BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis

Abstract

BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. Here, we used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, we found that the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, we carried out size-exclusion chromatography and analyzed Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions. We found that prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. Our results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling.

Keywords: BuGZ; Bub1; Bub3; BubR1; cell biology; cell division; checkpoint control; kinetochore mitosis; spindle assembly checkpoint.

Figure 1.

The core GLEBS domain of BuGZ is sufficient for kinetochore localization. A, domain map of the human BuGZ protein. ZF1 and ZF2 indicate zinc finger domains 1 and 2. The microtubule-binding domain overlaps with the zinc finger domains. The GLEBS core is the Gle2-binding sequence containing two highly conserved glutamic acid residues (indicated by asterisks in B). B, sequence alignments of Bub1 and BubR1 depicting the loop domain, GLEBS domain, and C-terminal extension (37). BuGZ's GLEBS domain and surrounding regions are aligned with analogous domains in Bub1 and BubR1. Putative loop and C-terminal extension domains in BuGZ are indicated based on the alignments with Bub1 and BubR1. C, graphic depicting the GFP–BuGZ constructs used to test for kinetochore localization. CTE, C-terminal extension; LGC, loop–GLEBS–CTE; LG, loop–GLEBS. D, immunofluorescence images of HeLa cells in early mitosis transfected with GFP–BuGZ constructs and analyzed for kinetochore localization. The cells are stained for DAPI and anti-centromere antibody (ACA). E, immunofluorescence images of HeLa cells in early mitosis transfected with “loop swap” constructs consisting of the core GLEBS domain of BuGZ fused to the loop domain of Bub1 (top) or BubR1 (bottom). The cells are stained for DAPI and ACA. Scale bars, 10 μm.

Figure 2.

Maximal BuGZ kinetochore loading occurs in early mitosis. A–D, representative immunofluorescence images of mitotic HeLa cells in prophase, early prometaphase, prometaphase, metaphase, and anaphase. The cells in B–D were stained with DAPI, ACA antibodies, and antibodies to either Bub3, Bub1, or BubR1. The cells in A were transfected with a full-length GFP–BuGZ construct and stained with DAPI and ACA antibodies. E–H, quantifications of GFP–BuGZ, BubR1, Bub1, and Bub3 at kinetochores during mitosis. For each graph, at least 20 kinetochores/cell were measured from at least 10 cells. Kinetochore intensities were normalized to ACA levels. Error bars represent standard deviation. Scale bars, 10 μm.

Figure 3.

BuGZ loads to kinetochores prior to Bub1 and BubR1 and is evicted from kinetochores prior to Bub1 and BubR1. Immunofluorescence images of GFP–BuGZ expressing HeLa cells in prophase, prometaphase (Prometa), and metaphase. The cells are stained with DAPI and antibodies to Bub1 and BubR1. Arrows point to the kinetochore pairs shown in the insets. Scale bars, 10 μm in whole-cell panels and 1 μm in insets.

Figure 4.

Bub3 preferentially complexes with BuGZ when kinetochore recruitment of checkpoint factors is blocked by Mps1 inhibition. A, schematic depicting the workflow for size-exclusion chromatography fractionation of HeLa cells arrested in mitosis with nocodazole. B, Western blots of nocodazole-arrested HeLa cells probed with antibodies to BubR1, Bub1, Mad1, Cdc20, BuGZ, Bub3, and Mad2. Molecular mass standards are shown; the blots and fractions are labeled by their elution volume. C, schematic depicting the workflow for size-exclusion chromatography fractionation of HeLa cells treated with reversine and MG132. D, Western blots of reversine/MG132-treated HeLa cells probed with antibodies to BubR1, Bub1, Mad1, Cdc20, BuGZ, Bub3, and Mad2. Molecular mass standards are shown; the blots and fractions are labeled by their elution volume. In B and D, dashed lines indicate where blots were cut to remove molecular mass markers.

Figure 5.

Bub3 and BuGZ form a stable complex. A, Coomassie-stained SDS-PAGE gel of Bub3 and BuGZ purified from ExpiSF9 insect cells. B, Coomassie-stained SDS-PAGE gel of SEC-MALS elution fractions for recombinant Bub3 alone (top panel), BuGZ alone (middle panel), and Bub3–BuGZ complex (bottom panel). C, SEC-MALS profiles of recombinant Bub3 alone (top panel), BuGZ alone (middle panel), and Bub3–BuGZ complex (bottom panel). Expected (Exp) molar mass and observed (Obs) molar mass are indicated. Expected molar mass for the complex was calculated for 1:1 stoichiometry.

Figure 6.

Model for BuGZ function in mitosis. BuGZ and Bub3 form a complex in the nucleus prior to mitotic entry. Early in prophase, BuGZ and Bub3 load to kinetochores. At nuclear envelope breakdown, BuGZ begins to exchange for Bub1 and BubR1 at kinetochores. By late prometaphase, BuGZ is largely evicted from kinetochores, whereas Bub1–Bub3–BubR1 complexes persist.