RESUMO
The nucleolus, for decades considered a ribosome factory and site for ribosomal RNA synthesis and processing, has recently acquired new fame. Analyses of proteins important for cell-cycle regulation have shown that this organelle is used to sequester proteins, thereby inhibiting their activity.
Assuntos
Ciclo Celular/fisiologia , Nucléolo Celular/metabolismo , Proteínas Nucleares , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Humanos , Meiose/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Proteolysis mediated by the anaphase-promoting complex (APC) triggers chromosome segregation and exit from mitosis, yet its regulation is poorly understood. The conserved Cdc20 and Cdh1 proteins were identified as limiting, substrate-specific activators of APC-dependent proteolysis. CDC20 was required for the degradation of the APC substrate Pds1 but not for that of other APC substrates, such as Clb2 and Ase1. Conversely, cdh1Delta mutants were impaired in the degradation of Ase1 and Clb2 but not in that of Pds1. Overexpression of either CDC20 or CDH1 was sufficient to induce APC-dependent proteolysis of the appropriate target in stages of the cell cycle in which substrates are normally stable.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina B , Proteínas Fúngicas/metabolismo , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Ciclinas/metabolismo , Proteínas Fúngicas/genética , Fase G1 , Mitose , Mutação , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Fase S , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Securina , Especificidade por Substrato , Ubiquitina-Proteína LigasesRESUMO
BACKGROUND: In eukaryotic cells, a specialized proteolysis machinery that targets proteins containing destruction-box sequences for degradation and that uses a ubiquitin ligase known as the anaphase-promoting complex/cyclosome (APC) plays a key role in the regulation of mitosis. APC-dependent proteolysis triggers the separation of sister chromatids at the metaphase-anaphase transition and the destruction of mitotic cyclins at the end of mitosis. Recently, two highly conserved WD40-repeat proteins, Cdc20 and Cdh1/Hct1, have been identified as substrate-specific regulators for APC-dependent proteolysis in the budding yeast Saccharomyces cerevisiae. Here, we have investigated the cell cycle regulation of Cdc20 and Cdh1/Hct1. RESULTS: Whereas the levels CDH1/HCT1 RNA and Cdh1/Hct1 protein are constant throughout the cell cycle, CDC20 RNA and Cdc20 protein are present only during late S phase and mitosis and Cdc20 protein is unstable throughout the entire cell cycle. The instability of Cdc20 depends on CDC23 and CDC27, which encode components of the APC. During the G1 phase, a destruction box within Cdc20 mediates its instability, but during S phase and mitosis, although Cdc20 destruction is still dependent on CDC23 and CDC27, it does not depend on the Cdc20 destruction box. CONCLUSIONS: There are remarkable differences in the regulation of Cdc20 and Cdh1/Hct1. Furthermore, the APC activator Cdc20 is itself a substrate of the Cdc27 have a role in the degradation of Cdc20 during S Phase and early mitosis that is not mediated by its destruction box.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclina B , Proteínas Fúngicas/fisiologia , Ligases/fisiologia , Mitose/fisiologia , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae , Complexos Ubiquitina-Proteína Ligase , Ciclossomo-Complexo Promotor de Anáfase , Antígenos CD , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase , Caderinas , Desidrogenases de Carboidrato/metabolismo , Proteínas Cdc20 , Proteínas Cdh1 , Ciclinas/genética , Proteínas Fúngicas/metabolismo , Fase G1/fisiologia , RNA Fúngico/metabolismo , Saccharomyces cerevisiae , Transcrição Gênica/genética , Ubiquitina-Proteína LigasesRESUMO
In budding yeast, the release of the protein phosphatase Cdc14 from its inhibitor Cfi1/Net1 in the nucleolus during anaphase triggers the inactivation of Clb CDKs that leads to exit from mitosis. The mitotic exit pathway controls the association between Cdc14 and Cfi1/Net1. It is comprised of the RAS-like GTP binding protein Tem1, the exchange factor Lte1, the GTPase activating protein complex Bub2-Bfa1/Byr4, and several protein kinases including Cdc15 and Dbf2. Here we investigate the regulation of the protein kinases Dbf2 and Cdc15. We find that Cdc15 is recruited to both spindle pole bodies (SPBs) during anaphase. This recruitment depends on TEM1 but not DBF2 or CDC14 and is inhibited by BUB2. Dbf2 also localizes to SPBs during anaphase, which coincides with activation of Dbf2 kinase activity. Both events depend on the mitotic exit pathway components TEM1 and CDC15. In cells lacking BUB2, Dbf2 localized to SPBs in cell cycle stages other than anaphase and telophase and Dbf2 kinase was prematurely active during metaphase. Our results suggest an order of function of mitotic exit pathway components with respect to SPB localization of Cdc15 and Dbf2 and activation of Dbf2 kinase. BUB2 negatively regulates all 3 events. Loading of Cdc15 on SPBs depends on TEM1, whereas loading of Dbf2 on SPBs and activation of Dbf2 kinase depend on TEM1 and CDC15.
Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/efeitos dos fármacos , Ativação Enzimática/fisiologia , Proteínas Fúngicas/farmacologia , Proteínas de Ligação ao GTP/efeitos dos fármacos , Mitose/fisiologia , Proteínas Serina-Treonina Quinases , Saccharomycetales/metabolismo , Fuso Acromático/ultraestruturaAssuntos
Hepatopatias , Porfirias , Dermatopatias , Protocolos Clínicos , Humanos , Hepatopatias/classificação , Hepatopatias/diagnóstico , Hepatopatias/enzimologia , Porfirias/classificação , Porfirias/diagnóstico , Porfirias/enzimologia , Dermatopatias/classificação , Dermatopatias/diagnóstico , Dermatopatias/enzimologiaRESUMO
Exit from mitosis must not occur prior to partitioning of chromosomes between daughter cells. We find that the GTP binding protein Tem1, a regulator of mitotic exit, is present on the spindle pole body that migrates into the bud during S phase and mitosis. Tem1's exchange factor, Lte1, localizes to the bud. Thus, Tem1 and Lte1 are present in the same cellular compartment (the bud) only after the nucleus enters the bud during nuclear division. We also find that the presence of Tem1 and Lte1 in the bud is required for mitotic exit. Our results suggest that the spatial segregation of Tem1 and Lte1 ensures that exit from mitosis only occurs after the genetic material is partitioned between mother and daughter cell.
Assuntos
Núcleo Celular/fisiologia , Proteínas Fúngicas/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Mitose/fisiologia , Proteínas Monoméricas de Ligação ao GTP/fisiologia , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiae , Fuso Acromático/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Fase G1 , Proteínas de Ligação ao GTP/metabolismo , Fuso Acromático/metabolismoRESUMO
In eukaryotes, the activation of mitotic cyclin-dependent kinases (CDKs) induces mitosis, and their inactivation causes cells to leave mitosis. In budding yeast, two redundant mechanisms induce the inactivation of mitotic CDKs. In one mechanism, a specialized ubiquitin-dependent proteolytic system (called the APC-dependent proteolysis machinery) degrades the mitotic (Clb) cyclin subunit. In the other, the kinase-inhibitor Sic1 binds to mitotic CDKs and inhibits their kinase activity. The highly conserved protein phosphatase Cdc14 promotes both Clb degradation and Sic1 accumulation. Cdc14 promotes SIC1 transcription and the stabilization of Sic1 protein by dephosphorylating Sicl and its transcription factor Swi5. Cdc14 activates the degradation of Clb cyclins by dephosphorylating the APC-specificity factor Cdh1. So how is Cdc14 regulated? Here we show that Cdc14 is sequestered in the nucleolus for most of the cell cycle. During nuclear division, Cdc14 is released from the nucleolus, allowing it to reach its targets. A highly conserved signalling cascade, critical for the exit from mitosis, is required for this movement of Cdc14 during anaphase. Furthermore, we have identified a negative regulator of Cdc14, Cfi1, that anchors Cdc14 in the nucleolus.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Nucléolo Celular/fisiologia , Mitose/fisiologia , Proteínas Nucleares/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Nucléolo Celular/enzimologia , Cromátides , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , LevedurasRESUMO
Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs) by an unknown mechanism. We show that the Cdc14 phosphatase triggers mitotic exit by three parallel mechanisms, each of which inhibits Cdk activity. Cdc14 dephosphorylates Sic1, a Cdk inhibitor, and Swi5, a transcription factor for SIC1, and induces degradation of mitotic cyclins, likely by dephosphorylating the activator of mitotic cyclin degradation, Cdh1/Hct1. Feedback between these pathways may lead to precipitous collapse of mitotic CDK activity and help coordinate exit from mitosis.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ciclina B , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA , Mitose/fisiologia , Proteínas Tirosina Fosfatases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Complexos Ubiquitina-Proteína Ligase , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdh1 , Proteínas de Ciclo Celular/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina , Ciclinas/metabolismo , Replicação do DNA/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas de Ligação ao GTP , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Ligases/metabolismo , Ligases/fisiologia , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Fosforilação , Proteínas Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ubiquitina-Proteína LigasesRESUMO
Cell cycle progression of somatic cells depends on net mass accumulation. In Saccharomyces cerevisiae the cAMP-dependent kinases (PKAs) promote cytoplasmic growth and modulate the growth-regulated mechanism triggering the begin of DNA synthesis. By altering the cAMP signal in budding yeast cells we show here that mitotic events can also depend on growth. In fact, the hyperactivation of PKAs permanently inhibited both anaphase and exit from mitosis when cell growth was repressed. In S. cerevisiae the anaphase promoting complex (APC) triggers entry into anaphase by mediating the degradation of Pds1p. The cAMP pathway activation was lethal together with a partial impairment of the Cdc16p APC subunit, causing a preanaphase arrest, and conversely low PKA activity suppressed the lethality of cdc16-1 cells. Deregulated PKAs partially prevented the decrease of Pds1p intracellular levels concomitantly with the anaphase inhibition, and the PKA-dependent preanaphase arrest could be suppressed in pds1(-) cells. Thus, the cAMP pathway and APC functionally interact in S. cerevisiae and Pds1p is required for the cAMP-mediated inhibition of chromosome separation. Exit from mitosis requires APC, Cdc15p, and the polo-like Cdc5p kinase. PKA hyperactivation and a cdc15 mutation were synthetically lethal and brought to a telophase arrest. Finally, a low cAMP signal allowed cell division at a small cell size and suppressed the lethality of cdc15-2 or cdc5-1 cells. We propose that mitosis progression and the M/G1 phase transition specifically depend on cell growth through a mechanism modulated by PKAs and interacting with the APC/CDC15/CDC5 mitotic system. A possible functional antagonism between PKAs and the mitosis promoting factor is also discussed.