RESUMEN
The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis, and asymmetric association of SIN proteins with the mitotic spindle pole bodies (SPBs) is important for its regulation. Here, we have used semi-automated image analysis to study SIN proteins in large numbers of wild-type and mutant cells. Our principal conclusions are: first, that the association of Cdc7p with the SPBs in early mitosis is frequently asymmetric, with a bias in favour of the new SPB; second, that the early association of Cdc7p-GFP to the SPB depends on Plo1p but not Spg1p, and is unaffected by mutations that influence its asymmetry in anaphase; third, that Cdc7p asymmetry in anaphase B is delayed by Pom1p and by activation of the spindle assembly checkpoint, and is promoted by Rad24p; and fourth, that the length of the spindle, expressed as a fraction of the length of the cell, at which Cdc7p becomes asymmetric is similar in cells dividing at different sizes. These data reveal that multiple regulatory mechanisms control the SIN in mitosis and lead us to propose a two-state model to describe the SIN.
Asunto(s)
GTP Fosfohidrolasas/genética , Puntos de Control de la Fase M del Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Schizosaccharomyces pombe/genética , Huso Acromático/genética , Cuerpos Polares del Huso/genética , Proteínas de Ciclo Celular/genética , Citocinesis/genética , Proteínas Fluorescentes Verdes/genética , Procesamiento de Imagen Asistido por Computador , Péptidos y Proteínas de Señalización Intracelular/genética , Mitosis/genética , Proteínas Quinasas/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Huso Acromático/fisiologíaRESUMEN
In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Prueba de Complementación Genética , Datos de Secuencia Molecular , Fenotipo , Pneumocystis carinii/química , Pneumocystis carinii/genética , Pneumocystis carinii/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/química , Saccharomycetales/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homología de Secuencia de Aminoácido , Telómero/química , Telómero/metabolismo , Factores de TranscripciónRESUMEN
Cytokinesis in Schizosaccharomyces pombe begins at mitotic entry, when the site of division is defined by formation of the contractile acto-myosin ring (CAR) at the cell cortex. Contraction of the CAR and formation of the division septum are triggered at the end of mitosis by septation initiation network (SIN) proteins associated with the spindle pole body (SPB). SIN signalling requires activation of the GTPase Spg1p, which is regulated by the bipartite GTPase-activating protein (GAP) Byr4p-Cdc16p. We show that, for Spg1p to associate with the SPB, it must be bound to its GAP or to its mitotic effector, the protein kinase Cdc7p. Analysis of the GAP proteins reveals that the steady-state level of Byr4p reflects that of Spg1p. Furthermore, if the interaction of Byr4p with Spg1p is compromised, the level of Byr4p decreases dramatically. The adaptation of the level of Byr4p to that of Spg1p requires the presence of Cdc16p and is mediated by proteasome-dependent destruction. It requires neither association with the SPB nor an active SIN. We propose a mechanism that limits the amount of the Byr4p-Cdc16p GAP to the amount required to inhibit Spg1p signalling.