TPR proteins as essential components of the yeast cell cycle.
Explore the source record for details and available documents.
Amino Acid Sequence↗
SEARCH · PubMed Health
Results for “Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast.
We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction. The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa. The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast. The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase. Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase. The truncated forms of Cut9 block entry into mitosis, however. The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcnl, similar to budding yeast Cdc26. Furthermore, deletion of protein kinase PKA (Pkal) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9. In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9. The Cut9 subunit is likely to be a target for regulating APC/ cyclosome function through protein-protein interactions and phosphorylation.
Amino Acid Sequence↗
Cell cycle. The only way out of mitosis.
The ubiquitination and destruction of cyclins provides the way out of mitosis. A multiprotein complex polyubiquitinates cyclin B and promotes the separation of sister chromatids.
Anaphase↗
A new twist to the cell cycle.
Explore the source record for details and available documents.
Anaphase-Promoting Complex-Cyclosome↗
Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis.
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, we demonstrate 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.
Amino Acid Sequence↗
NUC-2, a component of the phosphate-regulated signal transduction pathway in Neurospora crassa, is an ankyrin repeat protein.
In response to phosphorus limitation, the fungus Neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. The NUC-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway. The nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high pH and low phosphate concentration. We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of the nuc-2 mutation as determined by classical genetic mapping. The nuc-2' open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues. Its predicted amino acid sequence has high similarity to that of its homolog in Saccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of a nuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-induced nuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. The nuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of the nuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.
Amino Acid Sequence↗
Localization of the human homolog of the yeast cell division control 27 gene (CDC27) proximal to ITGB3 on human chromosome 17q21.3.
The human homolog of the Saccharomyces cerevisiae cell division control 27 gene (CDC27) was mapped to human chromosome 17q12-q21 using a panel of human/rodent somatic cell hybrids and localized distal to the breast cancer susceptibility gene, BRCA1, using a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of human CDC27 on human chromosomes 17 is between the marker D17S409 and the beta 3 subunit of integrin (ITGB3). Further confirmation of this localization comes from the sequence tagged site (STS) mapping of human CDC27 to the same yeast artificial chromosomes (YACs) positive for ITGB3. The estimated distance between ITGB3 and human CDC27 is less than 600 kb.
Animals↗
Snap helix with knob and hole: essential repeats in S. pombe nuclear protein nuc2+.
The S. pombe nuc2+ gene is required for mitotic chromosome disjunction. Its mutation arrests mitosis at the metaphase. The gene product is present in the nuclear scaffold-like fraction. The nuc2+ protein contains a domain, separated from ten 34 amino acid repeat segments, that is capable of binding AT-rich DNA in vitro. The ts mutation resides in one of the 34 amino acid repeats. Circular dichroism, limited proteolysis of the repeats, and model fitting indicate the presence of helical segments connected by protease-sensitive hinges. We propose that these repeats form a novel secondary structure (snap helix) having "knob and hole" helix-associating motifs. The packing of the snap helices would be stabilized by the bonding between the hydrophobic amino acids surrounding the knobs and holes. The nuc2+ protein may in one way bind to DNA and in another way mutually associate to form a part of the chromosome scaffold.
Amino Acid Sequence↗
CDC27Hs colocalizes with CDC16Hs to the centrosome and mitotic spindle and is essential for the metaphase to anaphase transition.
We have isolated cDNAs and raised antibodies 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. We find that 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. We conclude that CDC27 and CDC16 are evolutionarily conserved components of the centrosome and mitotic spindle that control the onset of postmetaphase events during mitosis.
Anaphase↗
A 20S complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B.
Cyclin B is degraded at the onset of anaphase by a ubiquitin-dependent proteolytic system. We have fractionated mitotic Xenopus egg extracts to identify components required for this process. We find that 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, we refer to this complex 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.
Anaphase↗
The yeast CDC16 and CDC27 genes restrict DNA replication to once per cell cycle.
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 overreplicated 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.
Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
Linking yeast genetics to mammalian genomes: identification and mapping of the human homolog of CDC27 via the expressed sequence tag (EST) data base.
We describe a strategy for quickly identifying and positionally mapping human homologs of yeast genes to cross-reference the biological and genetic information known about yeast genes to mammalian chromosomal maps. Optimized computer search methods have been developed to scan the rapidly expanding expressed sequence tag (EST) data base to find human open reading frames related to yeast protein sequence queries. These methods take advantage of the newly developed BLOSUM scoring matrices and the query masking function SEG. The corresponding human cDNA is then used to obtain a high-resolution map position on human and mouse chromosomes, providing the links between yeast genetic analysis and mapped mammalian loci. By using these methods, a human homolog of Saccharomyces cerevisiae CDC27 has been identified and mapped to human chromosome 17 and mouse chromosome 11 between the Pkca and Erbb-2 genes. Human CDC27 encodes an 823-aa protein with global similarity to its fungal homologs CDC27, nuc2+, and BimA. Comprehensive cross-referencing of genes and mutant phenotypes described in humans, mice, and yeast should accelerate the study of normal eukaryotic biology and human disease states.
Amino Acid Sequence↗
Efficient manipulation of the human adenovirus genome as an infectious yeast artificial chromosome clone.
A yeast artificial chromosome (YAC) containing a complete human adenovirus type 2 genome was constructed, and viral DNA derived from the YAC was shown to be infectious upon introduction into mammalian cells. The adenovirus YAC could be manipulated efficiently using homologous recombination-based methods in the yeast host, and mutant viruses, including a variant that expresses the human analog of the Saccharomyces cerevisiae CDC27 gene, were readily recovered from modified derivatives of the YAC. The application of powerful yeast genetic techniques to an infectious adenovirus clone promises to significantly enhance the genetic analysis of adenovirus and to simplify the construction of adenovirus-based vectors for vaccines or for gene transfer to mammalian cells or whole animals. The adenovirus YAC was produced by homologous recombination in vivo between adenovirus 2 virion DNA and YAC vector plasmids carrying segments of the viral left and right genomic termini. This recombinational cloning strategy is generally applicable to the construction of YACs containing other DNA segments, such as the genomes of other viruses. Further, it is very efficient and may permit the targeted cloning of segments of the genomes of higher organisms directly from genomic DNA.
Adenoviruses, Human↗
A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.
A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.
Amino Acid Sequence↗
Bypassing anaphase by fission yeast cut9 mutation: requirement of cut9+ to initiate anaphase.
A novel anaphase block phenotype was found in fission yeast temperature-sensitive cut9 mutants. Cells enter mitosis with chromosome condensation and short spindle formation, then block anaphase, but continue to progress into postanaphase events such as degradation of the spindle, reformation of the postanaphase cytoplasmic microtubule arrays, septation, and cytokinesis. The cut9 mutants are defective in the onset of anaphase and possibly in the restraint of postanaphase events until the completion of anaphase. The cut9+ gene encodes a 78-kD protein containing the 10 34-amino acid repeats, tetratricopeptide repeats (TPR), and similar to budding yeast Cdc16. It is essential for viability, and the mutation sites reside in the TPR. The three genes, namely, nuc2+, scn1+, and scn2+, genetically interact with cut9+. The nuc2+ and cut9+ genes share an essential function to initiate anaphase. The cold-sensitive scn1 and scn2 mutations, defective in late anaphase, can suppress the ts phenotype of cut9.
Amino Acid Sequence↗
Fission yeast TPR-family protein nuc2 is required for G1-arrest upon nitrogen starvation and is an inhibitor of septum formation.
Fission yeast nuc2+ gene encodes a protein of a tetratricopeptide repeat (TPR) family which is conserved throughout evolution. We previously showed that nuc2 is required for exit from the mitotic metaphase. In this study, we present evidence which shows that nuc2 has two additional roles in the cell cycle. We showed that the nuc2 mutant is sterile even at the permissive temperature and septation occurs in the absence of chromosome separation at the restrictive temperature. The nuc2 mutant fails to arrest at the G1 phase upon nitrogen starvation at the permissive temperature which is a prerequisite for conjugation. Upon starvation, however, the nuc2 mutant ceased division normally and induced starvation-dependent gene expression. Therefore, the nuc2 mutant is deficient only for failure to block DNA replication upon starvation. At the lower restrictive temperature, the nuc2 mutant showed a 'cut' phenotype where septation and cytokinesis takes place without the completion of mitosis. Ectopic overexpression of the nuc2+ gene caused multiple rounds of S and M phases in the complete absence of septum formation. We propose that nuc2 is a novel cell cycle regulator essential for three events; firstly for exit from mitosis, secondly for DNA replication restraint under nutrient starvation and thirdly for inhibition of septation and cytokinesis until the completion of mitosis.
Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
Higher-order chromosome structure in yeast.
Explore the source record for details and available documents.
Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome↗
The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.
cdc1+ is required for cell cycle progression in Schizosaccharomyces pombe. Cells carrying temperature-sensitive cdc1 mutants undergo cell cycle arrest when shifted to the restrictive temperature, becoming highly elongated. Here we describe the cloning and sequencing of cdc1+, which is shown to encode a 462 residue protein that displays significant sequence similarity to the small subunit of mammalian DNA polymerase delta. cdc1+ interacts genetically with pol3+, which encodes the large subunit of DNA polymerase delta in fission yeast, and the Cdc1 protein binds to Pol3 in vitro, strongly suggesting that Cdc1 is likely to be the small subunit of Pol delta. In addition, we show that cdc1+ overexpression is sufficient to rescue cells carrying temperature-sensitive cdc27 alleles and that the Cdc1 and Cdc27 proteins interact in vivo and in vitro. Deletion of either cdc1+ or cdc27+ results in cell cycle arrest with the arrested cells having a single nucleus with 2C DNA content. No evidence was obtained for a cut phenotype, indicating that neither cdc1+ nor cdc27+ is required for checkpoint function. cdc1 mutant cells are supersensitive to the DNA synthesis inhibitor hydroxyurea and to the DNA damaging agent MMS, display increased frequency of mini-chromosome loss and have an extended S phase.
Alleles↗