Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod

We report here the isolation and molecular characterization of the Drosophi la homolog of the mitotic checkpoint control protein Bub3. The Drosophila B ub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosoph ila Bub3 is also found at the kinetochore regions in mitotic larval neurobl asts and in meiotic primary and secondary spermatocytes, with the strong si gnal seen during prophase and prometaphase becoming increasingly weaker aft er the chromosomes have aligned at the metaphase plate. We further show tha t the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore l ocalization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multip rotein complex recruited as a unit to the kinetochore. Ln contrast, we demo nstrate that the kinetochore constituents Zw10 and Rod are not needed for t he binding of Bub3 to the kinetochore. This suggests that the kinetochore i s assembled in at least two relatively independent pathways.