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PMID: 12944469 Published · ppublish English Journal Article

Recognizing chromosomes in trouble: association of the spindle checkpoint protein Bub3p with altered kinetochores and a unique defective centromere.

Molecular and cellular biology ·Vol. 23 ·No. 18 ·2003-09-00 ·Pages 6406-18

Kerscher O, Crotti LB, Basrai MA

Abstract

Spindle checkpoint proteins monitor the interaction of the spindle apparatus with the kinetochores, halting anaphase even if the microtubule attachment of only a single chromosome is altered. In this study, we show that Bub3p of Saccharomyces cerevisiae, an evolutionarily conserved spindle checkpoint protein, exhibits distinct interactions with an altered or defective kinetochore(s). We show for the first time that green fluorescent protein-tagged S. cerevisiae Bub3p (Bub3-GFP) exhibits not only a diffuse nuclear localization pattern but also forms distinct nuclear foci in unperturbed growing and G(2)/M-arrested cells. As Bub3-GFP foci overlap only a subset of kinetochores, we tested a model in which alterations or defects in kinetochore or spindle integrity lead to the distinct enrichment of Bub3p at these structures. In support of our model, kinetochore-associated Bub3-GFP is enriched upon activation of the spindle checkpoint due to nocodazole-induced spindle disassembly, overexpression of the checkpoint kinase Mps1p, or the presence of a defective centromere (CEN). Most importantly, using a novel approach with the chromatin immunoprecipitation (ChIP) technique and genetically engineered defective CEN [CF/CEN6(Delta31)], we determined that Bub3-GFP can associate with a single defective kinetochore. Our studies represent the first comprehensive molecular analysis of spindle checkpoint protein function in the context of a wild-type or defective kinetochore(s) by use of live-cell imaging and the ChIP technique in S. cerevisiae.

MeSH Terms
Base Sequence Cell Cycle/drug effects,genetics Cell Cycle Proteins/genetics,metabolism Cell Nucleus/genetics,metabolism Centromere/genetics,metabolism Chromosomes, Fungal DNA-Binding Proteins/genetics,metabolism Gene Expression Regulation, Fungal Green Fluorescent Proteins Kinetochores/metabolism Luminescent Proteins/genetics,metabolism Molecular Sequence Data Nocodazole/pharmacology Protein Kinases/genetics,metabolism Protein Serine-Threonine Kinases/genetics,metabolism Protein-Tyrosine Kinases/genetics,metabolism Recombinant Proteins/genetics,metabolism Saccharomyces cerevisiae/drug effects,genetics Saccharomyces cerevisiae Proteins/genetics,metabolism Spindle Apparatus/metabolism,physiology
Chemicals
BUB3 protein, S cerevisiae CBF2 protein, S cerevisiae Cell Cycle Proteins DNA-Binding Proteins Luminescent Proteins Recombinant Proteins Saccharomyces cerevisiae Proteins Green Fluorescent Proteins Protein Kinases Protein-Tyrosine Kinases Bub1 spindle checkpoint protein Protein Serine-Threonine Kinases MPS1 protein, S cerevisiae Nocodazole
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Kerscher Oliver
Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 8901 Wisconsin Avenue, Bethesda, MD 20889-5101, USA.
Crotti Luciana B
Basrai Munira A
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.23.18.6406-6418.2003
Published
2003-09-00
Pages
6406-18
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC193694
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.23.18.6406-6418.2003 PMC Full Text View journal details
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