Expression of six of them was individually ablated by RNA interference in both the procyclic and bloodstream forms of T. brucei. Only the CDC27- and APC1-depleted cells were enriched in the G2/M phase with inhibited growth. Further studies indicated that T. brucei APC1 and CDC27 failed to complement the corresponding deletion mutants of budding yeast. However, their depletion from procyclic-form T. brucei enriched cells with two kinetoplasts and an enlarged nucleus possessing short metaphase-like mitotic spindles, suggesting that APC1 and CDC27 may play essential roles in promoting anaphase in the procyclic form. Their depletion from the bloodstream form, however, enriched cells with two kinetoplasts and two nuclei connected through a microtubule bundle, suggesting a late anaphase arrest. This is the first time functional APC/C subunit homologs were identified in T. brucei. The apparent differential activities of this putative APC/C in two distinct developmental stages suggest an unusual function. The apparent lack of functional involvement of some of the other individual structural subunit homologs of APC/C may indicate the structural uniqueness of T. brucei APC/C.     

Three-dimensional structure of the anaphase-promoting complex

The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin-protein ligase, composed of at least 11 subunits, that controls progression through mitosis and G1. Using cryo-electron microscopy and angular reconstitution, we have obtained a three-dimensional model of the human APC at a resolution of 24 A. The APC has a complex asymmetric structure 140 A x 140 A x 135 A in size, in which an outer protein wall surrounds a large inner cavity. We discuss the possibility that this cavity represents a reaction chamber in which ubiquitination reactions take place, analogous to the inner cavities formed by other protein machines such as the 26S proteasome and chaperone complexes. This cage hypothesis could help to explain the great subunit complexity of the APC.