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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↗

BIMAAPC3, a component of the Aspergillus anaphase promoting complex/cyclosome, is required for a G2 checkpoint blocking entry into mitosis in the absence of NIMA function.

Temperature sensitive (ts) nimA mutants of Aspergillus nidulans arrest at a unique point in G2 which is post activation of CDC2. Here we show that this G2 arrest is due to loss of nimA function and that it is dependent on BIMAAPC3, a component of the anaphase promoting complex/cyclosome (APC/C). Whereas nimA single mutants arrested in G2 with decondensed chromatin and interphase microtubule arrays, nimA, bimAAPC3 double mutants arrested growth with condensed chromatin and aster-like microtubule arrays. nimA, bimAAPC3 double mutants entered mitosis with kinetics similar to bimAAPC3 single mutants and wild-type cells, indicating a checkpoint-like role for BIMAAPC3 in G2. Even cells which had been depleted for NIMA protein and which contained insignificant levels of NIMA kinase activity entered mitosis on inactivation of bimAAPC3. BIMAAPC3 was present in a >25S complex containing BIMEAPC1, and bimAAPC3 mutants were sensitive to elevated CYCLIN B expression, consistent with BIMAAPC3 being a component of the APC/C. Inactivation of bimAAPC3 had little affect on the steady state levels of the B-type cyclin, NIMECyclin B. Our results indicate that BIMAAPC3, and most likely the APC/C itself, is activated in G2 in nimA mutants. We propose that APC/C activation is part of a novel, late G2 checkpoint, which responds to a defective process or structure in nimA mutants, and which prevents inappropriate entry into mitosis.

Alleles↗

Viral inhibition of the anaphase promoting complex enhances replication by elevating nucleotide pools.

The anaphase promoting complex/cyclosome (APC/C) is a large, ubiquitin ligase and a central regulator of cell cycle progression. By targeting key substrates for degradation during mitosis and G1 phase, the APC/C coordinates metabolic fluctuations that occur during the cell cycle. A diverse range of viruses have convergently evolved mechanisms to bind and inhibit the APC/C; however, a molecular understanding for these interactions has never been demonstrated. Here, we use chicken anemia virus (CAV), a small single-stranded DNA virus encoding only three proteins, to demonstrate the importance of viral APC/C inhibition during replication. We show that the Vp3 protein of CAV inhibits the APC/C, causing a dramatic mitotic arrest during infection. The mutant virus lacking Vp3 is defective for replication and can be rescued by APC/C inhibition. Metabolomic profiling during CAV infection revealed that Vp3 expression mediates a broad increase in nucleotide pools. Moreover, viral inhibition of the APC/C resulted in stabilization of enzymes required for nucleotide biosynthesis. These findings suggest that the APC/C is a general target of many viruses to elevate nucleotide levels and facilitate viral genome replication.

Anaphase-Promoting Complex-Cyclosome↗

Genomic organization and expression of the ubiquitin-proteasome complex-associated protein Rbx1/ROC1/Hrt1.

Rbx1/ROC1/Hrt1 (Rbx1) has been recently shown to be involved in the regulation of protein turn-over. Here, we report the organization of the human Rbx1 gene, established by both a cloning and a functional genomics approach. The human gene, composed of five exons, encompasses 22.3 kb on chromosome 22q 13. The expression of Rbx1 transcripts (0.5 kb) appears developmentally regulated during mouse embryonic development and is prevalent in the adult mouse genital tract. A Genbank database search for Rbx1 related sequences in various species, from plants to mammals, is indicative of a high degree of evolutionary conservation in mouse rat and zebra fish and also, for the main functional part of the molecule, in other living species, although their gene structures can be significantly altered.

Amino Acid Sequence↗

The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1.

The ordered progression through the cell cycle depends on regulating the abundance of several proteins through ubiquitin-mediated proteolysis. Degradation is precisely timed and specific. One key component of the degradation system, the anaphase promoting complex (APC), is a ubiquitin protein ligase. It is activated both during mitosis and late in mitosis/G(1), by the WD repeat proteins Cdc20 and Cdh1, respectively. These activators target distinct sets of substrates. Cdc20-APC requires a well-defined destruction box (D box), whereas Cdh1-APC confers a different and as yet unidentified specificity. We have determined the sequence specificity for Cdh1-APC using two assays, ubiquitination in a completely defined and purified system and degradation promoted by Cdh1-APC in Xenopus extracts. Cdc20 is itself a Cdh1-APC substrate. Vertebrate Cdc20 lacks a D box and therefore is recognized by Cdh1-APC through a different sequence. By analysis of Cdc20 as a substrate, we have identified a new recognition signal. This signal, composed of K-E-N, serves as a general targeting signal for Cdh1-APC. Like the D box, it is transposable to other proteins. Using the KEN box as a template, we have identified cell cycle genes Nek2 and B99 as additional Cdh1-APC substrates. Mutation in the KEN box stabilizes all three proteins against ubiquitination and degradation.

Amino Acid Sequence↗

A site-directed approach for constructing temperature-sensitive ubiquitin-conjugating enzymes reveals a cell cycle function and growth function for RAD6.

We have determined the gene sequence of a temperature-sensitive allele of the cell cycle-related ubiquitin-conjugating enzyme CDC34 (UBC 3) from Saccharomyces cerevisiae. The basis of temperature sensitivity is a missense mutation resulting in a proline to serine substitution at a residue that is conserved in all ubiquitin-conjugating enzymes identified thus far. This observation raised the possibility that other temperature-sensitive ubiquitin-conjugating enzymes could be generated in the same way. We therefore created the corresponding substitution in the DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), and examined the effect of temperature on the cell proliferation and DNA repair-related functions of this altered polypeptide. Yeast strains carrying this mutation proved to be temperature-sensitive with respect to cell proliferation but not with respect to the DNA damage-processing phenotypes exhibited by other rad6 mutants. Upon further investigation of the proliferation defect exhibited by this mutant, we discovered that other rad6 gene mutants deleted for the gene undergo cell cycle arrest at the nonpermissive temperature, whereas the engineered temperature-sensitive allele showed no evidence of a cell cycle defect. From these findings, we conclude that the proliferation function of RAD6 can be subdivided into a growth component and a cell division cycle component and that the growth component is unrelated to the DNA repair functions of RAD6. A reasonable interpretation of these results is that different proteins are targeted for ubiquitination in each case. The conserved proline residue of RAD6 and CDC34 is part of a turn motif common to all ubiquitin-conjugating enzymes. It is therefore likely that site-directed substitution of prolines located in turns can be generally applied for the creation of other temperature-sensitive ubiquitin-conjugating enzymes and possibly other proteins as well.

Alleles↗

Molecular cloning of a cDNA with a novel domain present in the tre-2 oncogene and the yeast cell cycle regulators BUB2 and cdc16.

In an effort to identify genes that are differentially regulated during mast cell development, subtracted cDNA prepared from wild-type murine P815 mastocytoma cells and a P815 subline that exhibits properties of mast cell differentiation was used to screen mast cell cDNA libraries. Several known mast cell-specific cDNAs were isolated including mast cell carboxypeptidase A (MC-CPA), murine mast cell protease-5 (MMCP-5), and gp49. A novel cDNA, designated Tbc1, was identified that showed differential expression in the two mast cell lines. The amino acid sequence predicted from the cDNA contains a 200 amino acid domain that is homologous to regions in the tre-2 oncogene and the yeast regulators of mitosis, BUB2 and cdc16. The N-terminal region contains a number of cysteine and histidine residues, potentially encoding a zinc finger domain. Tbc1 is a nuclear protein and is expressed in highest levels in hematopoietic cells, testis and kidney. Within these tissues, expression of Tbc1 is cell- and stage-specific. Based on sequence similarity, pattern of expression and subcellular localization, Tbc1 may play a role in the cell cycle and differentiation of various tissues.

3T3 Cells↗

Functional and physical characterization of the cell cycle ubiquitin-conjugating enzyme CDC34 (UBC3). Identification of a functional determinant within the tail that facilitates CDC34 self-association.

Like several other ubiquitin-conjugating enzymes, the yeast cell cycle enzyme CDC34 (UBC3) has a carboxyl-terminal extension or tail. These tails appear to carry out unique functions that can vary from one ubiquitin-conjugating enzyme to the next. Using biophysical techniques we have determined that the tail of CDC34 constitutes a highly structured and extended domain. Although the tail of CDC34 is the largest tail identified to date (125 residues), we have found that only 39 residues lying adjacent to the catalytic domain are necessary and sufficient for full cell cycle function and that this region fulfills a novel function that may be common to the tails of other ubiquitin-conjugating enzymes. Cross-linking studies demonstrate that this region facilitates a physical interaction between CDC34 monomers in vitro. Furthermore, phenotypic analysis of various CDC34 derivatives expressed in different cdc34 mutant strains indicates that this region facilitates the same interaction in vivo. Based on these findings, it appears that the cell cycle function of CDC34 is dependent upon the ability of CDC34 monomers to interact with one another and that this interaction is mediated by a small region of the CDC34 tail. The similarity of this region with sequences contained within the tails of the UBC1 and UBC6 enzymes suggests that these tails may function in a similar manner.

Anaphase-Promoting Complex-Cyclosome↗

The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein.

The CDC34 gene of Saccharomyces cerevisiae encodes a 295-residue ubiquitin-conjugating enzyme (E2). The function of this ubiquitin-conjugating activity remains to be defined as its in vivo substrates are presently unknown. The bacterially expressed and purified Cdc34 protein is shown here to catalyze its own ubiquitination via an intramolecular transfer of its thiol ester-linked ubiquitin to a lysine. In this process, multiple ubiquitin groups are added to Cdc34, and these ubiquitin groups were shown to be arranged predominantly in the form of a single Lys48-specific multiubiquitin chain. Analysis of the hydroxylamine-dependent cleavage of ubiquitin-Cdc34 conjugates at the single Asn-Gly sequence in Cdc34 placed the major ubiquitin linkage site within the C-terminal 215-295 residues of Cdc34. The 4 Lys residues (Lys273, Lys277, Lys293, and Lys294) in this region of CDC34 were substituted by arginine either singly or in combination to produce a set of Cdc34 mutants. Analysis of these Cdc34 mutants for autoubiquitination revealed that the multiubiquitin chain can be formed on any one of these 4 lysines although most Cdc34 conjugates contain a single multiubiquitin chain. Since the presence of a Lys48-specific multiubiquitin chain in protein conjugates is known to target acceptor proteins for degradation in the ubiquitin-mediated proteolytic pathway, the present result raises the possibility that one function of the ubiquitin-conjugating activity in CDC34 may be used to target its own degradation.

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↗