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

Abstract

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 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. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that 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 localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.

The RZZ Complex and the Spindle Assembly Checkpoint

“…Bub1, like Mad1, is not likely a component of the diffusible wait-anaphase signal because a large fraction is stable at unattached kinetochores. A major role for Bub1, like Mad1, may be to provide a protein scaffold at the kinetochores to support the binding (Taylor et al, 1998;Chen et al, 1998;Basu et al, 1998;Sharp-Baker and Chen, 2001), and cycling of Bub1 free Bub3 and BubR1. Although BubR1-Bub1 and Bub3-Bub1 are constitutive complexes in the cytoplasm, these complexes are not dynamically exchanging at the kinetochores (Musacchio and Hardwick, 2002).…”

“…BubR1 is postulated to act as a mechanosensor that monitors the activity of CENP-E (Chan et al, 1999). This suggests that the checkpoint activity of BubR1 might be regulated by conformational changes in CENP-E when it interacts with MTs (Basu et al, 1998). Kinetochore binding of Mad2 or BubR1 complexes activates the kinetochore formation of Cdc20, BubR1, or Mps1, which will induce more MCC to be produced (Howell et al, 2004).…”

Human MPS1 Kinase Is Required for Mitotic Arrest Induced by the Loss of CENP-E from Kinetochores

“…Many of the mitotic checkpoint genes in yeast are evolutionarily conserved, because orthologues of MAD1, MAD2, MAD3, BUB1, and BUB3 have been identified in worms, flies, and mammals (Chen et al, 1996;Li and Benezra, 1996;Taylor and McKeon, 1997;Basu et al, 1998;Cahill et al, 1998;Chan et al, 1998;Gorbsky et al, 1998;Jablonski et al, 1998;Jin et al, 1998;Taylor et al, 1998;Basu et al, 1999;Kitagawa and Hieter, 2001). Importantly, many of these mitotic checkpoint proteins bind preferentially to unattached kinetochores, where they are postulated to function in generating the "wait anaphase signal" (Hoffman et al, 2001, and references therein).…”

“…MPS1, BUB1, BUB3, MAD1, MAD2, and MAD3 monitor kinetochore microtubule attachments and prevent premature chromosome segregation by inhibiting degradation of securin/Pds1 and mitotic cyclins (Wassmann and Benezra, 2001;Peters, 2002). BUB2 acts along a different pathway that monitors spindle integrity and orientation and prevents premature cytokinesis by inhibiting the degradation of the mitotic cyclin Clb2 (Alexandru et al, 1999;Fesquet et al, 1999;Fraschini et al, 1999;Li, 1999;Bardin et al, 2000;Bloecher et al, 2000;Pereira et al, 2000).Many of the mitotic checkpoint genes in yeast are evolutionarily conserved, because orthologues of MAD1, MAD2, MAD3, BUB1, and BUB3 have been identified in worms, flies, and mammals (Chen et al, 1996;Li and Benezra, 1996;Taylor and McKeon, 1997;Basu et al, 1998;Cahill et al, 1998;Chan et al, 1998;Gorbsky et al, 1998;Jablonski et al, 1998;Jin et al, 1998;Taylor et al, 1998;Basu et al, 1999;Kitagawa and Hieter, 2001). Importantly, many of these mitotic checkpoint proteins bind preferentially to unattached kinetochores, where they are postulated to function in generating the "wait anaphase signal" (Hoffman et al, 2001, and references therein).…”

CENP-meta, an Essential Kinetochore Kinesin Required for the Maintenance of Metaphase Chromosome Alignment in Drosophila

“…These include rod (Karess and Glover 1989; Starr et al, 1998), the MAP kinases ppERK (Shapiro et al 1998; Zecevic et al 1998) and ppMEK (Shapiro et al 1998), and an APC activator protein fizzy (called cdc20/cdh1 in other organisms; Kallio et al 1998). Likewise, the protein kinase polo (Logarinho and Sunkel 1998) and a number of components of the highly conserved spindle assembly checkpoint, including Bub1 (Taylor and McKeon 1997; Taylor et al 1998; Basu et al 1999), Bub3 (Taylor et al 1998; Basu et al 1998; Martinez-Exposito et al 1999), Mad1 (Jin et al 1998; Chen et al 1998), and Mad2 (Waters et al 1998; Chen et al 1996, Chen et al 1998), are transiently kinetochore-bound.…”