Original Article

Spindle Checkpoint Protein Bub1 Is Required for Kinetochore Localization of Mad1, Mad2, Bub3, and CENP-E, Independently of Its Kinase Activity

Correspondence to: Rey-Huei Chen, Department of Molecular Biology and Genetics, 258 Biotechnology Bldg., Cornell University, Ithaca, NY 14583. Tel:(607) 255-6542 Fax:(607) 255-6249 E-mail:rc70{at}cornell.edu.

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1–Mad2. However, Bub1 differs from Mad1–Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

Key Words: spindle checkpoint, Xenopus, kinetochore, Bub1, Mad1

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