CDC27 of plants and yeast

In plant meristems, dividing cells interpret positional information and translate it into patterned cell differentiation. The Arabidopsis HOBBIT gene is required for cell division and cell differentiation in meristems. It encodes a homolog of the CDC27 subunit of the anaphase-promoting complex (APC). HOBBIT partially complements a yeast nuc2/cdc27 mutant. Unlike other CDC27 homologs in Arabidopsis, its transcription is cell cycle regulated. Furthermore, hobbit mutants show a reduction in DR5 :: GUS auxin reporter gene expression and accumulate the AXR3/IAA17 repressor of auxin responses. HOBBIT activity may thus couple cell division to cell differentiation by regulating cell cycle progression in the meristem or by restricting the response to differentiation cues, such as auxin, to dividing cells (Blilou, 2002).

The cell division cycle gene cdc27+ of the fission yeast Schizosaccharomyces pombe is required for the transition from G2 into mitosis. Genetic and physiological experiments suggest a close relationship between cdc27+ and the cdc2+ gene, a key regulator of mitosis in yeast and also in higher eukaryotic cells. The cdc27+ gene was isolated by complementation of a temperature-sensitive cdc27 mutant. The DNA sequence of this gene predicts a 1116 nucleotide open reading frame split by five short introns, ranging in size from 49 to 74 nucleotides. Analysis of cDNA clones confirms the structure of the gene. The deduced cdc27+ gene product consists of 372 amino acids with a predicted Mr of 43 kDa. Northern analysis reveals two mRNAs of 1.4 and 2.2 kb transcribed from this gene, the smaller transcript being approximately tenfold more abundant than the larger. The level of cdc27+ mRNAs remains constant through the cell cycle, indicating that the time of action of the cdc27+ gene, which is known to be regulated by elements of the mitotic control, is not determined by periodic accumulation of its transcripts (Hughes, 1992).

Cdc16p, Cdc23p and Cdc27p are all essential proteins required for cell cycle progression through mitosis in Saccharomyces cerevisiae. All three proteins contain multiple tandemly repeated 34 amino acid tetratricopeptide repeats (TPRs). Using two independent assays, two-hybrid analysis in vivo and co-immunoprecipitation in vitro, it has been demonstrated that Cdc16p, Cdc23p and Cdc27p self associate and interact with one another to form a macromolecular complex. A temperature sensitive mutation in the most highly conserved TPR domain of Cdc27p results in a greatly reduced ability to interact with Cdc23p, but has no effect on interactions with wild-type Cdc27p or Cdc16p. The specificity of this effect indicates that TPRs can mediate protein-protein interactions and that this mutation may define an essential interaction for cell cycle progression in yeast. The conservation of at least two of the three proteins from yeast to man suggests that this protein complex is essential for mitosis in a wide range of eukaryotes (Lamb, 1994).

CDC16 and CDC27 were identified as genes in S. cerevisiae necessary to limit DNA replication to once per cell cycle. A screen for mutants that overreplicates their DNA uncovered new conditional alleles that cause accumulation of up to 8C DNA. DNA overreplication involves all chromosomes and does not require passage through mitosis or another START. It occurs within a single cell cycle and can cause arrest at the MEC1 checkpoint. Remarkably, Clb2-Cdc28 activity remains elevated in the overreplicating cells. These observations distinguish CDC16 and CDC27 from other mutants that accumulate extra DNA after completing an aberrent mitosis, or skipping mitosis altogether, and entering a second, inappropriate G1 and S phase. CDC16 and CDC27 may contribute to replication control by targeted proteolysis of an S phase initiator (Heichman, 1996).

Direct interaction between DNA polymerase delta and its processivity factor proliferating cell nuclear antigen (PCNA) is essential for effective replication of the eukaryotic genome, yet the precise manner by which this occurs is unclear. The 54 kDa subunit of DNA polymerase delta from Schizosaccharomyces pombe has been shown to interact directly with Pcn1 (PCNA) both in vivo and in vitro. Binding is effected via a short sequence at the C-terminus of Cdc27 with significant similarity to the canonical PCNA binding motif first identified in the mammalian p21(Cip1) protein. This motif is both necessary and sufficient for binding of Pcn1 by Cdc27 in vitro and is essential for Cdc27 function in vivo. The Pcn1 binding motif in Cdc27 is distinct from its binding site for Cdc1, the 55 kDa B-subunit of polymerase delta; evidence is presented that Cdc27 can bind to Pcn1 and Cdc1 simultaneously. Cdc27 is shown to perform at least two distinct essential functions, one of which is independent of Pcn1 binding (Reynolds, 2000).

Schizosaccharomyces pombe DNA polymerase (pol) delta contains four subunits: pol 3, Cdc1, Cdc27, and Cdm1. The role of Cdc27 on the structure and activity of pol delta has been examined. The four-subunit complex is shown in this study to be monomeric in structure. The shape of Cdc27 is markedly asymmetric. Cdc27 contains two critical domains that govern its role in activating pol delta. The N-terminal region [amino acids (aa) 1-160] binds to Cdc1 and its extreme C-terminal end (aa 362-369) interacts with proliferating cell nuclear antigen (PCNA). Mutants of S. pombe pol delta, containing truncated Cdc27 derivatives deficient in binding to PCNA, supported DNA replication less processively than the wild-type complex. Fusion of a minimal PCNA-binding motif (aa 352-372) to C-terminally truncated Cdc27 derivatives restores processive DNA synthesis in vitro. In vivo, the introduction of these fused Cdc27 derivatives into cdc27Delta cells confers viability. These data support the model in which Cdc27 plays an essential role in DNA replication by recruiting PCNA to the pol delta holoenzyme (Burmudez, 2000).

Budding yeast initiates anaphase by activating the Cdc20-dependent anaphase-promoting complex (APC). The mitotic activity of Cdc28 (Cdk1) is required to activate this form of the APC, and mutants that are impaired in mitotic Cdc28 function have difficulty leaving mitosis. This defect can be explained by a defect in APC phosphorylation, which depends on mitotic Cdc28 activity in vivo and can be catalyzed by purified Cdc28 in vitro. Mutating putative Cdc28 phosphorylation sites in three components of the APC, Cdc16, Cdc23, and Cdc27, makes the APC resistant to phosphorylation both in vivo and in vitro. The nonphosphorylatable APC has normal activity in G1, but its mitotic, Cdc20-dependent activity is compromised. These results show that Cdc28 activates the APC in budding yeast to trigger anaphase. Previous reports have shown that the budding yeast Cdc5 homolog, Plk, can also phosphorylate and activate the APC in vitro. Like cdc28 mutants, cdc5 mutants affect APC phosphorylation in vivo. However, although Cdc5 can phosphorylate Cdc16 and Cdc27 in vitro, this in vitro phosphorylation does not occur on in vivo sites of phosphorylation (Rudner, 2000).

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).

CDC27 function in invertebrates

In C. elegans, mutants in the anaphase-promoting complex or cyclosome (APC/C) exhibit defects in germline proliferation, the formation of the vulva and male tail, and the metaphase to anaphase transition of meiosis I. Oocytes lacking APC/C activity can be fertilized but arrest in metaphase of meiosis I and are blocked from further development. To examine the cell cycle and developmental consequences of reducing but not fully depleting APC/C activity, defects in embryos and larvae were analyzed for mat-1/cdc-27 mutants grown at semi-permissive temperatures. Hypomorphic embryos develop to the multicellular stage but are slow to complete meiosis I and display aberrant meiotic chromosome separation. More severely affected embryos skip meiosis II altogether and exhibit striking defects in meiotic exit. These latter embryos fail to produce normal eggshells or establish normal asymmetries prior to the first mitotic division. In developing larvae, extended M-phase delays in late-dividing cell lineages are associated with defects in the morphogenesis of the male tail. This study reveals the importance of dosage-specific mutants in analyzing molecular functions of a ubiquitously functioning protein within different cell types and tissues, and striking correlations between specific abnormalities in cell cycle progression and particular developmental defects (Shakes, 2003).

CDC27 function in vertebrates

Cyclin B is degraded at the onset of anaphase by a ubiquitin-dependent proteolytic system. Mitotic Xenopus egg extracts have been fractionated to identify components required for this process. UBC4 and at least one other ubiquitin-conjugating enzyme can support cyclin B ubiquitination. The mitotic specificity of cyclin ubiquitination is determined by a 20S complex that contains homologs of budding yeast CDC16 and CDC27. Because these proteins are required for anaphase in yeast and mammalian cells, this complex is referred to as the anaphase-promoting complex (APC). CDC27 antibodies deplete APC activity, while immunopurified CDC27 complexes are sufficient to complement either interphase extracts or a mixture of recombinant UBC4 and the ubiquitin-activating enzyme E1. These results suggest that APC functions as a regulated ubiquitin-protein ligase that targets cyclin B for destruction in mitosis (King, 1995).

cDNAs have been isolated and antibodies have been raised corresponding to the human homologs of the S. cerevisiae CDC27 and CDC16 proteins, which are tetratrico peptide repeat (TPR)-containing proteins essential for mitosis in budding yeast. The CDC27Hs and CDC16Hs proteins colocalize to the centrosome at all stages of the mammalian cell cycle, and to the mitotic spindle. Injection of affinity-purified anti-CDC27Hs antibodies into logarithmically growing HeLa cells causes a highly reproducible cell cycle arrest in metaphase with apparently normal spindle structure. It is concluded that CDC27 and CDC16 are evolutionarily conserved proteins that control the onset of postmetaphase events during mitosis (Tugendreich, 1995).

Activation of the mitotic checkpoint pathway in response to mitotic spindle damage in eukaryotic cells delays the exit from mitosis in an attempt to prevent chromosome missegregation. One component of this pathway, hsMad2, has been shown in mammalian cells to physically associate with components of a ubiquitin ligase activity (termed the anaphase promoting complex or APC) when the checkpoint is activated, thereby preventing the degradation of inhibitors of the mitotic exit machinery. The inhibitory association between Mad2 and the APC component Cdc27 also takes place transiently during the early stages of a normal mitosis and is lost before mitotic exit. Mad2 associates with the APC regulatory protein p55Cdc in mammalian cells as has been reported in yeast. In contrast, however, this complex is present only in nocodazole-arrested or early mitotic cells and is associated with the APC as a Mad2/p55Cdc/Cdc27 ternary complex. Evidence for a Mad2/Cdc27 complex that forms independent of p55Cdc also is presented. These results suggest a model for the regulation of the APC by Mad2 and may explain how the spindle assembly checkpoint apparatus controls the timing of mitosis under normal growth conditions (Wassmann, 1998).

An essential eukaryotic DNA polymerase, DNA polymerase delta (pol delta), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). A 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 has been identified as a component of mouse and calf thymus pol delta. p66 interacts tightly with other subunits of pol delta during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human pol delta can be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrate that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrate that p66 increases the overall affinity of pol delta for PCNA. These results indicate that p66 is a functionally important subunit of human pol delta that stabilizes the pol delta complex and increases the affinity of pol delta for PCNA (Shikata, 2001).

Cell cycle regulated protein ubiquitination and degradation within subcellular domains may be essential for the normal progression of mitosis. Cdc27 is a conserved component of an essential M-phase ubiquitin-protein ligase called the anaphase-promoting complex/cyclosome. The subcellular distribution of Cdc27 was examined in greater detail in mammalian cells; Cdc27 is concentrated at spindle poles and on spindle microtubules, and is also found at kinetochores and along chromosome arms. This localization is not dependent on intact microtubules. While the great majority of Cdc27 protein in M phase cells is highly phosphorylated, only the dephosphorylated form of Cdc27 was found associated with isolated chromosomes. Kinases that also associate with isolated chromosomes catalyze the in vitro phosphorylation of the chromosome-associated Cdc27. Microinjection of anti-Cdc27 antibody into cells causes arrest at metaphase. Microinjection of cells with anti-Mad2 antibody normally induces premature anaphase onset resulting in catastrophic nondisjunction of the chromosomes. However, coinjection of anti-Cdc27 antibody with anti-Mad2 antibody results in metaphase arrest. The association of dephosphorylated APC/C components with mitotic chromosomes suggests mechanisms by which the spindle checkpoint may regulate APC/C activity at mitosis (Topper, 2002).

The anaphase-promoting complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and Cdc20 binding. Forty-three phospho-sites on APC have been identified of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro, at least 15 of the mitotic phospho-sites can be generated by cyclin-dependent kinase 1 (Cdk1), and 3 by Polo-like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased Cdc20 binding and APC activation. Immunofluorescence microscopy using phospho-antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase, phospho-APC accumulates on centrosomes where cyclin B ubiquitination is initiated, and then appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo. These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells (Kraft, 2003).

Hox proteins are transcription factors involved in controlling axial patterning, leukemias and hereditary malformations. HOXC10 oscillates in abundance during the cell cycle, being targeted for degradation early in mitosis by the ubiquitin-dependent proteasome pathway. Among abdominal-B subfamily members, the mitotic proteolysis of HOXC10 appears unique, since the levels of the paralogous HOXD10 and the related homeoprotein HOXC13 are constant throughout the cell cycle. When two destruction box motifs (D-box) are mutated, HOXC10 is stabilized and cells accumulate in metaphase. HOXC10 appears to be a new prometaphase target of the anaphase-promoting complex (APC), since its degradation coincides with cyclin A destruction and is suppressed by expression of a dominant-negative form of UbcH10, an APC-associated ubiquitin-conjugating enzyme. Moreover, HOXC10 co-immunoprecipitates the APC subunit CDC27; its in vitro degradation is reduced in APC-depleted extracts or by competition with the APC substrate cyclin A. These data imply that HOXC10 is a homeoprotein with the potential to influence mitotic progression, and might provide a link between developmental regulation and cell cycle control (Gabellini, 2003).

Cdc27: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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