Abstract
The BubR1/Bub3 complex is an important regulator of chromosome segregation as it facilitates proper kinetochore–microtubule interactions and is also an essential component of the spindle assembly checkpoint (SAC). Whether BubR1/Bub3 localization to kinetochores in human cells stimulates SAC signalling or only contributes to kinetochore–microtubule interactions is debated. Here we show that two distinct pools of BubR1/Bub3 exist at kinetochores and we uncouple these with defined BubR1/Bub3 mutants to address their function. The major kinetochore pool of BubR1/Bub3 is dependent on direct Bub1/Bub3 binding and is required for chromosome alignment but not for the SAC. A distinct pool of BubR1/Bub3 localizes by directly binding to phosphorylated MELT repeats on the outer kinetochore protein KNL1. When we prevent the direct binding of BubR1/Bub3 to KNL1 the checkpoint is weakened because BubR1/Bub3 is not incorporated into checkpoint complexes efficiently. In conclusion, kinetochore localization supports both known functions of BubR1/Bub3.
Highlights
The BubR1/Bub[3] complex is an important regulator of chromosome segregation as it facilitates proper kinetochore–microtubule interactions and is an essential component of the spindle assembly checkpoint (SAC)
We measured the relative ratio of the two proteins in a mitotic cell extract using immunoprecipitated Venus–BubR1 and Bub1–Venus to normalize the respective antibodies to a green fluorescent protein (GFP) antibody by Li-cor quantitative western blotting
Here we show that BubR1/Bub[3] can localize independently of Bub[1] to kinetochores by binding directly to MELTp repeats on KNL1
Summary
The BubR1/Bub[3] complex is an important regulator of chromosome segregation as it facilitates proper kinetochore–microtubule interactions and is an essential component of the spindle assembly checkpoint (SAC). The major kinetochore pool of BubR1/Bub[3] is dependent on direct Bub1/Bub[3] binding and is required for chromosome alignment but not for the SAC. A distinct pool of BubR1/Bub[3] localizes by directly binding to phosphorylated MELT repeats on the outer kinetochore protein KNL1. Preventing BubR1/Bub[3] kinetochore localization by blocking Bub[1] interaction increases checkpoint strength, but prevents stable kinetochore–microtubule attachment[28,29] This and the fact that overexpressed cytoplasmic fragments of BubR1 can partially support the SAC have led to suggestions that BubR1/Bub[3] kinetochore localization is not important for the checkpoint but instead contributes to checkpoint silencing through PP2A–B56 (refs 29–33). Mutation of the GLEBS motif in BubR1 that abolish Bub[3] binding prevents BubR1 kinetochore localization and strongly impairs checkpoint signalling. We have uncovered two functionally distinct pools of BubR1 bound to KNL1 at kinetochores that combined ensures accurate chromosome segregation
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