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

Abstract

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.

SUPPLEMENTAL MATERIAL

We particularly thank David Millband for yeast strains and preliminary data and Sheila Kadura and Shelley Sazer for communicating results prior to publication. We are indebted to Robin Allshire and Alison Pidoux for their generous supply of reagents and advice relating to the ChIP protocol and silencing assays. In addition, we thank Shelley Sazer for pREP3X-mad2 and pREP41X-mph1; Ravi Dhar for rae1-167 and Rae1p reagents; Keith Gull, Iain Hagan, and Barbara Mellone for antibodies; Yasushi Hiraoka, Tomohiro Matsumoto, Jonathan Miller, Paul Nurse, and Shelley Sazer for yeast strains; all members of the Hardwick lab for their support and discussions; and Alison Pidoux for critical reading of the manuscript.

Work in the Hordwick lab is supported by the Wellcome Trust, of which K.G.H. is a Senior Research Fellow; J.P.J. is supported by the CNRS, the University of Bordeaux 2, and l’Association pour la Recherche sur le Cancer.