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Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes.

Rischitor PE, May KM, Hardwick KG - PLoS ONE (2007)

Bottom Line: Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors.Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1.We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

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Bub1-Tel is sufficient to recruit both Bub3p and Mad3p.(A) Bub1-Tel recruits and co-localises with Bub3-mCherry at telomeres. (B) Bub1-Tel recruits and co-localises with Mad3-tdTomato at telomeres. Scale bars are 5 µm. (C) Quantitation of the co-localisation observed between checkpoint proteins and telomeres (Pot1), or kinetochores (Ndc80). Full co-localisation was scored when all of the telomere (or kinetochore) foci observed in a given cell co-localised with the Bub1-Tel and either Bub3p (grey columns) or Mad3p (red columns). See supplementary data (Tables S1, S2, S3 and S4) for further details. (D) Speculative model of Bub1 scaffold action at a telomere (TEL). Note, due to low signal intensity, we have not yet demonstrated that Bub3p and Mad3p exchange rapidly at the telomeres.
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pone-0001342-g003: Bub1-Tel is sufficient to recruit both Bub3p and Mad3p.(A) Bub1-Tel recruits and co-localises with Bub3-mCherry at telomeres. (B) Bub1-Tel recruits and co-localises with Mad3-tdTomato at telomeres. Scale bars are 5 µm. (C) Quantitation of the co-localisation observed between checkpoint proteins and telomeres (Pot1), or kinetochores (Ndc80). Full co-localisation was scored when all of the telomere (or kinetochore) foci observed in a given cell co-localised with the Bub1-Tel and either Bub3p (grey columns) or Mad3p (red columns). See supplementary data (Tables S1, S2, S3 and S4) for further details. (D) Speculative model of Bub1 scaffold action at a telomere (TEL). Note, due to low signal intensity, we have not yet demonstrated that Bub3p and Mad3p exchange rapidly at the telomeres.

Mentions: To test whether this Bub1-scaffold was sufficient to recruit Bub3p and Mad3p to telomeres we crossed in either Mad3-tdTomato or Bub3-mCherry. The Mad3 and Bub3 fusion proteins co-localised very well with the telomere marker (Pot1-CFP) and the Bub1-Tel (GFP) (Fig. 3 and Supplementary Tables S1, S2, S3 and S4). This simple experiment demonstrates that targeting of Bub1p to an ectopic site on a chromosome, is sufficient to recruit other checkpoint proteins to that site.


Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes.

Rischitor PE, May KM, Hardwick KG - PLoS ONE (2007)

Bub1-Tel is sufficient to recruit both Bub3p and Mad3p.(A) Bub1-Tel recruits and co-localises with Bub3-mCherry at telomeres. (B) Bub1-Tel recruits and co-localises with Mad3-tdTomato at telomeres. Scale bars are 5 µm. (C) Quantitation of the co-localisation observed between checkpoint proteins and telomeres (Pot1), or kinetochores (Ndc80). Full co-localisation was scored when all of the telomere (or kinetochore) foci observed in a given cell co-localised with the Bub1-Tel and either Bub3p (grey columns) or Mad3p (red columns). See supplementary data (Tables S1, S2, S3 and S4) for further details. (D) Speculative model of Bub1 scaffold action at a telomere (TEL). Note, due to low signal intensity, we have not yet demonstrated that Bub3p and Mad3p exchange rapidly at the telomeres.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2147072&req=5

pone-0001342-g003: Bub1-Tel is sufficient to recruit both Bub3p and Mad3p.(A) Bub1-Tel recruits and co-localises with Bub3-mCherry at telomeres. (B) Bub1-Tel recruits and co-localises with Mad3-tdTomato at telomeres. Scale bars are 5 µm. (C) Quantitation of the co-localisation observed between checkpoint proteins and telomeres (Pot1), or kinetochores (Ndc80). Full co-localisation was scored when all of the telomere (or kinetochore) foci observed in a given cell co-localised with the Bub1-Tel and either Bub3p (grey columns) or Mad3p (red columns). See supplementary data (Tables S1, S2, S3 and S4) for further details. (D) Speculative model of Bub1 scaffold action at a telomere (TEL). Note, due to low signal intensity, we have not yet demonstrated that Bub3p and Mad3p exchange rapidly at the telomeres.
Mentions: To test whether this Bub1-scaffold was sufficient to recruit Bub3p and Mad3p to telomeres we crossed in either Mad3-tdTomato or Bub3-mCherry. The Mad3 and Bub3 fusion proteins co-localised very well with the telomere marker (Pot1-CFP) and the Bub1-Tel (GFP) (Fig. 3 and Supplementary Tables S1, S2, S3 and S4). This simple experiment demonstrates that targeting of Bub1p to an ectopic site on a chromosome, is sufficient to recruit other checkpoint proteins to that site.

Bottom Line: Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors.Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1.We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

Show MeSH