Structural analysis of Bub3 interactions in the mitotic spindle checkpoint

Abstract

The Mad3/BubR1, Mad2, Bub1, and Bub3 proteins are gatekeepers for the transition from metaphase to anaphase. Mad3 from Saccharomyces cerevisiae has homology to Bub1 but lacks a corresponding C-terminal kinase domain. Mad3 forms a stable heterodimer with Bub3. Negative-stain electron microscopy shows that Mad3 is an extended molecule (Ϸ200 Å long), whereas Bub3 is globular. The Gle2-binding-sequence (GLEBS) motifs found in Mad3 and Bub1 are necessary and sufficient for interaction with Bub3. The calorimetrically determined dissociation constants for GLEBS-motif peptides and Bub3 are Ϸ5 M. Crystal structures of these peptides with Bub3 show that the interactions for Mad3 and Bub1 are similar and mutually exclusive. In both structures, the GLEBS peptide snakes along the top surface of the ␤-propeller, forming an extensive interface. Mutations in either protein that disrupt the interface cause checkpoint deficiency and chromosome instability. We propose that the structure imposed on the GLEBS segment by its association with Bub3 enables recruitment to unattached kinetochores.␤-propeller ͉ crystal structures ͉ Rae1/Nup98 ͉ GLEBS motif

Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1

“…As predicted, the charge-reversal mutations E201K and E202K abolish the interaction between the two proteins. This result is in concurrence with previous mutagenesis experiments that identified the salt bridge formed between E382 of Mad3 and R197 of Bub3 as critical for complex formation in this system (33). Rae1•Nup98 GLEBS Binds to RNA in Vitro.…”

Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling

“…Two previous studies proposed that the first KEN box in the N-terminal Cdc20 binding site of BubR1 is responsible for BubR1 binding to Cdc20 (14,41). Based on this, it is plausible that Bub3 binding may alter the conformation of the initially rod-shaped BubR1 N terminus (34,50) in a way to either promote the initial assembly or stabilization of a BubR1-Cdc20 complex for generating the characteristic, selective inhibition of APC/C Cdc20 .…”

“…A role for Bub3 in mitotic checkpoint silencing has also been proposed in fission yeast (33). Bub3 binds to the Gle2-bindingsequence (GLEBS) motif of Bub1 and Mad3 (the yeast homolog of BubR1) in a mutually exclusive manner, with binding mediated through the top face of its β-propeller (34). Another GLEBS motif-containing protein, BugZ, was also shown to interact with Bub3, stimulating its mitotic function by promoting its stability and kinetochore loading (35)(36)(37).…”

Systematic Analysis inCaenorhabditis elegansReveals that the Spindle Checkpoint Is Composed of Two Largely Independent Branches

“…Because both BUB-1 and Mad3 use a similar and mutually exclusive interaction mechanism to associate with BUB-3 (Wang et al, 2001;Larsen et al, 2007), it is tempting to speculate that there are two pools of BUB-3: a population that enriches at kinetochores complexed with BUB-1 and a population that associates with Mad3 SAN-1 that acts cytoplasmically. It is unclear what effect there is, if any, of kinetochore cycling of BUB-3, presumably via its direct association with BUB-1.…”