title: 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.
abstract: 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.
authors.affiliation: Verna and Marrs McClean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Sheila
authors.initials: S
authors.lastname: Kadura
authors.forename: Xiangwei
authors.initials: X
authors.lastname: He
authors.forename: Vincent
authors.initials: V
authors.lastname: Vanoosthuyse
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Hardwick
authors.forename: Shelley
authors.initials: S
authors.lastname: Sazer
mesh_terms: D000409:Alanine / Q000737:chemistry; D000483:Alleles; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D005656:Fungal Proteins; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007399:Interphase; D018386:Kinetochores / Q000378:metabolism; D008677:Metaphase; D008856:Microscopy, Fluorescence; D008938:Mitosis; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins; D017354:Point Mutation; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D008941:Spindle Apparatus
chemical_list: C070352:BUB3 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; C406764:MAD3 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C512748:mad3 protein, S pombe; D049452:Green Fluorescent Proteins; D011494:Protein Kinases; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; D000409:Alanine