RESUMO
The fission yeast multiprotein-component Sim4 complex plays a fundamental role in the assembly of a functional kinetochore. It affects centromere association of the histone H3 variant CENP-A as well as kinetochore association of the DASH complex. Here, multicopy suppressor analysis of a mutant version of the Sim4 complex component Mal2 identified the essential Fta2 kinetochore protein, which is required for bipolar chromosome attachment. Kinetochore localization of Mal2 and Fta2 depends on each other, and overexpression of one protein can rescue the phenotype of the mutant version of the other protein. fta2 mal2 double mutants were inviable, implying that the two proteins have an overlapping function. This close interaction with Fta2 is not shared by other Sim4 complex components, indicating the existence of functional subgroups within this complex. The Sim4 complex seems to be assembled in a hierarchical way, because Fta2 is localized correctly in a sim4 mutant. However, Fta2 kinetochore localization is reduced in a spc7 mutant. Spc7, a suppressor of the EB1 family member Mal3, is part of the conserved Ndc80-MIND-Spc7 kinetochore complex.
Assuntos
Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Supressão Genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Cinetocoros/química , Mitose , Complexos Multiproteicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Fenótipo , Ligação Proteica , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/fisiologiaRESUMO
A critical aspect of mitosis is the interaction of the kinetochore with spindle microtubules. Fission yeast Mal3 is a member of the EB1 family of microtubule plus-end binding proteins, which have been implicated in this process. However, the Mal3 interaction partner at the kinetochore had not been identified. Here, we show that the mal3 mutant phenotype can be suppressed by the presence of extra Spc7, an essential kinetochore protein associated with the central centromere region. Mal3 and Spc7 interact physically as both proteins can be coimmunoprecipitated. Overexpression of a Spc7 variant severely compromises kinetochore-microtubule interaction, indicating that the Spc7 protein plays a role in this process. Spc7 function seems to be conserved because, Spc105, a Saccharomyces cerevisiae homolog of Spc7, identified by mass spectrometry as a component of the conserved Ndc80 complex, can rescue mal3 mutant strains.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Fuso Acromático/metabolismo , Centrômero/química , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/genética , Expressão Gênica , Imunoprecipitação , Cinetocoros/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mitose , Mutação/genética , Fenótipo , Ligação Proteica , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
We identified a truncated allele of dam1 as a multicopy suppressor of the sensitivity of cdc13-117 (cyclin B) and mal3-1 (EB-1) cells to thiabendazole, a microtubule poison. We find that Dam1 binds to the plus end of spindle microtubules and kinetochores as cells enter mitosis and this is dependent on other components of the fission yeast DASH complex, including Ask1, Duo1, Spc34 and Dad1. By contrast, Dad1 remains bound to kinetochores throughout the cell cycle and its association is dependent on the Mis6 and Mal2, but not Mis12, Nuf2 or Cnp1, kinetochore proteins. In cells lacking Dam1, or other components of the DASH complex, anaphase is delayed due to activation of the spindle assembly checkpoint and lagging sister chromatids are frequently observed and occasionally sister chromatid pairs segregate to the same spindle pole. We find that the mitotic centromere-associated Klp5/Klp6 kinesin complex is essential in cells lacking components of the DASH complex. Cells lacking both Dam1 and Klp5 undergo a first cell cycle arrest in mitosis due to a failure to establish bipolar chromosome attachment.