Two WD repeat-containing TATA-binding protein-associated factors in fission yeast that suppress defects in the anaphase-promoting complex.

The general transcription factor IID consists of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here we report the isolation of two related TAF genes from the fission yeast Schizosaccharomyces pombe as multicopy suppressors of a temperature-sensitive mutation in the ubiquitin-conjugating enzyme gene ubcP4(+). The ubcP4(ts) mutation causes cell cycle arrest in mitosis, probably due to defects in ubiquitination mediated by the anaphase-promoting complex/cyclosome. One multicopy suppressor is the previously reported gene taf72(+), whereas the other is a previously unidentified gene named taf73(+). We show that the taf73(+) gene, like taf72(+), is essential for cell viability. The taf72(+) and taf73(+) genes encode proteins homologous to WD repeat-containing TAFs such as human TAF100, Drosophila TAF80/85, and Saccharomyces cerevisiae TAF90. We demonstrate that TAF72 and TAF73 proteins are present in the same complex with TBP and other TAFs and that TAF72, but not TAF73, is associated with the putative histone acetylase Gcn5. We also show that overexpression of TAF72 or TAF73 suppresses the cell cycle arrest in mitosis caused by a mutation in the anaphase-promoting complex/cyclosome subunit gene cut9(+). These results suggest that TAF72 and TAF73 may regulate the expression of genes involved in ubiquitin-dependent proteolysis during mitosis. Our study thus provides evidence for a possible role of WD repeat-containing TAFs in the expression of genes involved in progression through the M phase of the cell cycle.

Ubiquitin system: selectivity and timing of protein destruction.

A growing number of cellular functions have been shown to be regulated through protein degradation. The selective degradation of many short-lived proteins in eukaryotic cells is mediated by the ubiquitin system, by which proteins covalently ligated to ubiquitin are targeted for degradation. The selectivity of the destruction is ensured by the substrate specificity in the ubiquitination steps composed of a series of enzymatic reactions. Ubiquitin-ligase (E3), in conjunction with ubiquitin-conjugating enzyme (E2), has been implicated as playing an essential role in the substrate recognition. The substantial character, however, of the ligase was not clear until several recent studies demonstrated ligases that exert key roles in irreversible steps of the cell-cycle control. In this review, attention is focused on the molecular basis of target recognition of ubiquitination, particularly as exemplified in the ubiquitin-ligases in the cell-cycle control mechanisms.

An essential function of Grr1 for the degradation of Cln2 is to act as a binding core that links Cln2 to Skp1.

In budding yeast, SCF complexes, composed of Skp1, Cdc53 and one of the F-box proteins, have been implicated in Cdc34-dependent ubiquitination. Grr1, which is required for degradation of G1 cyclins Cln1 and Cln2 as well as for regulation of glucose repression, is an F-box protein and interacts with Skp1 through the F-box motif. Grr1 also interacts in vitro with phosphorylated Cln1 and Cln2. However, ubiquitination of Cln1 has not been successful in an in vitro reconstituted system. In this study, domain analysis was performed to understand the role of Grr1 in the degradation of Cln2. Grr1 has another motif, leucine-rich repeats (LRR), in addition to the F-box. We found that the LRR is a domain for Cln2 binding. A deletion of half of the LRR abolished the interaction of Grr1 with phosphorylated Cln2 but not with Skp1 in vivo, and a deletion of the F-box abolished the interaction of Grr1 with Skp1 but not with phosphorylated Cln2 in vivo. Based on these results, we constructed grr1 mutants that are defective in association with either Skp1 or Cln2. Cln2 was highly stabilized and accumulated in the phosphorylated forms in the mutant cells. Furthermore, Skp1 associated in vivo with phosphorylated Cln2 in a Grr1-dependent manner. These data suggest that Grr1 is required for degradation of Cln2 through linking phosphorylated Cln2 to Skp1 in a SCFGrr1 complex.

Aphidicolin induces alterations in Golgi complex and disorganization of microtubules of HeLa cells upon long-term administration.

Treatment of HeLa cells with aphidicolin at 5 or 0.5 microg/ml induced cell cycle arrest at G1/S or G2/M phase, respectively, and was accompanied by unbalanced cell growth. Long-term administration of aphidicolin (more than 48 h) resulted in noticeable loss of reproductive capacity though cells were viable at the time of treatment. Immunofluorescence with anti-Golgi membrane protein monoclonal antibody (mAbG3A5) showed disfigurement of the characteristic mesh-like configuration when cells were treated for more than 48 h. Interestingly, we found that the fragmented Golgi complex formed a ring around the nucleus in more than 20% of the cells. Immunoelectron microscopy using mAbG3A5 antibody demonstrated that the stack structure of the fragmented Golgi complex in aphidicolin-arrested cells appeared partially broken up and seemed to have converted to a vesicle-like structure. Analysis using an antibody to tubulin and anticentrosome human autoimmune serum showed that alterations in the Golgi complex were induced even by the lower 0.5 microg/ml dose. These alterations were accompanied by both changes in the distribution of microtubules and an increase in the number of centrosomes. These cells lost their distinct perinuclear microtubule organizing center (MTOC). On the other hand, treatment with aphidicolin at 5 microg/ml did not induce multiplication of the centrosome although the loss of distinct MTOC was still evident. No changes took place in the Golgi complex, microtubule, or centrosome of cells treated with 0.5 microg/ml aphidicolin when cycloheximide was added simultaneously to the culture.

Grr1 functions in the ubiquitin pathway in Saccharomyces cerevisiae through association with Skp1.

Cdc34, a ubiquitin-conjugating enzyme in Saccharomyces cerevisiae, is required for cell cycle progression. sic1, an S-phase cyclin-dependent kinase (CDK) inhibitor, is a critical target of Cdc34-mediated ubiquitination. Other essential target protein(s) could be defined since cdc34 sic1 double mutants still arrest in G2 phase. To identify proteins which function in the Cdc34-dependent ubiquitin pathway, a series of extragenic suppressors of the cdc34-1 sic1 double mutations was isolated. One of them was found to be defective in GRR1, which is involved not only in glucose repression but also in G1 cyclin destabilization. However, neither lack of glucose repression nor stabilization of G1 cyclin caused the suppression of cdc34-1 sic1. Conversely, Grr1 overproduction in cdc34-1 sic1 cells impaired colony formation, even at the permissive temperature. A multicopy suppressor, MGO1, which rescued the growth defect associated with Grr1 overproduction was isolated, and found to be identical to SKP1. Furthermore, Grr1 bound Skp1 directly in vitro. These results strongly suggest that Grr1 functions in the ubiquitin pathway through association with Skp1.

Inhibition of nuclear envelope reconstitution in Xenopus interphase egg extract by hemin.

Addition of hemin to the nuclear reconstitution system of Xenopus interphase egg extract using sperm head chromatin resulted in abnormal pseudonuclei exhibiting flattened membrane patches randomly distributed both on the surface and inside the nuclei. The structures that resembled nuclear pores were observed on these flattened membrane patch structures. Although the nucleosome structure was formed as revealed by the micrococcal nuclease digestion, the B-type lamin uptake into the nuclei was inhibited by hemin. Using heminagarose affinity chromatography, we isolated several hemin-binding proteins from fully reconstituted pseudonuclei. Some of the hemin-binding proteins bound concanavalin A (Con A). Comparison of hemin-binding proteins with those isolated from both fractions of supernatant and pellet separated by high speed centrifugation of the egg extract showed that the hemin-binding proteins of pseudonuclei were supplied from both fractions. The uptake of nuclear hemin-binding proteins did not occur in the incompletely reconstituted nuclei resulting from addition of excess sperm chromatin to the system. These results suggest that the hemin-binding proteins participate in the late steps of nuclear reconstitution during formation of the nuclear envelope.

Induction of ubiquitin conjugating enzyme activity for degradation of topoisomerase II alpha during adenovirus E1A-induced apoptosis.

Topoisomerase (topo) II alpha is degraded via polyubiquitination during adenovirus E1A-induced apoptosis in MA1 cells, a derivative of the human epidermoid carcinoma cell line KB. Topo II alpha ubiquitination activity in MA1 cells increased nearly 10-fold after induction of E1A in response to dexamethasone. To identify a topo II alpha ubiquitination factor(s), the S100 fractions prepared from apoptosis-induced (42 h) and uninduced (0 h) MA1 cells were first fractionated by ubiquitin-Sepharose columns. The ubiquitination activity induced by E1A was predominantly eluted with 20 mM AMP. Further fractionation of the AMP eluates on Resource-Q columns and the thiolester formation of the proteins resolved by electrophoresis with biotinylated ubiquitin revealed that a species of E2 isozyme recovered in the QFT2 fraction increased markedly in MA1 cells after E1A expression. These results indicate that a ubiquitination factor(s) specific to topo II alpha is induced during E1A-induced apoptosis in MA1 cells.

A ubiquitin-conjugating enzyme in fission yeast that is essential for the onset of anaphase in mitosis.

A cDNA encoding a ubiquitin-conjugating enzyme designated UbcP4 in fission yeast was isolated. Disruption of its genomic gene revealed that it was essential for cell viability. In vivo depletion of the UbcP4 protein demonstrated that it was necessary for cell cycle progression at two phases, G2/M and metaphase/anaphase transitions. The G2 arrest of UbcP4-depleted cells was dependent upon chk1, which mediates checkpoint pathway. UbcP4-depleted cells arrested at metaphase had condensed chromosomes but were defective in separation. However, septum formation and cytokinesis were not restrained during the metaphase arrest. Overexpression of UbcP4 specifically rescued the growth defect of cut9ts cells at a restrictive temperature. cut9 encodes a component of the anaphase-promoting complex (APC) which is required for chromosome segregation at anaphase and moreover is defined as cyclin-specific ubiquitin ligase. Cdc13, a mitotic cyclin in fission yeast, was accumulated in the UbcP4-depleted cells. These results strongly suggested that UbcP4 is a ubiquitin-conjugating enzyme working in conjunction with APC and mediates the ubiquitin pathway for degradation of "sister chromatid holding protein(s)" at the onset of anaphase and possibly of mitotic cyclin at the exit of mitosis.

Incubation at the nonpermissive temperature induces deficiencies in UV resistance and mutagenesis in mouse mutant cells expressing a temperature-sensitive ubiquitin-activating enzyme (E1).

In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.

Degradation of topoisomerase IIalpha during adenovirus E1A-induced apoptosis is mediated by the activation of the ubiquitin proteolysis system.

The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A12S in response to dexamethasone. The level of topoisomerase IIalpha begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncogene 10, 651-662). Topoisomerase IIalpha prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP- and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase IIalpha was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 x g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase IIalpha could be detected by incubating it in the S70 or S100 extract, which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42-h extract was 4- to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component(s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase IIalpha, is activated or induced during the latent phase of E1A-induced apoptosis.

Cloning and expression of cDNA encoding a human ubiquitin-conjugating enzyme similar to the Drosophila bendless gene product.

cDNA encoding a novel human ubiquitin-conjugating enzyme has been cloned from an epidermoid carcinoma KB cDNA library. This clone encodes a protein of 152 amino acids with a calculated M(r) of 17,137. The amino acid sequence showed 80% identity with the Drosophila's bendless gene product (ubiquitin-conjugating enzyme E2). The corresponding transcripts are highly expressed in heart, skeletal muscle, and testis. The product expressed in Escherichia coli exhibited the ability to form a thiol ester linkage with ubiquitin in a ubiquitin-activating enzyme E1-dependent manner. These results suggest that the obtained cDNA encodes a novel human E2 which may be involved in protein degradation mainly in the muscles and testis.

Ubiquitin-activating enzyme, E1, is phosphorylated in mammalian cells by the protein kinase Cdc2.

The ubiquitin-activating enzyme (E1) is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. E1 was found to be phosphorylated in cells of a mouse mammary carcinoma cell line, FM3A. Peptide mapping of trypsin digests of labeled E1 indicated that two oligopeptides were mainly phosphorylated in vivo. The same oligopeptides were also labeled in vitro on Cdc2 kinase-mediated phosphorylation of E1, affinity-purified from the same cell line. The Cdc2 kinase is a key enzyme playing a pivotal role in G2/M transition in the cell cycle. The phosphorylation of one of the two oligopeptides was prominent at the G2/M phase of the cell cycle, and dependent upon the Cdc2 kinase activity in vivo since it was significantly reduced in tsFT210, a mutant cell line deficient in Cdc2 kinase. Mutation analysis indicated that the serine residue at the fourth position of the E1 enzyme was a phosphorylation site of Cdc2 kinase. These findings suggest that E1 is a target of Cdc2 kinase in the cell, implying that the ubiquitin system may be dynamically involved in cell cycle control through phosphorylation of this key enzyme.

Abnormal integrity of the nucleolus associated with cell cycle arrest owing to the temperature-sensitive ubiquitin-activating enzyme E1.

A mouse cell mutant, ts85, containing the temperature-sensitive ubiquitin-activating enzyme was arrested in G2 phase at the non-permissive temperature. In the arrested cells, azure C, a nucleolus-specific stain, revealed a U-shaped or ring-shaped arrangement of nucleolar lobes with an unstained region in the center. Silver staining of the nucleolar organizer region (NOR) and fluorescence in situ hybridization (FISH) with rDNA both gave signals in azure C-positive regions. Electron microscopic examination revealed a cloud of unidentified electron-dense particles (diameter approximately 70 nm) in the azure C-negative center space. When the arrested cells were released into M-phase, we observed the association of NOR-bearing chromosomes with a pulverization-like abnormality. FISH with rDNA and NOR silver staining demonstrated that the pulverization-like abnormality was restricted to NORs. The frequent occurrence of persistent nucleolar material in prophase and prometaphase of the stressed cells after release indicated a delayed dissociation of the nucleolus that brought about the abnormal chromosomes in M-phase. ts85 cells transfected with the mouse E1 cDNA recovered growth at the non-permissive temperature and no longer showed abnormal nucleolar morphology. It seems that the ubiquitin system plays a role in the dissolution of the nucleolus, possibly involving the NOR-bearing chromosomes.

Conditional resistance to thymineless death predominantly selects DNA synthesis-deficient mutants of mammalian cells.

Temperature-sensitive growth mutants of the mouse mammary carcinoma cell line FM3A were isolated by selecting survivors of thymidylate starvation for a limited time at the restrictive temperature (39.5 degrees C). Nineteen lines of independent isolates were established and all were found to be deficient in DNA synthesis. Cell-cell hybridization with authentic mutant lines of FM3A demonstrated that the mutants fell into three complementation groups, which were deficient in DNA polymerase alpha or ubiquitin-activating enzyme E1 or both.

Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1.

A cDNA encoding the ubiquitin-activating enzyme, E1, was isolated from the mouse mammary carcinoma cell line, FM3A, and shown to complement mutant mouse cells deficient in the enzyme. The 3495-bp cDNA encodes 1058 amino acids (aa), and shares extensive homology with the human E1 enzyme at both the nucleotide and aa sequence levels.

tsBN75 and tsBN423, temperature-sensitive x-linked mutants of the BHK21 cell line, can be complemented by the ubiquitin-activating enzyme E1 cDNA.

tsBN75 and tsBN423 are independently isolated temperature-sensitive (ts) mutants of the BHK21 cell line for cell growth. Both tsBN75 and tsBN423 belong to the same complementation group and show G2 block at the nonpermissive temperature. Both were efficiently transformed to ts+ cells with the mouse and human cDNA encoding the ubiquitin-activating enzyme, E1. While no transformants of tsBN423 cells had a DNA content greater than the parental 2C, several ts+ transformants of tsBN75 cells acquired a multiploid DNA content. These data thus demonstrate the function of the human and mouse E1 cDNAs and further suggest that E1 functions in more than one step in cell cycle progression.