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Results for “Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome”

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PubMed1999

Identification of human APC10/Doc1 as a subunit of anaphase promoting complex.

Anaphase-promoting complex or cyclosome (APC) is a ubiquitin ligase which specifically targets mitotic regulatory factors such as Pds1/Cut2 and cyclin B. Identification of the subunits of multiprotein complex APC in several species revealed the highly conserved composition of APC from yeast to human. It has been reported, however, that vertebrate APC is composed of at least eight subunits, APC1 to APC8, while budding yeast APC is constituted of at least 12 components, Apc1 to Apc13. It has not yet been clearly understood whether additional components found in budding yeast, Apc9 to Apc13, are actually composed of mammalian APC. Here we isolated and characterized human APC10/Doc1, and found that APC10/Doc1 binds to APC core subunits throughout the cell cycle. Further, it was found that APC10/Doc1 is localized in centrosomes and mitotic spindles throughout mitosis, while it is also localized in kinetochores from prophase to anaphase and in midbody in telophase and cytokinesis. These results strongly support the notion that human APC10/Doc1 may be one of the APC core subunits rather than the transiently associated regulatory factor.

Amino Acid Sequence↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2005

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.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2002

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.

Anaphase↗

PubMed1999

Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex.

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin-protein ligase whose activity is essential for progression through mitosis. The vertebrate APC is thought to be composed of 8 subunits, whereas in budding yeast several additional APC-associated proteins have been identified, including a 33-kDa protein called Doc1 or Apc10. Here, we show that Doc1/Apc10 is a subunit of the yeast APC throughout the cell cycle. Mutation of Doc1/Apc10 inactivates the APC without destabilizing the complex. An ortholog of Doc1/Apc10, which we call APC10, is associated with the APC in different vertebrates, including humans and frogs. Biochemical fractionation experiments and mass spectrometric analysis of a component of the purified human APC show that APC10 is a genuine APC subunit whose cellular levels or association with the APC are not cell cycle-regulated. We have further identified an APC10 homology region, which we propose to call the DOC domain, in several protein sequences that also contain either cullin or HECT domains. Cullins are present in several ubiquitination complexes including the APC, whereas HECT domains represent the catalytic core of a different type of ubiquitin-protein ligase. DOC domains may therefore be important for reactions catalyzed by several types of ubiquitin-protein ligases.

Amino Acid Sequence↗

PubMed2003

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.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2001

Expression of CCAAT/enhancer binding protein family genes in monolayer and sandwich culture of hepatocytes: induction of stress-inducible GADD153.

Removal of hepatocytes from their physiological environment for experimentation in vitro activates loss of liver-specific phenotype. Hepatocytes cultured in a sandwich configuration reportedly maintain greater expression of certain liver-specific genes than hepatocytes in monolayer cultures. We show that sandwich culture of rat hepatocytes improves retention of expression of a liver-enriched transcription factor, C/EBPalpha (CCAAT/enhancer binding protein alpha), which regulates many liver-specific genes. However, we also demonstrate increased expression of a stress-responsive C/EBP homologue, GADD153 (growth arrest and DNA damage gene 153), during monolayer culture, which may promote dedifferentiation. Induction of GADD153 was not prevented in sandwich cultured hepatocytes. Activation of a homologue of the mouse GADD153 target gene, doc1, was observed in monolayer and sandwich culture, suggesting that GADD153 was transcriptionally active. We suggest that the capability of sandwich cultures to maintain hepatocyte phenotype may be limited by the altered profile of transcription factor activity.

Animals↗

PubMed2001

Disruption of Apc10/Doc1 in three alleles of oligosyndactylism.

Oligosyndactylism (Os) is a radiation-induced mouse mutation associated with recessive lethality and a dominant effect on limb and kidney development. The lethal effect of the mutation is due to a cell-autonomous block in the transition from metaphase to anaphase. We have previously characterized two transgene-induced mutations, 94-A and 94-K, which are allelic with Os. These mutations facilitated the identification of genomic segments and transcribed sequences in the affected region. One of the transcripts in this region corresponds to the mouse homolog of the anaphase-promoting complex component APC10/DOC1. The disruption of this gene can explain the mitotic arrest phenotype of all three alleles of Os.

Abnormalities, Radiation-Induced↗

PubMed1997

A novel yeast screen for mitotic arrest mutants identifies DOC1, a new gene involved in cyclin proteolysis.

B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37 degrees C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.

Amino Acid Sequence↗

PubMed2003

TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1.

BACKGROUND: 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. RESULTS: We have biochemically characterized subcomplexes of the human APC. 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. CONCLUSIONS: 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. In 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.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2006

Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C.

The temporal control of mitotic protein degradation remains incompletely understood. In particular, it is unclear why the mitotic checkpoint prevents the anaphase-promoting complex/cyclosome (APC/C)-mediated degradation of cyclin B and securin in early mitosis, but not cyclin A. Here, we show that another APC/C substrate, NIMA-related kinase 2A (Nek2A), is also destroyed in pro-metaphase in a checkpoint-independent manner and that this depends on an exposed carboxy-terminal methionine-arginine (MR) dipeptide tail. Truncation of the Nek2A C terminus delays its degradation until late mitosis, whereas Nek2A C-terminal peptides interfere with APC/C activity in an MR-dependent manner. Most importantly, we show that Nek2A binds directly to the APC/C, also in an MR-dependent manner, even in the absence of the adaptor protein Cdc20. As similar C-terminal dipeptide tails promote direct association of Cdc20, Cdh1 and Apc10-Doc1 with core APC/C subunits, we propose that this sequence also allows a substrate, Nek2A, to directly bind the APC/C. Thus, although Cdc20 is required for the degradation of Nek2A, it is not required for its recruitment and this renders its degradation insensitive to the mitotic checkpoint.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1999

Fission yeast APC/cyclosome subunits, Cut20/Apc4 and Cut23/Apc8, in regulating metaphase-anaphase progression and cellular stress responses.

BACKGROUND: The 20S cyclosome/APC complex promotes metaphase-anaphase transition by ubiquitinating its specific substrates such as mitotic cyclins and anaphase inhibitor Cut2/Pds1/securin. The complex has been shown to contain more than 10 proteins in budding yeast and frog. In fission yeast, however, only five (Cut4, Cut9, Nuc2, Apc10, Hcn1) have been identified. RESULTS: More than five hundred temperature-sensitive mutants were screened for identifying those defective in mitotic anaphase. Fifty-five showed the cut (cell untimely torn) phenotype or metaphase-arrest phenotypes, 27 of them locating at new loci. Their extracts were run in sucrose gradient centrifugation, and four showed alterations in the sedimentation profiles. The gene products of cut20+ and cut23+ were thus identified. Phenotypes of cut20-100 mutant highly resemble cut4-533 in many ways: they are hypersensitive to canavanine and CdCl2, and suppressed by PKA-inactivating regulators, cAMP-dependent phosphodiesterase and PKA regulatory subunits. Cut20 interacts closely with Cut4 in the assembly process of cyclosome. But cut20 mutant differs from cut4, as a novel gene stw1+ suppresses cut20 mutant but not cut4. cut23-194 mutant cells are sterile and blocked at metaphase, but does not show sensitivity to the stress and cAMP. TPR repeat-containing Cut23 may not be the stable component of APC/cyclosome, and its level significantly fluctuates during cell cycle. Cut23 may be ubiquitinated and degraded in a cell cycle dependent fashion. CONCLUSIONS: We identified two new subunits of fission yeast cyclosome/APC complex. Our observations indicate that cyclosome components are divided into several subgroups with distinctly different roles.

Adenomatous Polyposis Coli Protein↗

PubMed2003

A brute force postgenome approach to identify temperature-sensitive mutations that negatively interact with separase and securin plasmids.

BACKGROUND: The fission yeast Schizosaccharomyces pombe separase/Cut1 and securin/Cut2 are required for anaphase-specific activation of proteolysis that leads to proper sister chromatid separation. We intended to identify ts (temperature sensitive) strains whose growth was inhibited by multicopy plasmid pCUT1 or pCUT2 at the permissive temperature. RESULTS: After a one-by-one transformation of 1015 randomly isolated ts strains, 18 transformants that retarded in colony formation at the permissive or semipermissive temperature were isolated. Six of them, in the absence of pCUT1 or pCUT2, produced mitotic phenotypes with condensed chromosomes at the restrictive temperature. Gene cloning established that these mutants were defective in either the subunits (Cut9, Cut23, Cut20 or Apc10) of APC (anaphase promoting complex)/cyclosome or Cut8, a regulator for 26S proteasome localization. The inhibitory effect of separase against APC/cyclosome mutations was abolished when the catalytic site mutation C1730A was introduced and overproduced, indicating that inhibition needs an active separase. Securin/Cut2 overproduction also caused a negative effect on these mutants. Surprisingly, the phenotypes of cut9 and cut23 in the presence of pCUT1 or pCUT2 were not the mitotic arrest, and they were strikingly different depending on pCUT1 or pCUT2. CONCLUSIONS: This study shows the functional link between separase/Cut1 and APC/cyclosome in a separase activity-dependent manner. The negative effect of active separase overproduction on APC/cyclosome mutations is possibly due to the direct inhibition of APC/cyclosome. In addition, the manner of the inhibition by high copy securin and separase plasmids were quite different each other and did not result in the mitotic block.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Direct interaction between Smad3, APC10, CDH1 and HEF1 in proteasomal degradation of HEF1.

BACKGROUND: The Transforming Growth Factor-beta (TGF-beta) regulates myriad cellular events by signaling through members of the Smad family signal transducers. As a key signal transducer of TGF-beta, Smad3 exhibits the property of receptor-activated transcriptional modulator and also the novel ability of regulating the proteasomal degradation of two Smad3 interacting proteins, SnoN and HEF1. It has been shown that Smad3 recruits two types of Ub E3 ligases, Smurf2 and the Anaphase Promoting Complex (APC), to mediate SnoN ubiquitination, thereby enhancing SnoN degradation. The molecular mechanisms underlying Smad3-regulated HEF1 degradation are not well understood. Furthermore, it is not clear how Smad3 recruits the APC complex. RESULTS: We detected physical interaction between Smad3 and an APC component APC10, as well as the interaction between HEF1 and CDH1, which is the substrate-interacting component within APC. Detailed domain mapping studies revealed distinct subdomains within the MH2 domain of Smad3 for binding to APC10 and HEF1 and suggests the formation of a complex of these four proteins (Smad3, HEF1, APC10 and CDH1). In addition, the protein levels of HEF1 are subjected to the regulation of overexpressed APC10 and CDH1. CONCLUSIONS: Our data suggests that Smad3 may recruit the APC complex via a direct interaction with the APC subunit APC10 to regulate the ubiquitination and degradation of its interactor HEF1, which is recognized as an ubiquitination substrate by the CDH1 subunit of the APC complex.

Activin Receptors, Type I↗