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A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint.

Overlack K, Primorac I, Vleugel M, Krenn V, Maffini S, Hoffmann I, Kops GJ, Musacchio A - Elife (2015)

Bottom Line: Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment.This gain-of-function BubR1 mutant cannot sustain a functional checkpoint.Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.

ABSTRACT
The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. Bub1 and BubR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment. Because both Bub1 and BubR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that Bub1, but not BubR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bub1 onto BubR1 promotes Bub1-independent kinetochore recruitment of BubR1. This gain-of-function BubR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubR1 relies on direct hetero-dimerization with Bub1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

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The TPR domain of BubR1 influences kinetochore binding affinity in addition to the loop region.(A) Representative images of HeLa cells transfected with the indicated GFP-BubR1362–571 constructs, showing that the B1-loop enhances kinetochore localization. However in the absence of endogenous Bub1 the short loop mutant, which lacks the N-terminal TPR domain, is less efficient than the full-length BubR1 in its localization to kinetochores (Figure 3E). Cells were treated as described in Figure 3E. Scale bar: 10 µm. (B) Representative images of HeLa cells transfected with the indicated GFP-BubR11–571 constructs, showing that the TPR domain is contributing together with the loop region to the ability to stay at KTs in the absence of endogenous Bub1. Cells were treated as described in Figure 3E. Scale bar: 10 µm. (C) Quantification of BubR1 KT levels in cells treated as in (A–B). The graph shows mean intensity from at least two independent experiments. Error bars represent SEM. Values for BubR1FL in non-depleted cells are set to 1.DOI:http://dx.doi.org/10.7554/eLife.05269.012
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fig4s3: The TPR domain of BubR1 influences kinetochore binding affinity in addition to the loop region.(A) Representative images of HeLa cells transfected with the indicated GFP-BubR1362–571 constructs, showing that the B1-loop enhances kinetochore localization. However in the absence of endogenous Bub1 the short loop mutant, which lacks the N-terminal TPR domain, is less efficient than the full-length BubR1 in its localization to kinetochores (Figure 3E). Cells were treated as described in Figure 3E. Scale bar: 10 µm. (B) Representative images of HeLa cells transfected with the indicated GFP-BubR11–571 constructs, showing that the TPR domain is contributing together with the loop region to the ability to stay at KTs in the absence of endogenous Bub1. Cells were treated as described in Figure 3E. Scale bar: 10 µm. (C) Quantification of BubR1 KT levels in cells treated as in (A–B). The graph shows mean intensity from at least two independent experiments. Error bars represent SEM. Values for BubR1FL in non-depleted cells are set to 1.DOI:http://dx.doi.org/10.7554/eLife.05269.012

Mentions: In IP experiments both in the presence and absence of endogenous Bub1, GFP-Bub1209–409 interacted with BubR1 at levels that were only modestly lower than those of full-length GFP-Bub1 (Figure 4E–F). Conversely, GFP-Bub1209–270 did not interact with BubR1, in agreement with the inability of this construct to promote BubR1 localization. In this context, it should be noted that the tetratrico peptide repeats (TPRs) near the N-terminus of Bub1 and BubR1 (Figure 1A) had been initially identified as primary determinants of kinetochore recruitment (Kiyomitsu et al., 2007; Bolanos-Garcia et al., 2011), but later shown to be dispensable (Krenn et al., 2012, 2014), a result confirmed here. In our IP experiments, however, we observe that Bub1 interacts with the outer kinetochore more strongly when the TPRs are present, in agreement with our previous studies (Krenn et al., 2012, 2014). The Bub1 TPR region interacts with a short sequence motif of Knl1 named KI1 motif (Kiyomitsu et al., 2007; Krenn et al., 2012, 2014). This interaction, whose precise significance is unclear, enhances the SAC response (Krenn et al., 2014). Additional evidence of a modest additional role of the TPR in the interaction of Bub1 and BubR1 with kinetochores is presented in Figure 4—figure supplement 2 and Figure 4—figure supplement 3.


A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint.

Overlack K, Primorac I, Vleugel M, Krenn V, Maffini S, Hoffmann I, Kops GJ, Musacchio A - Elife (2015)

The TPR domain of BubR1 influences kinetochore binding affinity in addition to the loop region.(A) Representative images of HeLa cells transfected with the indicated GFP-BubR1362–571 constructs, showing that the B1-loop enhances kinetochore localization. However in the absence of endogenous Bub1 the short loop mutant, which lacks the N-terminal TPR domain, is less efficient than the full-length BubR1 in its localization to kinetochores (Figure 3E). Cells were treated as described in Figure 3E. Scale bar: 10 µm. (B) Representative images of HeLa cells transfected with the indicated GFP-BubR11–571 constructs, showing that the TPR domain is contributing together with the loop region to the ability to stay at KTs in the absence of endogenous Bub1. Cells were treated as described in Figure 3E. Scale bar: 10 µm. (C) Quantification of BubR1 KT levels in cells treated as in (A–B). The graph shows mean intensity from at least two independent experiments. Error bars represent SEM. Values for BubR1FL in non-depleted cells are set to 1.DOI:http://dx.doi.org/10.7554/eLife.05269.012
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4337726&req=5

fig4s3: The TPR domain of BubR1 influences kinetochore binding affinity in addition to the loop region.(A) Representative images of HeLa cells transfected with the indicated GFP-BubR1362–571 constructs, showing that the B1-loop enhances kinetochore localization. However in the absence of endogenous Bub1 the short loop mutant, which lacks the N-terminal TPR domain, is less efficient than the full-length BubR1 in its localization to kinetochores (Figure 3E). Cells were treated as described in Figure 3E. Scale bar: 10 µm. (B) Representative images of HeLa cells transfected with the indicated GFP-BubR11–571 constructs, showing that the TPR domain is contributing together with the loop region to the ability to stay at KTs in the absence of endogenous Bub1. Cells were treated as described in Figure 3E. Scale bar: 10 µm. (C) Quantification of BubR1 KT levels in cells treated as in (A–B). The graph shows mean intensity from at least two independent experiments. Error bars represent SEM. Values for BubR1FL in non-depleted cells are set to 1.DOI:http://dx.doi.org/10.7554/eLife.05269.012
Mentions: In IP experiments both in the presence and absence of endogenous Bub1, GFP-Bub1209–409 interacted with BubR1 at levels that were only modestly lower than those of full-length GFP-Bub1 (Figure 4E–F). Conversely, GFP-Bub1209–270 did not interact with BubR1, in agreement with the inability of this construct to promote BubR1 localization. In this context, it should be noted that the tetratrico peptide repeats (TPRs) near the N-terminus of Bub1 and BubR1 (Figure 1A) had been initially identified as primary determinants of kinetochore recruitment (Kiyomitsu et al., 2007; Bolanos-Garcia et al., 2011), but later shown to be dispensable (Krenn et al., 2012, 2014), a result confirmed here. In our IP experiments, however, we observe that Bub1 interacts with the outer kinetochore more strongly when the TPRs are present, in agreement with our previous studies (Krenn et al., 2012, 2014). The Bub1 TPR region interacts with a short sequence motif of Knl1 named KI1 motif (Kiyomitsu et al., 2007; Krenn et al., 2012, 2014). This interaction, whose precise significance is unclear, enhances the SAC response (Krenn et al., 2014). Additional evidence of a modest additional role of the TPR in the interaction of Bub1 and BubR1 with kinetochores is presented in Figure 4—figure supplement 2 and Figure 4—figure supplement 3.

Bottom Line: Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment.This gain-of-function BubR1 mutant cannot sustain a functional checkpoint.Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

View Article: PubMed Central - PubMed

Affiliation: Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.

ABSTRACT
The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. Bub1 and BubR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment. Because both Bub1 and BubR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that Bub1, but not BubR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bub1 onto BubR1 promotes Bub1-independent kinetochore recruitment of BubR1. This gain-of-function BubR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubR1 relies on direct hetero-dimerization with Bub1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

Show MeSH
Related in: MedlinePlus