RESUMEN
In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway. One of these encodes the protein phosphatase 2A (PP2A) B'-regulatory subunit par1p. Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants. Our data suggest that PP2A-par1p acts as a negative regulator of SIN signalling.
Asunto(s)
División Celular/fisiología , Proteínas Fúngicas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Schizosaccharomyces/fisiología , Clonación Molecular , Colorantes Fluorescentes/metabolismo , Proteínas Fúngicas/genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteínas de Unión a Poli(A) , Proteína Fosfatasa 2 , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe , Transducción de Señal/fisiología , TemperaturaRESUMEN
BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic. RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis. CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Huso Acromático/metabolismo , Ciclo Celular/fisiología , Clonación Molecular/métodos , Genes Fúngicos/genética , Mutación/genética , Mutación/fisiología , Fosforilación , Proteínas/metabolismo , Schizosaccharomyces/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
In Saccharomyces cerevisiae, the phosphoprotein phosphatase Cdc14p plays a central role in exit from mitosis, by promoting B-type cyclin degradation and allowing accumulation of the cyclin-dependent kinase inhibitor Sic1p. Cdc14p is sequestered in the nucleolus during interphase, from where it is released at the end of mitosis, dependent upon mitotic exit network function. The CDC14 gene is essential and loss-of-function mutants arrest at the end of mitosis. We have identified a fission yeast orthologue of CDC14 through database searches. A Schizosaccharomyces pombe flp1 (cdc fourteen-like-phosphatase) null mutant is viable, divides at a reduced size and shows defects in septation. flp1p is not the essential effector of the S. pombe septation initiation network, but may potentiate signalling of the onset of septation. In contrast to S. cerevisiae Cdc14p, flp1p is not required for the accumulation or destruction of the B-type cyclin cdc13p, the cyclin-dependent kinase inhibitor rum1p, or for dephosphorylation of the APC/C specificity factor ste9p in G1. Like its budding yeast counterpart, flp1p is restricted to the nucleolus until mitosis, when it is dispersed through the nucleus. In contrast to S. cerevisiae Cdc14p, flp1p is also present on the mitotic spindle and contractile ring. The potential roles of flp1p in cell cycle control are discussed.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclina B/metabolismo , Proteínas Fúngicas/metabolismo , Mitosis/fisiología , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/fisiología , Nucléolo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Genes cdc/fisiología , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Mutagénesis/fisiología , Fosforilación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiologíaRESUMEN
We identified an Arabidopsis thaliana gene, AtMAP3Kepsilon1, and a Brassica napus cDNA, BnMAP3Kepsilon1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT-PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kepsilon1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Brassica/genética , Proteínas de Ciclo Celular/metabolismo , División Celular , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Brassica/enzimología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Genes de Plantas , Datos de Secuencia Molecular , Proteínas de Plantas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Homología de Secuencia de AminoácidoRESUMEN
The onset of septum formation in the fission yeast Schizosaccharomyces pombe is signaled via the spglp GTPase-switch, which is part of the septation initiation network. This is negatively regulated by the two-component GTPase-activating protein (GAP) comprised of the products of the cdc16 and byr4 genes. Loss-of-function mutations in either of these genes result in multiple rounds of septum formation without cell cleavage. In this work, we demonstrate that attenuation of the protein kinase cdc7p can rescue the lethality of a null allele of cdc16. This observation provides the basis for selection of chromosomal mutations and multicopy suppressors that attenuate the signaling of septation. Using this screen, mutations in all the previously described septation initiation network genes were obtained, with the exception of byr4, sid4 and plo1. We also demonstrate that increased expression of the dma1 gene can rescue the lethality of a null allele of cdc16. The implications for the regulation of septum formation in fission yeast are discussed.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Activadoras de GTPasa/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Alelos , Proteínas Bacterianas/genética , Ciclo Celular , Proteínas de Ciclo Celular/fisiología , División Celular , GTP Fosfohidrolasas/metabolismo , Eliminación de Gen , Mutación , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras/genética , Schizosaccharomyces/fisiologíaRESUMEN
In order to identify additional components important for cell division in the fission yeast Schizosaccharomyces pombe we have screened a bank of conditional cold-sensitive mutants for cytokinesis defects. One of these mutants showed a delay in cell cleavage, and strong genetic interactions with other genes implicated in medial ring formation. Cloning of the corresponding gene indicates that it encodes a protein with significant homology to the regulatory light chain of non-muscle myosins. We have named the gene rlc1 (regulatory light chain 1). The gene is not essential for division, but null mutants display a cell cleavage defect and form an aberrant F-actin ring. Two myosin-II heavy chains have been identified in fission yeast: Co-immunoprecipitation experiments indicate that rlc1p associates more strongly with myo3p than myo2p.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Cadenas Ligeras de Miosina/genética , Cadenas Ligeras de Miosina/metabolismo , Miosina Tipo II , Miosina Tipo V , Miosinas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Clonación Molecular , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Mutagénesis/fisiología , Fenotipo , Fosforilación , Unión Proteica/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SchizosaccharomycesRESUMEN
Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis, a phenomenon called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature and is suppressed by a single recessive mutant, mcs3-12. Mcs3 acts independently of the Wee1 kinase and Cdc25 phosphatase, two major regulators of Cdc2. We have isolated multicopy suppressors of the cell cycle arrest phenotype of mcs3-12 wee1-50 cdc25-22 cells, but did not identify the mcs3 gene itself. Instead several known mitotic regulators were isolated, including the Cdc25 phosphatase, Wis2 cyclophilin, Cek1 kinase, and an Hsp90 homologue, Swo1. We also isolated clones encoding non-functional, truncated forms of the Wee1 kinase and Dis2 type 1 phosphatase. In addition we identified a multicopy suppressor that encodes a structural homologue of the budding yeast SPO12 gene. We find that overexpression of fission yeast spo12 not only suppresses the phenotype of the mcs3-12 wee1-50 cdc25-22 strain, but also that of a win1-1 wee1-50 cdc25-22 strain at high temperature, indicating that the function of spo12 is not directly related to mcs3. We show that spo12 mRNA is periodically expressed during the fission yeast cell cycle, peaking at the G2/M transition coincidently with cdc15. Deletion of spo12, however, has no overt effect on either the mitotic or meiotic cell cycles, except when the function of the major B type cyclin, Cdc13, is compromised.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Fúngicas/genética , Genes Supresores , Proteína Quinasa 3 Activada por Mitógenos , Mitosis/genética , Proteínas Nucleares , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Alelos , Secuencia de Aminoácidos , Proteína Quinasa CDC2/metabolismo , Ciclina B/genética , Elementos Transponibles de ADN , Eliminación de Gen , Proteínas HSP90 de Choque Térmico/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Fenotipo , Fosfoproteínas Fosfatasas/genética , Plásmidos/metabolismo , Proteína Fosfatasa 1 , Proteínas Tirosina Quinasas/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Supresión Genética , Temperatura , Factores de TiempoRESUMEN
The Schizosaccharomyces pombe sep1 gene encodes a putative transcription factor that is required for cell separation. Among the genes required for septum formation and cytokinesis in fission yeast examined to date, the only one whose mRNA fluctuates significantly during the cell cycle is cdc15. In this study we have examined cdc15 mRNA levels in sep1 mutant and null backgrounds and have found that sep1p function is required for periodic accumulation of cdc15 mRNA. We have also localised sep1p and find that it is a nuclear protein, consistent with its proposed role as a transcription factor.
Asunto(s)
Proteínas de Ciclo Celular/genética , Exorribonucleasas/metabolismo , Proteínas de Unión al GTP/genética , Regulación Fúngica de la Expresión Génica , Proteínas Nucleares/metabolismo , Periodicidad , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Ciclo Celular , Núcleo Celular/química , Clonación Molecular , Exorribonucleasas/genética , Eliminación de Gen , Genes Esenciales , Proteínas Nucleares/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimologíaRESUMEN
Analysis of Schizosaccharomyces pombe mutants that are defective in septum formation and cytokinesis has identified the product of the cdc15 gene as a key element in formation of a division septum. S. pombe cells lacking cdc15p function cannot assemble a functional medial ring, and do not make a division septum. cdc15 mRNA accumulates periodically during the cell cycle, peaking after entry into mitosis, and increased expression of the gene in G2-arrested cells can promote F-actin ring formation. Here, we have investigated the effects of mutations that block cell division upon the expression of cdc15 in synchronised cell populations, and analysed the expression of cdc15 when septum formation is induced by ectopic activation of the septation signalling network. We concluded the following: (i) the septation signalling network genes are not required for periodic accumulation of cdc15 mRNA; (ii) induction of septum formation in G2-arrested cells by activation of the septation signalling network does not result in accumulation of cdc15 mRNA, which is therefore not a prerequisite for septum formation; (iii) failure to turn off septum formation at the end of mitosis results in continued expression of cdc15; and (iv) periodic accumulation of cdc15 mRNA is mediated by a 97 bp region 5' to the mRNA start site.
Asunto(s)
Proteínas de Ciclo Celular/genética , Ciclo Celular , Proteínas de Unión al GTP/genética , ARN de Hongos/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , División Celular , Codón Iniciador/genética , Fase G2 , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Genes cdc/fisiología , Mutación/genética , Estabilidad del ARN , ARN de Hongos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
We have isolated the Schizosaccharomyces pombe orthologue of the Saccharomyces cerevisiae MOB1 gene in a screen designed to enrich for septation mutants. The gene is essential, and cells lacking it display a phenotype typical of septation signalling network mutants. mob1p is located on both spindle pole bodies throughout mitosis. In addition it is also co-localised with the medial ring later in mitosis, and flanks the septum as the medial ring contracts. We also demonstrate that mob1p can be precipitated from cells in a complex with the septation regulating kinase sid2p.
Asunto(s)
Proteínas de Ciclo Celular/genética , Membrana Celular/fisiología , Proteínas de Unión al ADN , Mitosis/fisiología , Fosfoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces/citología , Saccharomyces/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Transducción de Señal/fisiología , Huso Acromático/fisiología , Alelos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/análisis , Frío , Regulación Fúngica de la Expresión Génica/fisiología , Mutación , Fosfoproteínas/análisis , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismoRESUMEN
The past year has seen significant advances in our understanding of how the events which occur at the end of mitosis, such as cytokinesis and the inactivation of mitotic cyclin dependent kinases are triggered, and also how they are prevented from occurring prematurely or inappropriately. This control is achieved through a combination of temporally ordered proteolytic events and changes in the subcellular localisation of proteins. These studies have also revealed that the nucleolus and spindle pole bodies play a key role in this regulation.
Asunto(s)
Genes cdc/fisiología , Mitosis/genética , Complejos de Ubiquitina-Proteína Ligasa , Ciclosoma-Complejo Promotor de la Anafase , Animales , Humanos , Ligasas/metabolismo , Mitosis/fisiología , Ubiquitina-Proteína Ligasas , LevadurasRESUMEN
The COP9/signalosome complex is conserved from plant to mammalian cells. In Arabidopsis, it regulates the nuclear abundance of COP1, a transcriptional repressor of photomorphogenic development [1] [2]. All COP (constitutive photomorphogenesis) mutants inappropriately express genes that are normally repressed in the dark. Eight subunits (Sgn1-Sgn8) of the homologous mammalian complex have been purified [3] [4]. Several of these have been previously identified through genetic or protein interaction screens. No coherent model for COP9/signalosome function has yet emerged, but a relationship with cell-cycle progression by transcriptional regulation, protein localisation or protein stability is possible. Interestingly, the COP9/signalosome subunits possess domain homology to subunits of the proteasome regulatory lid complex [5] [6]. Database searches indicate that only Sgn5/JAB1 is present in Saccharomyces cerevisiae, precluding genetic analysis of the complex in cell-cycle regulation. Here we identify a subunit of the signalosome in the fission yeast Schizosaccharomyces pombe through an analysis of the DNA-integrity checkpoint. We provide evidence for the conservation of the COP9/signalosome complex in fission yeast and demonstrate that it functions during S-phase progression.
Asunto(s)
Proteínas de Plantas/análisis , Proteínas de Plantas/fisiología , Proteínas , Fase S/fisiología , Schizosaccharomyces/química , Schizosaccharomyces/citología , Transducción de Señal , Complejo del Señalosoma COP9 , División Celular , Núcleo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Secuencia Conservada , ADN de Hongos/análisis , Genes cdc , Humanos , Immunoblotting , Microscopía Fluorescente , Complejos Multiproteicos , Mutagénesis , Péptido Hidrolasas , Plantas , Proteínas Quinasas/genética , Schizosaccharomyces/genéticaRESUMEN
The fission yeast gene cps1, which encodes the catalytic subunit of beta-glucan synthase, was isolated in a screen for mutants that show an increase in ploidy at the restrictive temperature. cps1 mutants display defects in both polarity and septation at the permissive temperature, and become swollen and multinucleate at the restrictive temperature. Analysis of the interaction of cps1 with other mutations suggests the existence of a septation checkpoint, which requires the activity of the protein kinase weel for function.
Asunto(s)
Proteínas de Ciclo Celular , Pared Celular/metabolismo , Genes Fúngicos , Glucosiltransferasas/genética , Proteínas de la Membrana/genética , Proteínas Nucleares , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/citología , Actinas/aislamiento & purificación , División Celular , ADN de Hongos/aislamiento & purificación , Citometría de Flujo , Glucosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Ploidias , Proteínas Tirosina Quinasas/metabolismo , Schizosaccharomyces/genética , Temperatura , Tubulina (Proteína)/aislamiento & purificaciónRESUMEN
The fission yeast Schizosaccharomyces pombe provides a simple eukaryotic model for the study of cytokinesis. S. pombe cells are rod-shaped, grow mainly by elongation at their tips, and divide by binary fission after forming a centrally placed division septum. Analysis of mutants has begun to shed light upon how septum formation and cytokinesis are regulated both spatially and temporally. Some of the proteins involved in these events have been functionally conserved throughout eukaryotic evolution, suggesting that aspects of this control will be common to all eukaryotic cells.
Asunto(s)
División Celular/genética , Schizosaccharomyces/genética , Regulación de la Expresión Génica , Schizosaccharomyces/citología , Factores de TiempoRESUMEN
Infection by some types of human papillomavirus (HPV) is associated with the development of cervical cancer. Analysis of viral DNA from cervical tumours shows that the E2 gene is frequently disrupted during integration into the host cell's DNA. It has therefore been suggested that loss of E2p is an important step in malignant transformation. Expression of E2p in the fission yeast Schizosaccharomyces pombe retards the G2-M transition, by delaying activation of Cdc2p kinase. In contrast, S phase progression, and commitment to cell division in late G1 are not affected. The delay is independent of the transcriptional trans-activation function of E2p, and does not result from E2p DNA binding mimicking DNA damage. Increased expression of E2p also delays mitotic initiation in mammalian cells. S. pombe may thus provide a simple model for the analysis of E2p function.
Asunto(s)
Proteínas de Unión al ADN , Inhibidores de Crecimiento/genética , Mitosis/genética , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Papillomaviridae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/virología , ras-GRF1 , Adenosina Trifosfatasas/fisiología , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular Transformada , ADN Helicasas/fisiología , Proteínas Fúngicas/genética , Fase G1/genética , Fase G2/genética , Vectores Genéticos , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/fisiología , Humanos , Riñón/citología , Mutación , Proteínas Oncogénicas Virales/fisiología , Papillomaviridae/fisiología , Ratas , Fase S/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/citología , Transcripción GenéticaRESUMEN
Schizosaccharomyces pombe cdc16p is required to limit the cell to forming a single division septum per cell cycle; the heat-sensitive loss-of-function mutant cdc16-116 completes mitosis, and then undergoes multiple rounds of septum formation without cell cleavage. cdc16p is a homologue of Saccharomyces cerevisiae BUB2p, and has also been implicated in the spindle assembly checkpoint function in S. pombe. To identify other proteins involved in regulating septum formation, we have screened for multicopy suppressors of the cdc16-116 mutation. In this paper, we describe one of these suppressors, zfs1. The null allele (zfs1-D1) is viable. However, at low temperatures it divides at a reduced size, while at higher temperatures, it partially suppresses heat sensitive mutants in genes signalling the onset of septum formation. Zfs1-D1 cells show an increased rate of chromosome loss during exponential growth. Moreover, if assembly of the spindle is prevented, zfs1-D1 cells do not arrest normally, but the activity of cdc2p kinase decays, and cells form a division septum without completing a normal mitosis. We conclude that zfs1 function is required to prevent septum formation and exit from mitosis if the mitotic spindle is not assembled. The suppression of cdc16-116 by zfs1 is independent of dma1 function and the spindle assembly checkpoint genes mad2 and mph1. The genetic interactions of zfs1 with genes regulating septum formation suggest that it may be a modulator of the signal transduction network controlling the onset of septum formation and exit from mitosis.
Asunto(s)
Proteínas Portadoras , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Actinas/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , División Celular/genética , Eliminación de Gen , Expresión Génica , Genes Supresores , Proteínas Mad2 , Mitosis/genética , Mutación , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/metabolismo , Transducción de SeñalRESUMEN
In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is induced by a signal transduction network involving several protein kinases and a GTPase switch. One of the roles of the spg1p GTPase is to localise the cdc7p protein kinase to the poles of the mitotic spindle, from where the onset of septation is thought to be signalled at the end of mitosis. Immunofluorescence studies have shown that cdc7p is located on both spindle pole bodies early in mitosis, but only on one during the later stages of anaphase. This is mediated by inactivation of spg1p on one pole before the other. The GAP for spg1p is a complex of two proteins, cdc16p and byr4p. Localisation of cdc16p and byr4p by indirect immunofluorescence during the mitotic cell cycle showed that both proteins are present on the spindle pole body in interphase cells. During mitosis, byr4p is seen first on both poles of the spindle, then on only one. This occurs prior to cdc7p becoming asymmetric. In contrast, the signal due to cdc16p decreases to a low level during early mitosis, before being seen strongly on the same pole as byr4p. Double staining indicates that this is the opposite pole to that which retains cdc7p in late anaphase. Examination of the effect of inactivating cdc16p at various stages of the cell cycle suggests that cdc16p, together with cdc2p plays a role in restraining septum formation during interphase. The asymmetric inactivation of spg1p is mediated by recruitment of the cdc16p-byr4p GAP to one of the poles of the spindle before the other, and the asymmetry of the spindle pole bodies may be established early during mitosis. Moreover, the spindle pole bodies appear to be non-equivalent even after division has been completed.
Asunto(s)
Mitosis/fisiología , Proteínas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Huso Acromático/metabolismo , Secuencia de Bases , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente Indirecta , Fase G2/fisiología , Proteínas Activadoras de GTPasa , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/fisiologíaRESUMEN
Schizosaccharomyces pombe divides by means of a centrally placed division septum. The initiation of septation must be tightly coordinated with events in mitosis, as premature formation of the septum can lethally cut the undivided nucleus. The Spg1p GTPase and the Cdc7p kinase, with which it interacts, play a central role in signaling the initiation of septum formation. Loss-of-function mutations in either gene prevent septation, whereas inappropriate activation of Spg1p can induce septum formation from G1 or G2 interphase cells. Increased expression of either gene leads to multiple rounds of septation without cell cleavage, emphasizing the need for precise cell cycle regulation of their activity. To understand the mechanisms underlying this regulation, we have investigated whether these key initiators of septum formation are controlled by changes in their activity and/or location during mitosis and cytokinesis. We demonstrate that Spg1p localizes to the spindle pole body in interphase and to both spindle poles during mitosis. In contrast, Cdc7p shows no discrete localization during interphase, but early in mitosis it associates with both spindle pole bodies and, as the spindle extends, is seen on only one pole of the spindle during anaphase B. Spg1p activity is required for localization of Cdc7p in vivo but not for its kinase activity in vitro. Staining with an antiserum that recognizes preferentially GDP-Spg1p indicates that activated GTP-Spg1p predominates during mitosis when Cdc7p is associated with the spindle pole body. Furthermore, staining with this antibody shows that asymmetric distribution of Cdc7p may be mediated by inactivation of Spg1p on one spindle pole. Deregulated septation in mutant cells correlates with segregation of Cdc7p to both spindle poles.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Huso Acromático/metabolismo , Guanosina Trifosfato/metabolismo , MitosisRESUMEN
In the fission yeast Schizosaccharomyces pombe, the cdc11 gene is required for the initiation of septum formation at the end of mitosis. The sce3 gene was cloned as a multi-copy suppressor of the heat-sensitive mutant cdc11-136. When over-expressed, it rescues all mutants of cdc11 and also a heat-sensitive allele of cdc14, but not the cdc14 null mutant. Deletion shows that sce3 is not essential for cell proliferation. It encodes a putative RNA-binding protein which shows homology to human eIF4B. Immunolocalisation indicates that Sce3p is located predominantly in the cytoplasm. Elevated expression of sce3 increases the steady-state level of cdc14 mRNA. Possible mechanisms of its action are discussed.