The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. immediate hetero-dimerization with Bub1 at a pseudo-symmetric user interface. This pseudo-symmetric relationship underpins a template-copy romantic relationship essential for kinetochore-microtubule connection and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network. DOI: http://dx.doi.org/10.7554/eLife.05269.001 (Primorac et Caudatin al. 2013 In human cells Bub3 is required Caudatin for kinetochore recruitment of Bub1 and BubR1 and consistently the B3BDs of Bub1 and BubR1 are necessary and in the case of Bub1 also sufficient for kinetochore targeting of Bub1 and BubR1 (Taylor et al. 1998 Logarinho et al. 2008 Malureanu et al. 2009 Elowe et al. 2010 Lara-Gonzalez et al. 2011 Krenn et al. 2012 The subordination of BubR1 kinetochore recruitment to the presence Caudatin of Bub1 suggests that Bub3 may operate differently when bound to Bub1 or BubR1. In this study we set out to investigate the molecular basis of this phenomenon and its implications for spindle checkpoint signaling and kinetochore-microtubule attachment. Results Mps1 and Bub1 are required for kinetochore localization of BubR1 The SAC kinase Mps1 has been shown to phosphorylate MELT repeats of Knl1 to promote kinetochore recruitment of Bub1 and BubR1 (Heinrich et al. 2012 London et al. 2012 Shepperd et al. 2012 Yamagishi et al. 2012 Primorac et al. 2013 Vleugel et al. 2013 Krenn et al. 2014 We precipitated Bub1 or Knl1 (Vleugel et al. 2013 from mitotic lysates of HeLa cells treated with or without the Mps1 inhibitor Reversine (Santaguida et al. 2010 Quantitative mass spectrometry (observe ‘Materials and methods’) of proteins associated with Bub1 or Knl1 confirmed the crucial role of Mps1 as we observed a strong suppression of the conversation of Bub1 BubR1 and Bub3 with kinetochores in the presence of Reversine (Physique 1C-D. Large deviations from a value Rabbit polyclonal to Caspase 7. of 1 1 for the Reversine/DMSO ratio show suppression of Caudatin binding). In HeLa cells treated with nocodazole which depolymerizes microtubules and activates the SAC Bub1 decorated kinetochores at essentially normal levels after the depletion of BubR1 (Physique 1E quantified in Physique 1F. Quantifications of RNAi-based depletions are shown in Physique 1-figure product 1A-B). Conversely BubR1 did not decorate kinetochores after Bub1 depletion (Physique 1G-H). These results confirm that BubR1 requires Bub1 for kinetochore recruitment in line with previous studies (Millband and Hardwick 2002 Gillett et al. 2004 Johnson et al. 2004 Perera et al. 2007 Logarinho et al. 2008 Klebig et al. 2009 By monitoring the localization of a GFP-Bub1 reporter construct we had previously exhibited that Bub1209-270 encompassing the B3BD may be the minimal Bub1 localization area (Taylor et al. 1998 Krenn et al. 2012 Bub1209-270 Caudatin targeted kinetochores extremely efficiently even following the depletion of endogenous Bub1 (Body 1I). We asked if an similar GFP reporter build encompassing the B3BD of BubR1 BubR1362-431 was also recruited to kinetochores. BubR1362-431 had not been recruited to kinetochores also in the current presence of Bub1 (Body 1J. Diagrams of Bub1 and BubR1 deletions found in this research are in Body 1-figure dietary supplement 1C-D). Thus even if Bub1 and BubR1 share a related B3BD to interact with the same kinetochore-targeting subunit (Bub3) and interact in a phosphorylation-dependent manner with Knl1 the mechanisms of their kinetochore recruitment are different. This raises two crucial questions: (1) why is the B3BD region of Bub1 sufficient for kinetochore recruitment while the comparative region of BubR1 is not? And (2) if binding to Bub3 is not sufficient for strong kinetochore recruitment of BubR1 how is usually BubR1 recruited to kinetochores? Caudatin We will focus sequentially on these questions. The loop regions of Bub1 and BubR1 modulate the conversation of Bub3 with phosphorylated MELT motifs To investigate if and how Bub1209-270 and BubR1362-431 modulate the binding affinity of Bub3 for the MELTP repeats of Knl1 we immobilized on amylose beads a.