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 APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27

The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.     

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.     

Molecular Structure of the N-terminal Domain of the APC/C Subunit Cdc27 Reveals a Homo-dimeric Tetratricopeptide Repeat Architecture

The anaphase promoting complex/cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that targets specific cell cycle regulatory proteins for ubiquitin-dependent degradation, thereby controlling cell cycle events such as the metaphase to anaphase transition and the exit from mitosis. Biochemical and genetic studies are consistent with the notion that subunits of APC/C are organised into two distinct sub-complexes; a catalytic sub-complex including the cullin domain and RING finger subunits Apc2 and Apc11, respectively, and a tetratricopeptide repeat (TPR) sub-complex composed of the TPR subunits Cdc16, Cdc23 and Cdc27 (Apc3). Here, we describe the crystal structure of the N-terminal domain of Encephalitozoon cuniculi Cdc27 (Cdc27^Nterm), revealing a homo-dimeric structure, composed predominantly of successive TPR motifs. Mutation of the Cdc27^Nterm dimer interface destabilises the protein, disrupts dimerisation in solution, and abolishes the capacity of E. cuniculi Cdc27 to complement Saccharomyces cerevisiae Cdc27 in vivo. These results establish the existence of functional APC/C genes in E. cuniculi, the evolutionarily conserved dimeric properties of Cdc27, and provide a framework for understanding the architecture of full-length Cdc27.     

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.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.     

Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation

The ubiquitin ligase activity of the anaphase-promoting complex (APC)/cyclosome needs to be tightly regulated for proper cell cycle progression. Substrates are recruited to the APC by the Cdc20 and Cdh1 accessory proteins. The Cdh1-APC interaction is inhibited through phosphorylation of Cdh1 by Cdc28, the major cyclin-dependent protein kinase in budding yeast. More recently, Acm1 was reported to be a Cdh1-binding and -inhibitory protein in budding yeast. We found that although Acm1 is an unstable protein and contains the KEN-box and D-box motifs typically found in APC substrates, Acm1 itself is not an APC substrate. Rather, it uses these motifs to compete with substrates for Cdh1 binding, thereby inhibiting their recruitment to the APC. Mutation of these motifs prevented Acm1-Cdh1 binding in vivo and rendered Acm1 inactive both in vitro and in vivo. Acm1 stability was critically dependent on phosphorylation by Cdc28, as Acm1 was destabilized following inhibition of Cdc28, mutation of consensus Cdc28 phosphorylation sites in Acm1, or deletion of the Bmh1 and Bmh2 phosphoprotein-binding proteins. Thus, Cdc28 serves dual roles in inhibiting Cdh1-dependent APC activity during the cell cycle: stabilization of the Cdh1 inhibitor Acm1 and direct phosphorylation of Cdh1 to prevent its association with the APC.     

Cdc28 activates exit from mitosis in budding yeast

The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast.     

Structural analysis of the anaphase-promoting complex reveals multiple active sites and insights into polyubiquitylation

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase composed of approximately 13 distinct subunits required for progression through meiosis, mitosis, and the G1 phase of the cell cycle. Despite its central role in these processes, information concerning its composition and structure is limited. Here, we determined the structure of yeast APC/C by cryo-electron microscopy (cryo-EM). Docking of tetratricopeptide repeat (TPR)-containing subunits indicates that they likely form a scaffold-like outer shell, mediating assembly of the complex and providing potential binding sites for regulators and substrates. Quantitative determination of subunit stoichiometry indicates multiple copies of specific subunits, consistent with a total APC/C mass of approximately 1.7 MDa. Moreover, yeast APC/C forms both monomeric and dimeric species. Dimeric APC/C is a more active E3 ligase than the monomer, with greatly enhanced processivity. Our data suggest that multimerisation and/or the presence of multiple active sites facilitates the APC/C's ability to elongate polyubiquitin chains.     

The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23

Properly regulated cyclin proteolysis is critical for normal cell cycle progression. A nine-amino acid peptide motif called the destruction box (D box) is present at the N terminus of the yeast mitotic cyclins. This short sequence is required for cyclin ubiquitination and subsequent proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 required for cyclin ubiquitination. We have tested the D box of five mitotic cyclins for interaction with six APC/C subunits. The APC/C subunit Cdc23, but not five other subunits tested, interacted by two-hybrid analysis with the N terminus of wild-type Clb2. None of these subunits interacted with the N termini of the cyclins Clb1, Clb3, or Clb5. Mutations in the D box sequences of Clb2 inhibited interaction with Cdc23 both in vivo and in vitro. Our results provide the first evidence for a direct interaction between an APC/C substrate (Clb2) and an APC/C subunit (Cdc23).     

Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complex.

Both chromosome segregation and the final exit from mitosis require a ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome. The ubiquitination by APC is dependent on the presence of a destruction-box sequence in the N-terminus of target proteins. Recent reports have strongly suggested that Cdc20, a WD40 repeat-containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC-mediated proteolysis. To understand the function of CDC20, we have studied its regulation in some detail. The expression of the CDC20 gene is cell-cycle regulated such that it is transcribed only during late S phase and mitosis. Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1. Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1. Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected. This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction-box containing proteins, such as Cdc20, in an APC-independent manner.     

Cdc28 and Cdc14 control stability of the anaphase-promoting complex inhibitor Acm1

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC(Cdh1) inhibitor. Acm1 disappears in late mitosis when APC(Cdh1) becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G(1)/S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC 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.     

Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14

BACKGROUND: Exit from mitosis requires inactivation of mitotic cyclin-dependent kinases (CDKs). A key mechanism of CDK inactivation is ubiquitin-mediated cyclin proteolysis, which is triggered by the late mitotic activation of a ubiquitin ligase known as the anaphase-promoting complex (APC). Activation of the APC requires its association with substoichiometric activating subunits termed Cdc20 and Hct1 (also known as Cdh1). Here, we explore the molecular function and regulation of the APC regulatory subunit Hct1 in Saccharomyces cerevisiae. RESULTS: Recombinant Hct1 activated the cyclin-ubiquitin ligase activity of APC isolated from multiple cell cycle stages. APC isolated from cells arrested in G1, or in late mitosis due to the cdc14-1 mutation, was more responsive to Hct1 than APC isolated from other stages. We found that Hct1 was phosphorylated in vivo at multiple CDK consensus sites during cell cycle stages when activity of the cyclin-dependent kinase Cdc28 is high and APC activity is low. Purified Hct1 was phosphorylated in vitro at these sites by purified Cdc28-cyclin complexes, and phosphorylation abolished the ability of Hct1 to activate the APC in vitro. The phosphatase Cdc14, which is known to be required for APC activation in vivo, was able to reverse the effects of Cdc28 by catalyzing Hct1 dephosphorylation and activation. CONCLUSIONS: We conclude that Hct1 phosphorylation is a key regulatory mechanism in the control of cyclin destruction. Phosphorylation of Hct1 provides a mechanism by which Cdc28 blocks its own inactivation during S phase and early mitosis. Following anaphase, dephosphorylation of Hct1 by Cdc14 may help initiate cyclin destruction.     

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.     

Cdc20 in S-phase: the Banquo at replication's banquet

The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase that covalently attaches ubiquitins onto proteins to target them for proteolysis by the 26S proteasome. During mitosis, the APC/C is instrumental in allowing the cell to enter and exit from mitosis. The APC/C accomplishes this by using different specificity factors to recognize, interact with, and ubiquitylate key proteins that block cell cycle progression. The specificity factors, Cdc20p and Cdh1p, are not always associated with the APC/C and indeed they have the ability to interact with substrates in isolation. The molecular events that take place in order for Cdc20p and Cdh1p to couple substrates and APC/C are currently being resolved. Meanwhile, evidence has emerged suggesting that at least one of the specificity factors, Cdc20p, might be capable of functioning independently of the APC/C.     

Suppression of Cdc27B expression induces plant defence responses

Non-host resistance is the most general form of disease resistance in plants because it is effective against most phytopathogens. The importance of hypersensitive responses (HRs) in non-host resistance of Nicotiana species to the oomycete Phytophthora is clear. INF1 elicitin, an elicitor obtained from the late-blight pathogen Phytophthora infestans , is sufficient to induce a typical HR in Nicotiana species. The molecular mechanisms that underlie the non-host resistance component of plant defence responses have been investigated using differential-display polymerase chain reaction (PCR) in a model HR system between INF1 elicitin and tobacco BY-2 cells. Differential-display PCR has revealed that Cdc27B is down-regulated in tobacco BY-2 cells after treatment with INF1 elicitin. Cdc27B is one of 13 essential components of the anaphase-promoting complex or cyclosome (APC/C)-type E3 ubiquitin ligase complex in yeast. This APC/C-type E3 ubiquitin ligase complex regulates G2-to-M phase transition of the cell cycle by proteolytic degradation. In this study, we investigated the roles of this gene, NbCdc27B , in plant defence responses using virus-induced gene silencing. Suppression of NbCdc27B in Nicotiana benthamiana plants induced defence responses and a gain of resistance to Colletotrichum lagenarium fungus. Elicitin-induced hypersensitive cell death (HCD) was inhibited mildly in plants silenced with tobacco rattle virus::Cdc27B. Cdc27B could manage the signalling pathways of plant defence responses as a negative regulator without HCD.     

Cdc20: a WD40 activator for a cell cycle degradation machine

Cdc20 is an essential cell-cycle regulator required for the completion of mitosis in organisms from yeast to man and contains at its C terminus a WD40 repeat domain that mediates protein-protein interactions. In mitosis, Cdc20 binds to and activates the ubiquitin ligase activity of a large molecular machine called the anaphase-promoting complex/cyclosome (APC/C) and enables the ubiquitination and degradation of securin and cyclin B, thus promoting the onset of anaphase and mitotic exit. APC/C(Cdc20) is temporally and spatially regulated during the somatic and embryonic cell cycle by numerous mechanisms, including the spindle checkpoint and the cytostatic factor (CSF). Therefore, Cdc20 serves as an integrator of multiple intracellular signaling cascades that regulate progression through mitosis. This review summarizes recent progress toward the understanding of the functions of Cdc20, the mechanisms by which it activates APC/C, and its regulation by phosphorylation and by association with its binding proteins.     

The Four Canonical TPR Subunits of Human APC/C Form Related Homo-Dimeric Structures and Stack in Parallel to Form a TPR Suprahelix

The anaphase-promoting complex or cyclosome (APC/C) is a large E3 RING-cullin ubiquitin ligase composed of between 14 and 15 individual proteins. A striking feature of the APC/C is that only four proteins are involved in directly recognizing target proteins and catalyzing the assembly of a polyubiquitin chain. All other subunits, which account for > 80% of the mass of the APC/C, provide scaffolding functions. A major proportion of these scaffolding subunits are structurally related. In metazoans, there are four canonical tetratricopeptide repeat (TPR) proteins that form homo-dimers (Apc3/Cdc27, Apc6/Cdc16, Apc7 and Apc8/Cdc23). Here, we describe the crystal structure of the N-terminal homo-dimerization domain of Schizosaccharomyces pombe Cdc23 (Cdc23^Nterm). Cdc23^Nterm is composed of seven contiguous TPR motifs that self-associate through a related mechanism to those of Cdc16 and Cdc27. Using the Cdc23^Nterm structure, we generated a model of full-length Cdc23. The resultant “V”-shaped molecule docks into the Cdc23-assigned density of the human APC/C structure determined using negative stain electron microscopy (EM). Based on sequence conservation, we propose that Apc7 forms a homo-dimeric structure equivalent to those of Cdc16, Cdc23 and Cdc27. The model is consistent with the Apc7-assigned density of the human APC/C EM structure. The four canonical homo-dimeric TPR proteins of human APC/C stack in parallel on one side of the complex. Remarkably, the uniform relative packing of neighboring TPR proteins generates a novel left-handed suprahelical TPR assembly. This finding has implications for understanding the assembly of other TPR-containing multimeric complexes.