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Molecular and Cellular Biology, April 2002, p. 2728-2742, Vol. 22, No. 8
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.8.2728-2742.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Fission Yeast Mad3p Is Required for Mad2p To Inhibit the Anaphase-Promoting Complex and Localizes to Kinetochores in a Bub1p-, Bub3p-, and Mph1p-Dependent Manner

David N. Millband and Kevin G. Hardwick*

Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland EH9 3JR, United Kingdom

Received 4 October 2001/ Returned for modification 19 November 2001/ Accepted 17 January 2002

The spindle checkpoint delays the metaphase-to-anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast identified the Mad and Bub proteins as key components of this conserved regulatory pathway. Here we present the fission yeast homologue of Mad3p. Cells devoid of mad3+ are unable to arrest their cell cycle in the presence of microtubule defects. Mad3p coimmunoprecipitates Bub3p, Mad2p, and the spindle checkpoint effector Slp1/Cdc20p. We demonstrate that Mad3p function is required for the overexpression of Mad2p to result in a metaphase arrest. Mad1p, Bub1p, and Bub3p are not required for this arrest. Thus, Mad3p appears to have a crucial role in transducing the inhibitory "wait anaphase" signal to the anaphase-promoting complex (APC). Mad3-green fluorescent protein (GFP) is recruited to unattached kinetochores early in mitosis and accumulates there upon prolonged checkpoint activation. For the first time, we have systematically studied the dependency of Mad3/BubR1 protein recruitment to kinetochores. We find Mad3-GFP kinetochore localization to be dependent upon Bub1p, Bub3p, and the Mph1p kinase, but not upon Mad1p or Mad2p. We discuss the implications of these findings in the context of our current understanding of spindle checkpoint function.

* Corresponding author. Mailing address: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Rd., Edinburgh, Scotland EH9 3JR, United Kingdom. Phone: 44 (0) 131 650 7091. Fax: 44 (0) 131 650 7037. E-mail: hardwick{at}holyrood.ed.ac.uk.

Molecular and Cellular Biology, April 2002, p. 2728-2742, Vol. 22, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.8.2728-2742.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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