RESUMO
Snf2SR, a suppressor of rna1(ts), which is a temperature-sensitive mutation in Schizosaccharomyces pombe RanGAP (GTPase activating protein), possesses both the SNF2 and the helicase domains conserved in the chromatin remodeling SNF2 ATPase/helicase protein family. We have now clarified a function of Snf2SR. Snf2SR indeed showed DNA-stimulated ATPase activity, proving that it is a member of the SNF2 ATPase/helicase family. Consistent with this role, Snf2SR was localized in the nucleus and cell fractionation analysis revealed that Snf2SR was tightly associated with the nuclear matrix. The disruption of snf2SR(+) was detrimental for a cell proliferation of S. pombe. Snf2SR that did not enhance RanGAP activity by itself, but abolished histone-H3-mediated RanGAP inhibition, as previously reported for the histone H3 methyltransferase, Clr4, another rna1(ts) suppressor. In contrast to Clr4, Snf2SR directly bound to the GDP-bound form of the S. pombe Ran homologue Spi1 and enhanced the nucleotide exchange activity of Pim1, the S. pombe RanGEF (guanine nucleotide exchange factor). Over-expression of Spi1-G18V, a Ran GTPase mutant fixed in the GTP-bound form, was lethal to S. pombe Deltasnf2SR. Together, our results indicate that Snf2SR is involved in the Ran GTPase cycle in vivo.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/genética , Núcleo Celular/química , Proteínas Cromossômicas não Histona/metabolismo , Deleção de Genes , Histonas/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/análise , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.
Assuntos
Metabolismo Energético/fisiologia , Proteínas de Membrana/fisiologia , Poro Nuclear/fisiologia , Proteínas Nucleares/fisiologia , Trifosfato de Adenosina/farmacologia , Retículo Endoplasmático/fisiologia , Células HeLa , Humanos , Proteínas de Membrana/efeitos dos fármacos , Lâmina Nuclear/efeitos dos fármacos , Lâmina Nuclear/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Sirolimo/farmacologia , Temperatura , Fatores de TempoRESUMO
Although the Ran GTPase-activating protein RanGAP mainly functions in the cytoplasm, several lines of evidence indicate a nuclear function of RanGAP. We found that Schizosaccharomyces pombe RanGAP, SpRna1, bound the core of histone H3 (H3) and enhanced Clr4-mediated H3-lysine 9 (K9) methylation. This enhancement was not observed for methylation of the H3-tail containing K9 and was independent of SpRna1-RanGAP activity, suggesting that SpRna1 itself enhances Clr4-mediated H3-K9 methylation via H3. Although most SpRna1 is in the cytoplasm, some cofractionated with H3. Sprna1(ts) mutations caused decreases in Swi6 localization and H3-K9 methylation at all three heterochromatic regions of S. pombe. Thus, nuclear SpRna1 seems to be involved in heterochromatin assembly. All core histones bound SpRna1 and inhibited SpRna1-RanGAP activity. In contrast, Clr4 abolished the inhibitory effect of H3 on the RanGAP activity of SpRna1 but partially affected the other histones. SpRna1 formed a trimeric complex with H3 and Clr4, suggesting that nuclear SpRna1 is reciprocally regulated by histones, especially H3, and Clr4 on the chromatin to function for higher order chromatin assembly. We also found that SpRna1 formed a stable complex with Xpo1/Crm1 plus Ran-GTP, in the presence of H3.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Heterocromatina/metabolismo , Heterocromatina/fisiologia , Histonas/metabolismo , Metiltransferases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/genética , Primers do DNA , Marcadores Genéticos , Histona-Lisina N-Metiltransferase , Cinética , Metiltransferases/genética , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Previously, we described a novel nucleolar protein, NOP132, which interacts with the small GTP binding protein RRAG A. To elucidate the function of NOP132 in the nucleolus, we identified proteins that interact with NOP132 using mass spectrometric methods. NOP132 associated mainly with proteins involved in ribosome biogenesis and RNA metabolism, including the DEAD-box RNA helicase protein, DDX47, whose yeast homolog is Rrp3, which has roles in pre-rRNA processing. Immunoprecipitation of FLAG-tagged DDX47 co-precipitated rRNA precursors, as well as a number of proteins that are probably involved in ribosome biogenesis, implying that DDX47 plays a role in pre-rRNA processing. Introduction of NOP132 small interfering RNAs induced a ring-like localization of DDX47 in the nucleolus, suggesting that NOP132 is required for the appropriate localization of DDX47 within the nucleolus. We propose that NOP132 functions in the recruitment of pre-rRNA processing proteins, including DDX47, to the region where rRNA is transcribed within the nucleolus.
Assuntos
Proteínas de Transporte/fisiologia , Nucléolo Celular/enzimologia , Proteínas Nucleares/fisiologia , RNA Helicases/análise , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/química , RNA Helicases DEAD-box , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/química , Ligação Proteica , RNA Helicases/química , RNA Helicases/fisiologia , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Ribossomos/metabolismoRESUMO
Mog1 is conserved from yeast to mammal, but its function is obscure. We isolated yeast genes that rescued a temperature-sensitive death of S. cerevisiae Scmog1Delta, and of S. pombe Spmog1(ts). Scmog1Delta was rescued by Opi3p, a phospholipid N-methyltransferase, in addition to S. cerevisiae Ran-homologue Gsp1p, and a RanGDP binding protein Ntf2p. On the other hand, Spmog1(ts) was rescued by Cid13 that is a poly (A) polymerase specific for suc22(+) mRNA encoding a subunit of ribonucleotide reductase, Ssp1 that is a protein kinase involved in stress response pathway, and Crp79 that is required for mRNA export, in addition to Spi1, S. pombe Ran-homologue, and Nxt2, S. pombe homologue of Ntf2p. Consistent with the identification of those suppressors, lack of ScMog1p dislocates Opi3p from the nuclear membrane and all of Spmog1(ts) showed the nuclear accumulation of mRNA. Furthermore, SpMog1 was co-precipitated with Nxt2 and Cid13.
Assuntos
Metabolismo dos Lipídeos , RNA/metabolismo , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Schizosaccharomyces pombe/antagonistas & inibidores , Proteínas de Schizosaccharomyces pombe/fisiologia , Transdução de Sinais , Proteína ran de Ligação ao GTP/antagonistas & inibidores , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/fisiologia , Sequência de Bases , Dados de Sequência Molecular , Fosfatidil-N-Metiletanolamina N-Metiltransferase/metabolismo , Polinucleotídeo Adenililtransferase/análise , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
We isolated 11 independent temperature-sensitive (ts) mutants of Schizosaccharomyces pombe RanGAP, SpRna1 that have several amino acid changes in the conserved domains of RanGAP. Resulting Sprna1ts showed a strong defect in mitotic chromosome segregation, but did not in nucleocytoplasmic transport and microtubule formation. In addition to Sprna1+ and Spksp1+, the clr4+ (histone H3-K9 methyltransferase), the S. pombe gene, SPAC25A8.01c, designated snf2SR+ (a member of the chromatin remodeling factors, Snf2 family with DNA-dependent ATPase activity), but not the spi1+ (S. pombe Ran homolog), rescued a lethality of Sprna1ts. Both Clr4 and Snf2 were reported to be involved in heterochromatin formation essential for building the centromeres. Consistently, Sprna1ts was defective in gene-silencing at the centromeres. But a silencing at the telomere, another heterochromatic region, was normal in all of Sprna1ts strains, indicating SpRna1 in general did not function for a heterochromatin formation. snf2SR+ rescued a centromeric silencing defect and Deltaclr4+ was synthetic lethal with Sprna1ts. Taken together, SpRna1 was suggested to function for constructing the centromeres, by cooperating with Clr4 and Snf2SR. Loss of SpRna1 activity, therefore, caused chromosome missegregation.
Assuntos
Centrômero/ultraestrutura , Proteínas Ativadoras de GTPase/biossíntese , Proteínas Ativadoras de GTPase/fisiologia , Inativação Gênica , Schizosaccharomyces/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Segregação de Cromossomos , Citoplasma/metabolismo , Análise Mutacional de DNA , Proteínas de Fluorescência Verde/metabolismo , Heterocromatina/metabolismo , Histonas , Microscopia de Fluorescência , Mitose , Modelos Biológicos , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proteínas de Schizosaccharomyces pombe , TemperaturaRESUMO
Yeast Gtr1p and its human homolog RRAG A belong to the Ras-like small G-protein superfamily and genetically interact with RCC1, a guanine nucleotide exchange factor for Ran GTPase. Little is known regarding the function of Gtr1p. We performed yeast two-hybrid screening using Gtr1p as the bait to find interacting proteins. Rpc19p, a shared subunit of RNA polymerases I and III, associated with Gtr1p. The association of Gtr1p with Rpc19p occurred in a GTP-form-specific manner. RRAG A associated with RPA16 (human Rpc19p homolog) in a GTP-form-specific manner, suggesting that the association is conserved during evolution. Ribosomal RNA and tRNA synthesis were reduced in the gtr1Delta strain expressing the GDP form of Gtr1p, but not the GTP form of Gtr1p. Gel-filtration studies revealed an accumulation of the smaller Rpc19p-containing complex, but not of A135, in the gtr1Delta strain. Here, we propose that Gtr1p is involved in RNA polymerase I and III assembly by its association with Rpc19p and could be a mediator that links growth regulatory signals with ribosome biogenesis.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , RNA Polimerase III/química , RNA Polimerase I/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/genética , Genes Fúngicos , Marcadores Genéticos , Glutationa Transferase/metabolismo , Glicosídeo Hidrolases/análise , Proteínas Monoméricas de Ligação ao GTP/genética , Subunidades Proteicas/química , RNA Ribossômico/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
We have isolated a temperature-sensitive alanyl-tRNA synthetase mutant from hamster BHK21 cells, designated as ts ET12. It has a single nucleotide mutation, converting the 321st amino acid residue, 321Gly, to Arg. The mutation was localized between two RNA-binding domains of alanyl-tRNA synthetase. Thus far, we have isolated two temperature-sensitive aminoacyl-tRNA synthetase mutants from the BHK21 cell line: ts BN250 and ts BN269. They are defective in histidyl- and lysyl-tRNA synthetase respectively. Both mutants rapidly undergo apoptosis at the nonpermissive temperature, 39.5 degrees C. ts ET12 cells, however, did not undergo apoptosis until 48 h after a temperature-shift to 39.5 degrees C, while mutated alanyl-tRNA synthetase of ts ET12 cells was lost within 4 h. Loss of the mutated alanyl-tRNA synthetase was inhibited by a ubiquitin-dependent proteasome inhibitor, MG132, and by a protein-synthesis inhibitor, cycloheximide. Cell-cycle related proteins were also lost in ts ET12 cells at 39.5 degrees C, as shown in ts BN250. In contrast, the mutated aminoacyl-tRNA synthetases of ts BN250 and ts BN269 were stable at 39.5 degrees C. However, the defects of these mutants released EMAPII, an inducer of apoptosis at 39.5 degrees C. No release of EMAPII occurred in ts ET12 cells at 39.5 degrees C, consistent with the delay of apoptosis in these cells.
Assuntos
Alanina-tRNA Ligase/genética , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mutação , Temperatura , Alanina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Humanos , Dados de Sequência MolecularRESUMO
ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.
Assuntos
Fase G1/genética , RNA Helicases/genética , RNA Helicases/fisiologia , Fase S/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , Ciclina A/biossíntese , Ciclina B/biossíntese , DNA/química , DNA/genética , DNA Complementar/genética , Fator de Iniciação 4E em Eucariotos/biossíntese , Fase G1/fisiologia , Teste de Complementação Genética , Genoma Humano , Células HeLa , Humanos , Isoenzimas , Dados de Sequência Molecular , Mutação Puntual , RNA Mensageiro/biossíntese , Fase S/fisiologia , Análise de Sequência de Proteína , Temperatura , TransfecçãoRESUMO
The Ran GTPase system regulates the direction and timing of several cellular events, such as nuclear-cytosolic transport, centrosome formation, and nuclear envelope assembly in telophase. To gain insight into the Ran system's involvement in chromatin formation, we investigated gene silencing at the telomere in several mutants of the budding yeast Saccharomyces cerevisiae, which had defects in genes involved in the Ran system. A mutation of the RanGAP gene, rna1-1, caused reduced silencing at the telomere, and partial disruption of the nuclear Ran binding factor, yrb2-delta2, increased this silencing. The reduced telomere silencing in rna1-1 cells was suppressed by a high dosage of the SIR3 gene or the SIT4 gene. Furthermore, hyperphosphorylated Sir3 protein accumulated in the rna1-1 mutant. These results suggest that RanGAP is required for the heterochromatin structure at the telomere in budding yeast.
Assuntos
Proteínas Ativadoras de GTPase/genética , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Telômero/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Imunoprecipitação da Cromatina , Proteínas Ativadoras de GTPase/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismoRESUMO
RanGTPase is involved in many cellular processes. It functions in nuclear-cytosolic transport and centrosome formation. Ran also localizes to chromatin as RCC1 does, its guanine nucleotide exchange factor, but Ran's function on chromatin is not known. We found that gsp1, a temperature-sensitive mutant of GSP1, a Saccharomyces cerevisiae Ran homologue, suppressed the hydroxyurea (HU) and ultra violet (UV) sensitivities of the mec1 mutant. In UV-irradiated mec1 gsp1 cells, Rad53 was phosphorylated despite the lack of Mec1. This suppression depended on the TEL1 gene, given that the triple mutant, mec1 gsp1 tel1, was unable to grow. The gsp1 mutations also suppressed the HU sensitivity of the rad9 mutant in a Tel1-dependent manner, but not the HU sensitivity of the rad53 mutant. These results indicated that Rad53 was activated by the Tel1 pathway in mec1 gsp1 cells, suggesting that Gsp1 helps regulate the role switching the ATM family kinases Mec1 and Tel1.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Proliferação de Células , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Temperatura , Proteína ran de Ligação ao GTP/genéticaRESUMO
RCC1, a conserved chromosomal protein with a seven-bladed propeller is a GDP/GTP nucleotide exchange factor for RanGTPase that mediates various cellular events. We isolated 16 temperature-sensitive (ts) mutants of S. pombeRCC1-homolog, pim1+, by error-prone PCR. Five pim1(ts) mutants had a single mutation. The obtained pim1(ts) mutations and previously reported mutations were localized on similar sites in seven RCC1 repeats. Those mutations resulted in a reduced binding of Pim1 with Spi1. All pim1(ts) mutants showed a defect in nucleocytoplasmic protein transports, whereas the majority of them showed a normal mRNA export. In all pim1(ts) examined, chromosomal DNA replication was completed. However, mitotic spindle formation was abrogated, the septum was formed being uncoupled with nuclear division and abnormally widened, thus resulting in chromosomal DNA mis-segregation and the accumulation of enucleated cells. As a result, a defect of RanGEF/Pim1 abolished the orchestration of sequential mitotic events, spindle formation, septation and cytokinesis that are essential to produce two identical daughter cells.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Mitose/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Temperatura Alta , Humanos , Dados de Sequência Molecular , Mutação , Transporte Proteico , RNA Mensageiro/biossíntese , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de SequênciaRESUMO
Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re-replication. We show here that the N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S-G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S-G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re-replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeo Hidrolases/química , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/farmacologia , Células Cultivadas , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/genética , Ciclina A/metabolismo , Ciclina E/metabolismo , Dano ao DNA , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Geminina , Inativação Gênica , Células HeLa , Humanos , Rim/embriologia , Rim/metabolismo , Camundongos , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/farmacologia , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/genética , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Proteínas Ligases SKP Culina F-Box/genética , Ubiquitina/metabolismo , Raios UltravioletaRESUMO
In this article, we identify a cold-sensitive mutant of Xpo1p designated as xop1-2 (but will be referred to from here on as xpo1-ok) that is synthetically lethal with srm1-1, a Saccharomyces cerevisiae RCC1 homolog. xpo1-ok was a novel mutated allele with a single point mutation, T283P. Suppressors of xpo1-ok were isolated, and one of them was found to encode a novel nuclear envelope integral membrane protein designated as Brl1p (Brr6 like protein no. 1). Brl1p is homologous with Brr6p at the C-terminal domain, which is well conserved in the Brr6/Brl1 family. To characterize the function of Brl1p, a series of temperature-sensitive mutants of Brl1p were isolated. All of brl1 mutations were localized to the conserved C-terminal domain that is essential for a function of Brl1p. Some brl1 alleles showed defects in nuclear export of either mRNA or protein, and nuclear pore clustering, similar to brr6-1. The cellular localization of Brl1p is also similar to that of Brr6p. The genetic analysis suggested that Brl1p functionally interacts with Brr6p. An interaction of Brl1p with Brr6p was shown by the two-hybrid method. We hypothesize that Brl1p functions for nuclear export as a complex with Brr6p.
Assuntos
Transporte Ativo do Núcleo Celular , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/fisiologia , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Hibridização In Situ , Carioferinas/fisiologia , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Mutação , Sinais de Localização Nuclear , Proteínas Nucleares/fisiologia , Plasmídeos/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Temperatura , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismo , Proteína Exportina 1RESUMO
A Gtr1p GTPase, the GDP mutant of which suppresses both temperature-sensitive mutants of Saccharomyces cerevisiae RanGEF/Prp20p and RanGAP/Rna1p, was presently found to interact with Yrb2p, the S. cerevisiae homologue of mammalian Ran-binding protein 3. Gtr1p bound the Ran-binding domain of Yrb2p. In contrast, Gtr2p, a partner of Gtr1p, did not bind Yrb2p, although it bound Gtr1p. A triple mutant: yrb2delta gtr1delta gtr2delta was lethal, while a double mutant: gtr1delta gtr2delta survived well, indicating that Yrb2p protected cells from the killing effect of gtr1delta gtr2delta. Recombinant Gtr1p and Gtr2p were purified as a complex from Escherichia coli. The resulting Gtr1p-Gtr2p complex was comprised of an equal amount of Gtr1p and Gtr2p, which inhibited the Rna1p/Yrb2 dependent RanGAP activity. Thus, the Gtr1p-Gtr2p cycle was suggested to regulate the Ran cycle through Yrb2p.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proliferação de Células , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase , Substâncias Macromoleculares/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Nucleares/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/fisiologia , Proteína ran de Ligação ao GTP/químicaRESUMO
Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Animais , Western Blotting , Células COS , Ciclo Celular , Proteínas de Ciclo Celular/química , Cromatina/metabolismo , Ciclina A/metabolismo , Proteínas Fúngicas/metabolismo , Geminina , Células HeLa , Humanos , Immunoblotting , Microscopia de Fluorescência , Modelos Genéticos , Complexo de Reconhecimento de Origem , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fase S , Fatores de Tempo , Ubiquitina/metabolismoRESUMO
We investigated the function of DDX3Y, the Y chromosome AZFa region encoding a putative DEAD-box RNA helicase protein, the loss of which results in oligozoospermia or azoospermia in humans. The human DDX3Y amino acid sequence is similar to that of the X chromosome gene DDX3X (91.7% homology). Here we report that human Y- and X-encoded DEAD box RNA helicase proteins DDX3Y and DDX3X are interchangeable and have an essential function: both proteins rescued a temperature-sensitive mutant hamster cell line (tsET24) that was otherwise incapable of growth at a nonpermissive temperature. Mouse homologues Ddx3y and D1Pas1-PL10 also rescued the mutant cell line at a nonpermissive temperature. In situ hybridization revealed that Ddx3x mRNA was expressed in almost every cell in mouse testis, suggesting that Ddx3x is involved in spermatogenesis. A comparative study of DDX3X and DDX3Y was performed to determine the significance of DDX3Y for cell growth and spermatogenesis. Both DDX3X and DDX3Y promoter DNAs produced a similar degree of transcription in vivo, whereas deletion studies of the promoter DNAs indicated that these genes are differentially regulated. DDX3Y, similar to DDX3X, shuttles between the nucleus and cytoplasm in a crm1-dependent manner.
Assuntos
Cromossomos Humanos Y/genética , Mutação/genética , Proteínas/genética , Proteínas/metabolismo , RNA/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Linhagem Celular , Cricetinae , RNA Helicases DEAD-box , Feminino , Genes Reguladores/genética , Células HeLa , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Masculino , Camundongos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/genética , RNA Helicases/genética , RNA Helicases/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Homologia de Sequência do Ácido Nucleico , Espermatócitos/enzimologia , Espermatogênese/genética , Temperatura , Transcrição Gênica/genética , Proteína Exportina 1RESUMO
BACKGROUND: Both caffeine and the inactivation of RCC1, the guanine-nucleotide exchange factor (GEF) of Ran, induce premature chromatin condensation (PCC) in hamster BHK21 cells arrested in the S-phase, suggesting that RCC1 is a target for caffeine. RESULTS: Caffeine inhibited the Ran-GEF activity of RCC1 by preventing the binary complex formation of Ran-RCC1. Inhibition of the Ran-GEF activity of RCC1 by caffeine and its derivatives was correlated with their ability to induce PCC. Since caffeine is a derivative of xanthine, the bases and nucleosides were screened for their ability to inhibit RCC1. Adenine, adenosine, and all of the 2'-deoxynucleosides inhibited the Ran-GEF activity of RCC1; however, only adenine and 2'-deoxyadenosine (2'-dA) induced PCC. A factor(s) other than RCC1, should therefore be involved in PCC-induction. We found that both adenine and 2'-dA, but none of the other 2'-deoxynucleosides, inhibited the kinase activity of ATR, similar to that of caffeine. The ATR pathway was also abrogated by the inactivation of RCC1 in tsBN2 cells. CONCLUSION: The effect of caffeine on cell-cycle control mimics the biological effect of adenine and 2'-dA, both of which inhibit ATR. dATP, a final metabolite of adenine and 2'-dA, is suggested to inhibit ATR, resulting in PCC.
Assuntos
Adenina/farmacologia , Cafeína/farmacologia , Proteínas de Ciclo Celular , Desoxiadenosinas/farmacologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteína ran de Ligação ao GTP/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Estimulantes do Sistema Nervoso Central/farmacologia , Cromatina/fisiologia , Cricetinae , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Nucleotídeos de Guanina/farmacologia , Humanos , Mimetismo Molecular , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Fase S/fisiologia , Transdução de Sinais , TemperaturaRESUMO
Dsk2p from Saccharomyces cerevisiae belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus. We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome. Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits, rpn1 and pre2. K48-linked ubiquitin-dependent degradation was impaired by disruption of the DSK2 gene. These results indicate that Dsk2p is K48-linked polyubiquitin-binding protein and also interacts with the proteasome. We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.
Assuntos
Proteínas de Ciclo Celular , Cisteína Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Complexos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Sítios de Ligação , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Supressores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Temperatura , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinas/química , Ubiquitinas/genéticaRESUMO
RRAG A (Rag A)/Gtr1p is a member of the Ras-like small G protein family that genetically interacts with RCC1, a guanine nucleotide exchange factor for RanGTPase. RRAG A/Gtr1p forms a heterodimer with other G proteins, RRAG C and RRAG D/Gtr2p, in a nucleotide-independent manner. To further elucidate the function of RRAG A/Gtr1p, we isolated a protein that interacts with RRAG A. This protein is a novel nucleolar protein, Nop132. Nop132 is associated with the GTP form, but not the GDP form, of RRAG A, suggesting that RRAG A might regulate Nop132 function. Nop132 is also associated with RRAG C and RRAG D. The Nop132 amino acid sequence is similar to the Saccharomyces cerevisiae nucleolar Nop8p, which is associated with Gtr1p, Gtr2p, and Nip7p. Nop132 also interacts with human Nip7 and is colocalized with RRAG A, RRAG C, and Nip7. RNA interference knockdown of Nop132 inhibited cell growth of HeLa cells.