Proc. Natl. Acad. Sci. USA Vol. 96, pp. 8493–8498, July 1999 Cell Biology Retention of the Bub3 checkpoint protein on lagging chromosomes MARIA J. MARTINEZ-EXPOSITO*, KENNETH B. KAPLAN*, JAY COPELAND, AND PETER K. SORGER† Department of Biology, 68–371, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 Communicated by Stephen C. Harrison, Harvard University, Cambridge, MA, April 14, 1999 (received for review December 16, 1998) ABSTRACT Accurate chromosome segregation at mitosis is kinetochores (11–14), and dominant negative mutants and ensured both by the intrinsic fidelity of the mitotic machinery antibody microinjection (14–16) have implicated the proteins and by the operation of checkpoints that monitor chromosome- in the control of chromosome segregation. In this paper we microtubule attachment. When unattached kinetochores are report the isolation and analysis of the murine Bub3 gene. We present, anaphase is delayed and the time available for chromo- find that murine Bub3, like yeast Bub3p, binds to Bub1 to form some-microtubule capture increases. Genes required for this an active kinase complex (17). mBub3 is present on unattached delay first were identified in budding yeast (the MAD and BUB kinetochores and, in cells treated with very low concentrations genes), but it is not yet known how the checkpoint senses of taxol, it is selectively retained on lagging chromosomes. unattached chromosomes or how it signals cell-cycle arrest. We report the isolation and analysis of a murine homologue of BUB3, MATERIALS AND METHODS a gene whose deletion abolishes mitotic checkpoint function in DNA, Cell Culture, and Protein Methods. A screen by Saccharomyces cerevisiae. mBub3 belongs to a small gene family hybridization of a day 10 mouse embryo cDNA library with that has been highly conserved through evolution. By expressing radio-labeled DNA from two murine expressed sequence tags recombinant proteins in insect cells, we show that mBub3, like (ESTs), Genbank accession nos. W11868 and W13647, yielded yeast Bub3p, binds to Bub1 to form a complex with protein 19 positive phages. The insert in one subclone of the phage kinase activity. During prophase and prometaphase, preceding (clone 20a) appeared to be full length, containing a 1,350-bp kinetochore-microtubule attachment, Bub3 localizes to kineto- insert with an ATG at bp 57, an ORF 30% identical to yBub3, chores. High levels of mBub3 remain associated with lagging and a poly(A) tail. Overall, mBub3 is identical to two recently chromosomes but not with correctly aligned chromosomes dur- described human Bub3 proteins over 325 of its first 326 ing metaphase, consistent with a role for Bub3 in sensing residues, but is unlike either hBUB3 in its final amino acid (two microtubule attachment. Intriguingly, the number of lagging independent mBUB3 clones were confirmed to have the same chromosomes with high Bub3 staining increases dramatically in C terminus). This similarity is not a consequence of cross- cells treated with low (and pharmacologically relevant) concen- species contamination in the libraries because 89 silent DNA trations of the chemotherapeutic taxol and the microtubule differences are present between human and mouse. poison nocodazole. HeLa cells were grown in media containing 10% FBS. Nocodazole and taxol were added fresh from stocks in DMSO. The accurate segregation of chromosomes requires that all pairs Mitotic cells were isolated by first arresting the cells at the ͞ of sister chromatids achieve a state of bivalent attachment to the G1 S boundary via a double thymidine block, releasing them mitotic spindle before the onset of anaphase (1). Bivalent attach- for 14–16 hr, and harvesting them by shake-off (18). ment occurs when one kinetochore on a pair of sister chromatids mBub1 and mBub3 genes were cloned into the pFastBac is attached to microtubules emanating from one spindle pole and baculovirus expression vector (Life Technologies, Grand Is- the other kinetochore is attached to microtubules emanating land, NY) and fused to an amino terminal His10HA epitope from the opposite pole. A checkpoint monitors kinetochore- tag. Recombinant viruses were obtained and used to infect HI5 microtubule interaction and delays cell cycle progression until all insect cells essentially as described (19). chromosome attachments are bivalent (2–4). Antibodies against mBub1 and mBub3 were obtained by It seems likely that checkpoint lesions play an important role immunizing rabbits with keyhole limpet hemocyanin- in tumorigenesis. The frequent occurrence of aneuploidy in conjugated 14 aa peptides comprising Bub3 residues 207–221 tumor cells and the association of whole-chromosome loss of (SPO14), 313–327 (SPO15), or Bub1 158–172 (each rabbit was heterozygosity with the development of several types of can- immunized with a single peptide). Western blots were quan- cers suggest that error-prone chromosome segregation may titated by using a PhosphorImager after incubation with 125 give rise to a mutator phenotype and thereby promote tumor primary antibodies and I protein A. development (5–7). This possibility is strengthened by the Microscopy Methods. For immunofluorescence, cells grown finding that a human checkpoint gene (Bub1; ref. 3) is mutated on coverslips were fixed with 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton in some human colorectal cancers (8). ͞ ͞ Genes required for the mitotic checkpoint first were iden- X-100 PBS for 7 min, blocked with 0.2% gelatin in PBS 0.02% Saccharomyces cerevisiae MAD1–3 NaN3 for 20 min, and incubated for 20 min with raw or tified in budding yeast. The ␤ and BUB1–3 genes are not required for cell viability, but affinity-purified anti-Bub3 antisera, mouse monoclonal anti- mutations in these genes abolish mitotic delay in response to tubulin (TUB 2.1 from Sigma), and human CREST serum at unattached kinetochores, increase the rate of chromosome 1:1,000. Cells then were washed three times with 0.2% gelatin- PBS and 0.02% sodium azide, incubated for 20 min, and then loss, and raise the sensitivity of cells to antimicrotubule drugs Ј (9, 10). The isolation of mammalian homologues of Mad2 and stained with 4 ,6-diamidino-2-phenylindole and with affinity- Bub1 has shown that these proteins are present on unattached purified FITC or Texas Red-conjugated goat anti-rabbit or The publication costs of this article were defrayed in part by page charge Abbreviations: EST, expressed sequence tag; HA, hemagglutinin; 3D, three-dimensional. payment. This article must therefore be hereby marked ‘‘advertisement’’ in *M.J.M.-E. and K.B.K. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: psorger@ PNAS is available online at www.pnas.org. mit.edu. 8493 Downloaded by guest on September 29, 2021 8494 Cell Biology: Martinez-Exposito et al. Proc. Natl. Acad. Sci. USA 96 (1999) goat anti-mouse, or FITC-conjugated goat anti-human (Santa Cruz Biotechnology). Alternatively, cells in suspension were recovered by centrifugation, fixed, and stained in solution, then applied to slides in mounting solution. The specificity of anti-Bub3 antibodies was demonstrated by incubation with a 70-fold excess of either immunizing or nonspecific peptides for 1 hr on ice before use. Immunostained specimens were examined by using a 63ϫ Plan-apochromat objective, 0.2-␮m thick Z-sections, and a Princeton Instruments Quantix cooled charge-coupled device camera on a Ziess-Applied Precision DeltaVision Restoration microscope. Exposures were chosen that yielded 200–1,800 gray scale units, remaining well above the camera dark current but below the 4,096-unit maximum. To create three-dimensional (3D) models, two-dimensional polygons were first constructed on each Z-section, one fluor at a time, based on intensity levels chosen to highlight structural boundaries. The stacks of two- dimensional polygons then were rendered into 3D objects, and images of the models were enhanced by adjusting the transpar- ency and false color of the rendered 3D objects. RESULTS Isolation of a Murine Bub3 Gene and its mRae1 Homologue. A search of Genbank for ESTs yielded one murine EST with significant sequence similarity to yeast Bub1 and two ESTs with similarity to yeast Bub3. When DNA from these ESTs was used to screen mouse embryonic cDNA libraries, we isolated a kinase identical in sequence to the mBub1 gene recently reported by Taylor and McKeon (14) and a second gene (clone 20a) encoding a 326-residue protein 29% identical to S. cerevisiae Bub3 (Fig. 1; ref. 17). BLAST analysis with clone 20a revealed a closely related murine gene (which we will call mRae1) encoding a 368-aa protein 30% identical to clone 20a and about 40% identical to Schizosaccharomyces pombe Rae1 and S. cerevisiae GLE2 (20, 21). Our murine sequence data, combined with Genbank sequences, yielded a phylogentic tree FIG. 1. Sequence analysis of Bub3 and Rae1. (a) A tree generated showing that all six eukaryotes examined contained one Bub3- by PHYLIP (45) comparing sequences listed below and aligned with a CLUSTAL. Branch lengths are arbitrary, but the connectivity was robust. like and one Rae1-like gene (Fig. 1 ); in no organism were Bub3 and Rae1 genes from different organisms were obtained as additional genes found that were more than 10% identical. follows: 1) Sc-Bub3, accession number M64707 (17); 2) Sc-Gle2, Although Bub3 and Rae1 contain WD40 motifs, the regions of U18839 (20), 3) Sp-23H3.08c, Z99163; 4) Sp-Rae1, U14951 (21); 5) similarity between the two proteins extends well beyond the m-Bub3 (this work); 6, m-Rae1 (this work and ref. 23); 7) h-Bub3, WD40 domains (Fig. 1 b and c). We therefore conclude that AF053304 for spleen and 8) AF047473 for testis; 9) h-Rae1, U84720 Rae1 and Bub3 are members of a gene family in which there (23); 10) An-SLDB, AF032988 (43); 11) An-SONA, AF069492 (44); are two branches.