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Molecular and Cellular Biology, November 2004, p. 9786-9801, Vol. 24, No. 22
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.22.9786-9801.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Kinetochore Targeting of Fission Yeast Mad and Bub Proteins Is Essential for Spindle Checkpoint Function but Not for All Chromosome Segregation Roles of Bub1p{dagger}

Vincent Vanoosthuyse,1,{ddagger} Rebekka Valsdottir,1,{ddagger} Jean-Paul Javerzat,2 and Kevin G. Hardwick1*

Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, United Kingdom,1 Institut de Biochimie et Génétique Cellulaires, Centre National de la Recherche Scientifique, Bordeaux, France2

Received 5 April 2004/ Returned for modification 20 May 2004/ Accepted 24 August 2004

Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

* Corresponding author. Mailing address: Wellcome Trust Centre for Cell Biology, ICMB, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom. Phone: 44 131 6507091. Fax: 44 131 6507037. E-mail: Kevin.Hardwick{at}ed.ac.uk.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} V.V. and R.V. contributed equally to this work.

Molecular and Cellular Biology, November 2004, p. 9786-9801, Vol. 24, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.22.9786-9801.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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