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Molecular and Cellular Biology, September 2003, p. 6406-6418, Vol. 23, No. 18
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.18.6406-6418.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Recognizing Chromosomes in Trouble: Association of the Spindle Checkpoint Protein Bub3p with Altered Kinetochores and a Unique Defective Centromere

Oliver Kerscher,{dagger} Luciana B. Crotti, and Munira A. Basrai*

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20889

Received 5 March 2003/ Returned for modification 16 April 2003/ Accepted 11 June 2003

Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G2/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested a model in which alterations or defects in kinetochore or spindle integrity lead to the distinct enrichment of Bub3p at these structures. In support of our model, kinetochore-associated Bub3-GFP is enriched upon activation of the spindle checkpoint due to nocodazole-induced spindle disassembly, overexpression of the checkpoint kinase Mps1p, or the presence of a defective centromere (CEN). Most importantly, using a novel approach with the chromatin immunoprecipitation (ChIP) technique and genetically engineered defective CEN [CF/CEN6({Delta}31)], we determined that Bub3-GFP can associate with a single defective kinetochore. Our studies represent the first comprehensive molecular analysis of spindle checkpoint protein function in the context of a wild-type or defective kinetochore(s) by use of live-cell imaging and the ChIP technique in S. cerevisiae.

* Corresponding author. Mailing address: NNMC, Bldg. 8, Room 5101, 8901 Wisconsin Ave., Bethesda, MD 20889-5101. Phone: (301) 402-2552. Fax: (301) 480-0380. E-mail: basraim{at}nih.gov.

{dagger} Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520.

Molecular and Cellular Biology, September 2003, p. 6406-6418, Vol. 23, No. 18
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.18.6406-6418.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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