Distinct chromosome segregation roles for spindle checkpoint proteins.
The spindle checkpoint plays a central role in the fidelity of chromosome transmission by ensuring that anaphase is initiated only after kinetochore-microtubule associations of all sister chromatid pairs are complete. In this study, we find that known spindle checkpoint proteins do not contribute equally to chromosome segregation fidelity in Saccharomyces cerevisiae. Loss of Bub1 or Bub3 protein elicits the largest effect. Analysis of Bub1p reveals the presence of two molecular functions. An N-terminal 608-amino acid (nonkinase) portion of the protein supports robust checkpoint activity, and, as expected, contributes to chromosome segregation. A C-terminal kinase-encoding segment independently contributes to chromosome segregation through an unknown mechanism. Both molecular functions depend on association with Bub3p. A 156-amino acid fragment of Bub1p functions in Bub3p binding and in kinetochore localization by one-hybrid assay. An adjacent segment is required for Mad1p binding, detected by deletion analysis and coimmunoprecipitation. Finally, overexpression of wild-type BUB1 or MAD3 genes leads to chromosome instability. Analysis of this activity indicates that the Bub3p-binding domain of Bub1p contributes to this phenotype through disruption of checkpoint activity as well as through introduction of kinetochore or spindle damage.
Warren
Cheryl D
CD
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Brady
D Michelle
DM
Johnston
Raymond C
RC
Hanna
Joseph S
JS
Hardwick
Kevin G
KG
Spencer
Forrest A
FA
D000483:Alleles; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology; D020090:Chromosome Segregation; D002875:Chromosomes / Q000502:physiology* / Q000648:ultrastructure; D005656:Fungal Proteins; D015151:Immunoblotting; D009154:Mutation; D009687:Nuclear Proteins; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011494:Protein Kinases / Q000502:physiology; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000502:physiology* / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques
C070352:BUB3 protein, S cerevisiae; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; C406764:MAD3 protein, S cerevisiae; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases