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.
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.
Kadura
Sheila
S
Verna and Marrs McClean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
He
Xiangwei
X
Vanoosthuyse
Vincent
V
Hardwick
Kevin G
KG
Sazer
Shelley
S
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
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