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Eukaryotic Cell, May 2005, p. 867-878, Vol. 4, No. 5
1535-9778/05/$08.00+0     doi:10.1128/EC.4.5.867-878.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Two Complexes of Spindle Checkpoint Proteins Containing Cdc20 and Mad2 Assemble during Mitosis Independently of the Kinetochore in Saccharomyces cerevisiae

Atasi Poddar, P. Todd Stukenberg, and Daniel J. Burke*

Department of Biochemistry and Molecular Genetics, University of Virginia Medical Center, University of Virginia, Charlottesville, Virginia 22908

Received 26 January 2005/ Accepted 10 March 2005

Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, University of Virginia Medical Center, 1300 Jefferson Park Avenue, Box 800733, Charlottesville VA 22908-07333. Phone: 434-982-5482. Fax: 434-982-4834. E-mail: djb6t{at}virginia.edu.

Eukaryotic Cell, May 2005, p. 867-878, Vol. 4, No. 5
1535-9778/05/$08.00+0     doi:10.1128/EC.4.5.867-878.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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