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Vol. 16, Issue 1, 385-395, January 2005

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The A78V Mutation in the Mad3-like Domain of Schizosaccharomyces pombe Bub1p Perturbs Nuclear Accumulation and Kinetochore Targeting of Bub1p, Bub3p, and Mad3p and Spindle Assembly Checkpoint Function

Sheila Kadura ^*, Xiangwei He  , Vincent Vanoosthuyse , Kevin G. Hardwick , and Shelley Sazer ^*  ||

Verna and Marrs McClean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030; ^* Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; and Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom

Submitted July 6, 2004; Revised October 19, 2004; Accepted October 21, 2004
Monitoring Editor: Mark Solomon

During mitosis, the spindle assembly checkpoint (SAC) responds to faulty attachments between kinetochores and the mitotic spindle by imposing a metaphase arrest until the defect is corrected, thereby preventing chromosome missegregation. A genetic screen to isolate SAC mutants in fission yeast yielded point mutations in three fission yeast SAC genes: mad1, bub3, and bub1. The bub1-A78V mutant is of particular interest because it produces a wild-type amount of protein that is mutated in the conserved but uncharacterized Mad3-like region of Bub1p. Characterization of mutant cells demonstrates that the alanine at position 78 in the Mad3-like domain of Bub1p is required for: 1) cell cycle arrest induced by SAC activation; 2) kinetochore accumulation of Bub1p in checkpoint-activated cells; 3) recruitment of Bub3p and Mad3p, but not Mad1p, to kinetochores in checkpoint-activated cells; and 4) nuclear accumulation of Bub1p, Bub3p, and Mad3p, but not Mad1p, in cycling cells. Increased targeting of Bub1p-A78V to the nucleus by an exogenous nuclear localization signal does not significantly increase kinetochore localization or SAC function, but GFP fused to the isolated Bub1p Mad 3-like accumulates in the nucleus. These data indicate that Bub1p-A78V is defective in both nuclear accumulation and kinetochore targeting and that a threshold level of nuclear Bub1p is necessary for the nuclear accumulation of Bub3p and Mad3p.

Abbreviations used: SAC, spindle assembly checkpoint; Bub, budding uninhibited by benomyl; Mad, mitotic arrest deficient; NLS, nuclear localization signal; MBC, carbendazim; TBZ, thiabendazole; HU, hydroxyurea; YE, yeast extract; EMM, Edinburgh minimal media.

Present address: Department of Human and Molecular Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

^|| Corresponding author. E-mail address: ssazer{at}bcm.tmc.edu.

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