RESUMEN

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1+ and wis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1+ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.

Asunto(s)

Genes Fúngicos , Quinasas Quinasa Quinasa PAM , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Ciclo Celular/genética , Mapeo Cromosómico , Activación Enzimática , Quinasas de Proteína Quinasa Activadas por Mitógenos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Concentración Osmolar , Fosforilación , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/química , Mapeo Restrictivo , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

The Schizosaccharomyces pombe wis1(+) gene is essential for cell survival under stress conditions. The MAPKK homologue Wis1 is required for activation of the MAPK homologue Spc1, and integrity of the Wis1-Spc1 pathway is required for survival in extreme conditions of heat, osmolarity, oxidation or limited nutrition. We show here that Wis4, a protein kinase of a new MAPKKK class, phosphorylates Wis1 in vitro and activates it in vivo. Win1 is also required for full activation of Wis1, and Win1 rather than Wis4 mediates the osmotic stress signal. Surprisingly, the pathway can still be activated by heat or oxidative stress independently of the phosphorylation of two conserved Wis1 residues. Evidence is presented that the Pyp1 protein tyrosine phosphatase, which dephosphorylates Spc1, is central to this alternative activation mechanism.

Asunto(s)

Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Regulación Fúngica de la Expresión Génica , Quinasas Quinasa Quinasa PAM , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Ciclo Celular/genética , Secuencia Conservada , Activación Enzimática , Respuesta al Choque Térmico , Datos de Secuencia Molecular , Presión Osmótica , Estrés Oxidativo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/enzimología , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

The 20S cyclosome complex (also known as the anaphase-promoting complex) has ubiquitin ligase activity and is required for mitotic cyclin destruction and sister chromatid separation. The formation and activation of the 20S cyclosome complex is regulated by an unknown mechanism. Here we show that Cut4 (ref. 6) is an essential component of the cyclosome in fission yeast. Cut4 shares sequence similarity with BimE, a protein that regulates mitosis in Aspergillus nidulans. Mutations in cut4 result in hypersensitivity to cyclic AMP and to stress-inducing heavy metals, inhibition of the onset of anaphase, disruption of the 20S complex, and inhibition of mitotic cyclin ubiquitination. These phenotypes are fully suppressed by cAMP phosphodiesterase and the protein kinase A (PKA) regulatory subunit and weakly suppressed by Sti1 (an activator of the Hsp70 and Hsp90 chaperones). Suppression correlates with the amount of 20S complex, indicating that cyclosome formation and activation is inhibited by the cAMP/PKA pathway.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Ligasas/metabolismo , Schizosaccharomyces/metabolismo , Anafase/efectos de los fármacos , Anafase/genética , Clonación Molecular , Ciclinas/metabolismo , Genes Fúngicos , Metales Pesados/farmacología , Mutación , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética

RESUMEN

Fission yeast cells either remain in the mitotic cell cycle or exit to meiotic sporulation from an uncommitted G1 state dependent on the presence or absence of nitrogen source in the medium (Nurse and Bissett, 1981). We examined how heterothallic haploid cells, which cannot sporulate, behave under nitrogen-starvation for longer than 25 days at 26 degrees C. These cells were shown to enter a stable state (designated the dormant G0) with nearly full viability. Maintaining the dormant cells required glucose, suggesting that the cells remained metabolically active although cell division had ceased. They differed dramatically from mitotic and uncommitted G1 cells in heat resistance, and also in cytoplasmic and nuclear morphologies. After nitrogen replenishment, the initial responses of dormant G0 cells were investigated. The kinetics for reentry into the proliferative state were delayed considerably, and the changes in cell shape were enhanced particularly for those recovering from extended nitrogen starvation. A part of the delay could be accounted for by the duration of nuclear decondensation and cell elongation for the first cell division.

Asunto(s)

Nitrógeno/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , División Celular , Núcleo Celular/ultraestructura , Cromatina/ultraestructura , ADN de Hongos/biosíntesis , Fase G1 , Cinética , Microscopía Electrónica , Fase de Descanso del Ciclo Celular , Fase S , Schizosaccharomyces/ultraestructura

RESUMEN

A novel anaphase block phenotype was found in fission yeast temperature-sensitive cut9 mutants. Cells enter mitosis with chromosome condensation and short spindle formation, then block anaphase, but continue to progress into postanaphase events such as degradation of the spindle, reformation of the postanaphase cytoplasmic microtubule arrays, septation, and cytokinesis. The cut9 mutants are defective in the onset of anaphase and possibly in the restraint of postanaphase events until the completion of anaphase. The cut9+ gene encodes a 78-kD protein containing the 10 34-amino acid repeats, tetratricopeptide repeats (TPR), and similar to budding yeast Cdc16. It is essential for viability, and the mutation sites reside in the TPR. The three genes, namely, nuc2+, scn1+, and scn2+, genetically interact with cut9+. The nuc2+ and cut9+ genes share an essential function to initiate anaphase. The cold-sensitive scn1 and scn2 mutations, defective in late anaphase, can suppress the ts phenotype of cut9.

Asunto(s)

Anafase/fisiología , Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Secuencia de Aminoácidos , Anafase/genética , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase , Secuencia de Bases , Genes Letales , Prueba de Complementación Genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Fenotipo , Secuencias Repetitivas de Ácidos Nucleicos , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Huso Acromático/fisiología , Relación Estructura-Actividad , Supresión Genética

RESUMEN

The fission yeast Schizosaccharomyces pombe [corrected] temperature sensitivity cut8-563 mutation causes chromosome overcondensation and short spindle formation in the absence of sister chromatid separation. The cut8-563 mutation allows cytokinesis before the completion of anaphase, thus producing cells with a cut phenotype. The cut8+ gene product may be required for normal progression of anaphase. Diploidization occurs at the restrictive temperature, and 60 to 70% of the cells surviving after two generations are diploid. These phenotypes are reminiscent of those of budding yeast (Saccharomyces cerevisiae) ctf13 and ctf14 (ndc10) mutations. The cut8+ gene, isolated by complementation of the mutant, predicts a 262-amino-acid protein; the amino and carboxy domains are hydrophilic, while the central domain contains several hydrophobic stretches. It has a weak overall similarity to the budding yeast DBF8 gene product. DBF8 is an essential gene whose mutations result in delay in mitotic progression and chromosome instability. Anti-cut8 antibodies detect a 33-kDa polypeptide. Two multicopy suppressor genes for cut8-563 are identified. They are the cut1+ gene essential for nuclear division, and a new gene (designated cek1+) which encodes a novel protein kinase. The cek1+ gene product is unusually large (1,309 amino acids) and has a 112-amino-acid additional sequence in the kinase domain. The cek1+ gene is not an essential gene. Protein phosphorylation by cek1 may facilitate the progression of anaphase through direct or indirect interaction with the cut8 protein.

Asunto(s)

Anafase , Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Quinasas/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , División Celular , Clonación Molecular , ADN de Hongos/genética , Genes Supresores , Datos de Secuencia Molecular , Peso Molecular , No Disyunción Genética , Oligodesoxirribonucleótidos/química , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Huso Acromático/ultraestructura

RESUMEN

Fission yeast cold-sensitive mutants nda1-376 and nda4-108 display a cell cycle block phenotype at the restrictive temperature (cell elongation with the single nucleus) accompanied by an alteration in the nuclear chromatin region. DNA content analysis shows that the onset of DNA synthesis is blocked or greatly delayed in both mutant cells, the block being reversible in nda4-108. Upon release to the permissive temperature, nda4-108 cells resumed replicating DNA, followed by mitosis and cytokinesis. The nda4 phenotype was partly rescued by the addition of Ca2+ to the medium; Ca2+ plays a positive role in the nda4+ function. The predicted protein sequences of nda1+ and nda4+ isolated by complementation are similar to each other and also, respectively, to those of the budding yeast, MCM2 and CDC46, both of which are members of the gene family required for the initiation of DNA replication. The central domains of these proteins are conserved, whereas the NH2- and COOH- domains are distinct. Results of the disruption of the nda1+ and nda4+ genes demonstrates that they are essential for viability.

Asunto(s)

Calcio/farmacología , Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Antígenos CD/genética , Secuencia de Bases , División Celular/genética , Cromatina/fisiología , Mapeo Cromosómico , Cromosomas Fúngicos , Clonación Molecular , ADN de Hongos/biosíntesis , ADN de Hongos/aislamiento & purificación , Proteínas de Unión al ADN , Proteínas Fúngicas/química , Prueba de Complementación Genética , Proteína Cofactora de Membrana , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Mutación , Fase S , Schizosaccharomyces/citología , Schizosaccharomyces/crecimiento & desarrollo , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Temperatura , Transformación Genética

RESUMEN

Fission yeast cut mutants cause cytokinesis in the absence of normal nuclear division. These mutants show abnormal uncoupled mitosis and are known to be the result of mutations in the genes encoding DNA topoisomerase II, proteins related to spindle pole duplication, and a kinesin-related mitotic motor. We have screened 717 temperature-sensitive (ts) mutants by individually observing their cytological phenotypes at the restrictive temperature, and have newly isolated 25 cut mutants. Genetic analyses indicate that 14 of them fall into five previously identified loci, namely, top2, cut1, cut5, cut7 and cut9, whereas nine have been mapped onto seven new loci, designated cut13 to cut19. The cytological phenotypes of the newly identified cut mutants can be classified into three groups. One group consists of mutants in which a portion of the nuclear chromatin is stretched by the elongated spindle but the entire nucleus is not separated, reminiscent of, but not identical to, the phenotypes of top2 and cut1; mutants cut14-208, cut15-85, cut16-267 and cut17-275 display such a phenotype. Another group exhibits non-disjunctioned and condensed chromosomes in the presence of the spindle; cut13-131 belongs to this group. The cut19-708 mutant has also been found to have condensed chromosomes. The remaining group has a mixed phenotype of the above two groups; namely, stretched chromatin and condensed chromosomes; cut18-447 exhibits such a phenotype. The isolation and characterization of the mutated genes will be the subjects of future investigations.

Asunto(s)

ADN-Topoisomerasas de Tipo II/genética , Mitosis/genética , Schizosaccharomyces/genética , División Celular/genética , Cromosomas , Fenotipo , Huso Acromático

RESUMEN

Mutations in the fission yeast cut1+, cut2+, and cut10+ genes uncouple normally coordinated mitotic events and deregulate, rather than arrest, mitosis. DNA synthesis continues, making polyploid nuclei with several spindles. Multiple, aberrant spindle pole bodies (SPBs) are produced in cut1 mutant cells. The cut1+ and cut2+ genes are cloned by transformation. High gene dosage of cut1+ also complements cut2 and cut10 mutants. The cut2+ gene, however, complements only cut2. The 210 kd cut1+ gene product contains putative ATP binding and helical coil regions followed by a COOH-terminal domain homologous to the S. cerevisiae gene ESP1. Mutations in the ESP1 gene also result in many SPBs. The cut1+ product is shown by anti-cut1 antibody to be a rare component of the insoluble nuclear fraction. It may play a key role in coupling chromosome disjunction with other cell cycle events and is potentially a component, regulator, or motor for the SPB and/or kinetochores.

Asunto(s)

Genes Fúngicos , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cinética , Microscopía Electrónica , Mitosis , Datos de Secuencia Molecular , Mutación , Schizosaccharomyces/citología , Schizosaccharomyces/ultraestructura , Homología de Secuencia de Ácido Nucleico , Temperatura