RESUMEN
The Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. Here, we isolate and characterise a new yeast mutant, mps1-3, that is severely defective in chromosome biorientation and SAC signalling. Through an unbiased screen for extragenic suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data argue that a common mechanism based on Knl1/Spc105 phosphorylation is critical for Mps1 function in error correction and SAC signalling, thus supporting the idea that a single sensory apparatus simultaneously elicits both pathways.
Asunto(s)
Segregación Cromosómica , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Cinetocoros , Puntos de Control de la Fase M del Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático/genéticaRESUMEN
Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1SPC105, MIS12MTW1, and NDC80NDC80 complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association.
Asunto(s)
Cinetocoros/metabolismo , Espectrometría de Masas/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/patogenicidad , Secuencia de AminoácidosRESUMEN
Nuclear protein LaeA is known as the global regulator of secondary metabolism in Aspergillus. LaeA connects with VeA and VelB to form a heterotrimeric complex, which coordinates fungal development and secondary metabolism. Here, we describe a new interaction partner of LaeA, the kinetochore protein Spc105, from the aflatoxin-producing fungus Aspergillus flavus. We showed that in addition to involvement in nuclear division, Spc105 is required for normal conidiophore development and sclerotia production of A. flavus. Moreover, Spc105 positively regulates the production of secondary metabolites such as aflatoxin and kojic acid, and negatively regulates the production of cyclopiazonic acid. Transcriptome analysis of the Δspc105 strain revealed that 23 backbone genes were differentially expressed, corresponding to 19 of the predicted 56 secondary metabolite gene clusters, suggesting a broad regulatory role of Spc105 in secondary metabolism. Notably, the reduced expression of laeA in our transcriptome data led to the discovery of the correlation between Spc105 and LaeA, and double mutant analysis indicated a functional interdependence between Spc105 and LaeA. Further, in vitro and in vivo protein interaction assays revealed that Spc105 interacts directly with the S-adenosylmethionine (SAM)-binding domain of LaeA, and that the leucine zipper motif in Spc105 is required for this interaction. The Spc105-LaeA interaction identified in our study indicates a cooperative interplay of distinct regulators in A. flavus, providing new insights into fungal secondary metabolism regulation networks.
RESUMEN
Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.
Asunto(s)
Drosophila melanogaster/metabolismo , Cinetocoros/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Drosophila melanogaster/genética , Humanos , Cinetocoros/química , Cinetocoros/ultraestructura , Espectrometría de Masas , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes , Alineación de SecuenciaRESUMEN
Nearly all cell division mutants in Drosophila were recovered in late larval/pupal lethal screens, with less than 10 embryonic lethal mutants identified, because larval development occurs without a requirement for cell division. Only cells in the nervous system and the imaginal cells that generate the adult body divide during larval stages, with larval tissues growing by increasing ploidy rather than cell number. Thus, most mutants perturbing mitosis or the cell cycle do not manifest a phenotype until the adult body differentiates in late larval and pupal stages. To identify cell-cycle components whose maternal pools are depleted in embryogenesis or that have specific functions in embryogenesis, we screened for mutants defective in cell division during embryogenesis. Five new alleles of Cyclin E were recovered, ranging from a missense mutation that is viable to stop codons causing embryonic lethality. These permitted us to investigate the requirements for Cyclin E function in neuroblast cell fate determination, a role previously shown for a null Cyclin E allele. The mutations causing truncation of the protein affect cell fate of the NB6-4 neuroblast, whereas the weak missense mutation has no effect. We identified mutations in the pavarotti (pav) and tumbleweed (tum) genes needed for cytokinesis by a phenotype of large and multinucleate cells in the embryonic epidermis and nervous system. Other mutations affecting the centromere protein CAL1 and the kinetochore protein Spc105R caused mitotic defects in the nervous system.