In yeast and animals, the anaphase-promoting complex or cyclosome (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as securin and the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. To gain more insight into the roles of the plant APC/C, one of its subunits, APC2, which is encoded by a single-copy gene in Arabidopsis, was characterized at the molecular level. The Arabidopsis gene is able to partially complement a budding yeast apc2ts mutant. By yeast two-hybrid assays, an interaction of APC2 is demonstrated with two other APC/C subunits: APC11 and APC8/CDC23. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. apc2 null mutants are impaired in female megagametogenesis and accumulate a cyclin-beta-glucuronidase reporter protein but do not display metaphase arrest, as observed in other systems. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. Finally, intriguing differences are reported in APC2 protein subcellular localization compared with that in other systems. These observations support a conserved function of the APC/C in plants but a different mode of regulation (Capron, 2003).
The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation (Yu, 1998).
Entry into anaphase and exit from mitosis depend on a ubiquitin-protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin-protein ligase complex SCFCdc4 required for the initiation of DNA replication (Zachariae, 1998).
Two highly conserved RING finger proteins, ROC1 and ROC2, have been identified that are homologous to APC11, a subunit of the anaphase-promoting complex. ROC1 and ROC2 commonly interact with all cullins while APC11 specifically interacts with APC2, a cullin-related APC subunit. YeastROC1 encodes an essential gene whose reduced expression resulted in multiple, elongated buds and accumulation of Sic1p and Cln2p. ROC1 and APC11 immunocomplexes can catalyze isopeptide ligations to form polyubiquitin chains in an E1- and E2-dependent manner. ROC1 mutations completely abolish their ligase activity without noticeable changes in associated proteins. Ubiquitination of phosphorylated I kappa B alpha can be catalyzed by the ROC1 immunocomplex in vitro. Hence, combinations of ROC/APC11 and cullin proteins potentially constitute a wide variety of ubiquitin ligases (Ohta, 1999).
The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. The architecture of the APC was analyzed using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. The largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). The three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Δ complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. The IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. This study has provided an updated model for the subunit architecture of the APC (Thornton, 2006).
Homozygous inv mice lack a functional inversin protein and exhibit situs inversus plus severe cystic changes in the kidney and pancreas. Although the inversin sequence has provided few clues to its function, calmodulin has been identified as a binding partner. Evidence is presented that inversin interacts with the anaphase promoting complex protein Apc2. As expected of an Apc2 target, inversin possesses D-boxes and site-directed mutagenesis of the well-conserved D-box residues abrogates inversin-Apc2 interaction. An inversin-specific antibody reveals a dynamic expression pattern throughout the cell cycle and strong expression in the primary cilia of renal epithelium. These data support a role for inversin in primary cilia and involvement in the cell cycle. Mutations of the proteins polaris, cystin and polycystin-2, which are expressed in renal epithelium primary cilia, lead to renal cystic changes. Aberrant cell proliferation is also involved in cyst development. The data reported here suggest that inversin may provide a link between these two mechanisms (Morgan, 2002).
In mitosis, the anaphase-promoting complex (APC) regulates the onset of sister-chromatid separation and exit from mitosis by mediating the ubiquitination and degradation of the securin protein and mitotic cyclins. With the use of a baculoviral expression system, the ubiquitin ligase activity of human APC has been reconstituted. In combination with Ubc4 or UbcH10, a heterodimeric complex of APC2 and APC11 is sufficient to catalyze the ubiquitination of human securin and cyclin B1. However, the minimal APC2/11 ubiquitin ligase module does not possess substrate specificity, because it also ubiquitinates the destruction box deletion mutants of securin and cyclin B1. Both APC11 and UbcH10 bind to the C-terminal cullin homology domain of APC2, whereas Ubc4 interacts with APC11 directly. Zn(2+)-binding and mutagenesis experiments indicate that APC11 binds Zn(2+) at a 1:3 M ratio. Unlike the two Zn(2+) ions of the canonical RING-finger motif, the third Zn(2+) ion of APC11 is not essential for its ligase activity. Surprisingly, with Ubc4 as the E2 enzyme, Zn(2+) ions alone are sufficient to catalyze the ubiquitination of cyclin B1. Therefore, the Zn(2+) ions of the RING finger family of ubiquitin ligases may be directly involved in catalysis (Tang, 2001).
To investigate the mechanism of APC, Hi5 insect cells were coinfected with multiple recombinant baculoviruses harboring 10 APC genes and the cofactors Cdc20 (homolog of Fizzy) or Cdh1 (homolog of Fizzy related). When Hi5 cells were infected with viruses encoding 10 APC subunits (APC1-11) and Cdc20, the expressed APC proteins contained a ubiquitin ligase activity that, in combination with UbcH10, efficiently ubiquitinated human cyclin B1. This activity also supported ubiquitination of human securin. To determine which subunits are required for this activity, each virus was omitted individually from the coinfection. Only APC2 and APC11 are required for the ligase activity. Similar results were obtained when the Cdh1 baculovirus was used in the coinfection instead of Cdc20. The fact that omission of APC2 or APC11 alone causes the loss of the reconstituted activity indicates that the purified ligase activity is not due to the presence of the endogenous APC from insect cells (Tang, 2001).
Because Cdc20 and Cdh1 are positive regulators of APC, it was surprising that Cdc20 or Cdh1 are not required for the ubiquitin ligase activity of the reconstituted APC. Therefore the reconstituted APC activity was compared with that of the intact APC from Xenopus egg extracts. Nearly all APC substrates contain the destruction box (D-box) or the KEN-box motifs, which are required for the efficient ubiquitination and degradation of these substrates. Cdc20 and Cdh1 have been shown to confer the D-box and KEN-box specificity of APC. The activities of the intact APCCdc20 and APCCdh1 from Xenopus were assayed with cyclin B1 as the substrate. Purified Cdc20 and Cdh1 proteins greatly stimulate the ligase activity of the intact interphase Xenopus APC with UbcH10 as the E2 enzyme and wild-type cyclin B1 as substrate. However, neither the intact APCCdc20 nor APCCdh1 significantly ubiquitinate a D-box deletion mutant of cyclin B1 (DeltaDB-cyclin B1). Similar results were obtained with Ubc4 as the E2 enzyme, although the patterns of cyclin B-ubiquitin conjugates formed by the two enzymes were different. Ubc4 appears to be more processive than UbcH10 in the presence of either intact APCCdc20 or APCCdh1 (Tang, 2001).
Because Cdc20 and Cdh1 were not essential for the reconstituted APC activity, tests were carried out to determine whether the ligase activity obtained with overexpressed APC proteins conferred D-box specificity, similar to the intact APC. Not surprisingly, the reconstituted human APC ubiquitinated DeltaDB-cyclin B1 equally efficiently, indicating that the reconstituted activity did not possess substrate specificity. This activity also required the presence of APC2 and APC11. Therefore, the minimal ligase activity of APC that lacked D-box dependency was reconstituted. At present, the exact cause for the lack of D-box dependency of the reconstituted APC is not known. However, several factors might contribute to this. (1) The reconstituted APC might not have the correct quaternary arrangement of all the relevant subunits. (2) The set of subunits used to reconstitute the APC is not complete. Additional human APC subunits are required for the proper function of the reconstituted APC. (3) It is also possible that the high concentrations of the reconstituted APC and the substrates in the in vitro reactions may have eliminated the need for high-affinity interactions between APC and substrates. Currently, these possibilities are being investigated (Tang, 2001).
Because both APC2 and APC11 are required for the reconstituted APC activity, test were performed to see whether they interact with each other in the absence of the rest of the APC subunits. To characterize the immediate binding partners of APC11 in the APC complex, the GST-APC11 virus was coinfected with each of the other viruses in a pairwise manner. GST-APC11 and its associated subunits were then purified with glutathione-Sepharose beads and analyzed by SDS-PAGE followed by Coomassie staining and immunoblotting. APC11 binds tightly to APC2 and weakly to APC6. The identities of APC2 and APC6 were verified by immunoblotting. It was not apparent from the GST-APC11 pull-down experiment whether GST-APC11 interacts with APC10 because the His6-tagged APC10 (26 kDa) comigrates with proteolytic fragments of GST-APC11 on SDS-PAGE. Therefore a His6-tagged APC10 virus was used to infect Sf9 cells together with the GST-APC11 virus. The APC10 protein was then purified with Ni2+-NTA beads and analyzed by SDS-PAGE. APC10 binds tightly to GST-APC11 as revealed by Coomassie staining. Several large proteins contain the so-called DOC domains that are similar in sequence to APC10; some of these proteins also contain cullin homology domains or are homologous to E6-AP C terminus (HECT) domains. E6-AP, the founding member of a family of proteins containing HECT domains, mediates the HPV E6-dependent degradation of p53. It is likely that the DOC domains of these large multidomain proteins might also be involved in binding to yet unidentified RING-finger proteins (Tang, 2001).
To identify subunits that interact with APC2, the His6-tagged APC2 virus was used to infect Sf9 cells together with other viruses. The APC2 protein was then purified with Ni2+-NTA beads and analyzed by SDS-PAGE. APC2 indeed interacts with APC11, but no other strong interactions were found between APC2 and the rest of APC subunits. Therefore, APC2 and APC11 form a complex in the absence of the other APC subunits (Tang, 2001).
Next it was tested whether the subcomplex of APC2 and APC11 (APC2/11) is sufficient to support ubiquitination of APC substrates. With the use of UbcH10 as E2, APC2/11 catalyzes the ubiquitination of human securin, whereas either APC2 or APC11 alone has no activity. APC11 alone expressed either in bacteria or insect cells is sufficient to ubiquitinate human securin in the presence of Ubc4. Similar data were obtained with the use of human cyclin B1 as the substrate. Therefore, APC2/11 represents the minimal ligase module of APC, because it supports the ubiquitination of APC substrates with the use of either Ubc4 or UbcH10 as E2s (Tang, 2001).
Both Ubc4 and UbcH10 support the ubiquitination reactions catalyzed by APC in an additive manner. It is unclear which enzyme is the physiological E2 of the APC pathway. Microinjection of a UbcH10 dominant-negative mutant protein into mammalian cells arrests cells in mitosis. In addition, mutation of the Schizosaccharomyces pombe homolog of UbcH10, UbcP4, caused accumulation of cells in mitosis, similar to mutations of APC subunits. These findings suggest that UbcH10 might be involved in the mitotic degradation system in living cells. However, in budding yeast, mutations of either the Ubc4/5 family E2s or the UbcH10 homolog Ubc11 do not cause obvious mitotic phenotype. To further clarify this issue, UbcX, the Xenopus homolog of UbcH10, was immunodepleted from the mitotic Xenopus egg extract that contains active APC and degrades APC substrates with fast kinetics. The anti-UbcX antibody beads effectively deplete the UbcX protein from the mitotic extract. Addition of the bacterially expressed UbcX restores the ability of the mitotic extract to degrade cyclin B1. Taken together, UbcX is required for proper degradation of cyclin B1, and possibly other APC substrates, in Xenopus egg extracts. Unfortunately, no antibody was available that could immunodeplete Ubc4 from these extracts, and thus similar experiments could not be carried out for Ubc4 (Tang, 2001).
Next to be examined was why APC2 is not required for the ubiquitination reactions of Ubc4. UbcH10 interacts strongly with APC2, whereas it does not bind to APC11. In contrast, Ubc4 associates directly with APC11. It does not exhibit significant binding toward APC2. This finding explains why APC2 is only required for UbcH10-catalyzed reactions. The two E2s may recognize different binding determinants within the APC2/11 ligase module. The fact that UbcH10 does not bind APC11 is consistent with previous structural studies on the interactions between the Cbl RING domain and UbcH7, an E2 of the Ubc4 subfamily. The two loops that are critical for binding to RING domains are conserved between Ubc4 and UbcH7. However , UbcH10 contains quite divergent amino acid sequences in these two loops. Therefore, Ubc4 and UbcH10 may be recruited to the intact APC with distinct mechanisms: Ubc4 recognize APC11 initially, whereas UbcH10 first interacts with APC2. However, it remains possible that, once they are bound to APC, Ubc4 and UbcH10 occupy a similar site on APC and use a similar mechanism for transferring ubiquitin (Tang, 2001).
The regions within APC2 that interact with APC11 and UbcH10 were mapped. A series of truncation mutants of APC2 were constructed and tested for binding to APC11 and UbcH10. A C-terminal fragment of APC2, APC2e, spanning residues 549-822, is sufficient for binding to APC11 and UbcH10. This region almost coincides with the cullin homology region of APC2 that includes residues 512-750. Interestingly, even although APC2 and APC11 formed an active complex when they were coexpressed in insect cells, the full-length APC2 protein did not bind to APC11 in this assay. This is consistent with the fact that, when APC2 and APC11 were expressed individually in insect cells and mixed after purification, no ubiquitin ligase activity is observed. Therefore, the full-length APC2 protein can not form a complex with APC11 post-translationally. However, smaller fragments of APC2 were able to bind to APC11 (Tang, 2001).
Because APC2e binds to both APC11 and UbcH10, whether the APC2e/11 complex is an active ubiquitin ligase was examined. The APC fragments were coexpressed with APC11 in insect cells, and assayed for their ability to ubiquitinate cyclin B1 in the presence of UbcH10. The APC2b and APC2e fragments ubiquitinate cyclin B1 efficiently; both of these fragments retain the ability to bind to APC11 and UbcH10. Therefore, a complex of the cullin domain of APC2 and the RING finger protein APC11 is sufficient to catalyze ubiquitination of APC substrates, albeit with decreased efficiency (Tang, 2001).
Chromosome segregation and mitotic exit depend on activation of the anaphase-promoting complex (APC) by the substrate adaptor proteins CDC20 and CDH1. The APC is a ubiquitin ligase composed of at least 11 subunits. The interaction of APC2 and APC11 with E2 enzymes is sufficient for ubiquitination reactions, but the functions of most other subunits are unknown. Subcomplexes of the human APC have been biochemically characterized. One subcomplex, containing APC2/11, APC1, APC4, and APC5, can assemble multiubiquitin chains but is unable to bind CDH1 and to ubiquitinate substrates. The other subcomplex contains all known APC subunits except APC2/11. This subcomplex can recruit CDH1 but fails to support any ubiquitination reaction. In vitro, the C termini of CDC20 and CDH1 bind to the closely related TPR subunits APC3 and APC7. Homology modeling predicts that these proteins are similar in structure to the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. APC activation by CDH1 depends on a conserved C-terminal motif that is also found in CDC20 and APC10. It is concluded that APC1, APC4, and APC5 may connect APC2/11 with TPR subunits. TPR domains in APC3 and APC7 recruit CDH1 to the APC and may thereby bring substrates into close proximity of APC2/11 and E2 enzymes. By analogy to PEX5, the different TPR subunits of the APC might function as receptors that interact with the C termini of regulatory proteins such as CDH1, CDC20, and APC10 (Vodermaier, 2003).
The isolation and characterization of two subcomplexes of the human APC have provided first insight into the molecular interactions between APC's many subunits. The cullin subunit APC2 and its binding partner, the RING finger protein APC11, are found in a subcomplex with APC1, APC4, and APC5 and are essential for the assembly of multiubiquitin chains from ubiquitin residues donated by E2 enzymes. Substrate ubiquitination requires the activator proteins CDH1 and CDC20, which interact via their C termini with the TPR subunits APC3 and APC7. APC's TPR subunits are predicted to form structures that are similar to the one of the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. The APC may therefore contain multiple TPR subunits to allow modular interactions with different regulatory proteins. These results reveal a function for the TPR subunits of the APC, and they provide insight into how substrates are recruited to the ubiquitin ligase (Vodermaier, 2003).
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