Purification and characterisation of the fission yeast Ndc80 complex
Protein Expression and Purification 135 巻
61-69 頁
2017-07 発行
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| ファイル情報(添付) | |
| タイトル ( eng ) |
Purification and characterisation of the fission yeast Ndc80 complex
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| 作成者 |
Matsuo Yuzy
Maurer Sebastian P.
Surrey Thomas
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| 収録物名 |
Protein Expression and Purification
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| 巻 | 135 |
| 開始ページ | 61 |
| 終了ページ | 69 |
| 抄録 |
The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment. However, the purification of the Ndc80 complex from this yeast has not been achieved, which hampers the in-depth investigation as to how the outer kinetochore attaches to the plus end of the spindle microtubule. Here we report the two-step purification of the fission yeast Ndc80 holo complex from bacteria. First, we purified separately two sub-complexes consisting of Ndc80-Nuf2 and Spc24-Spc25. Then, these two sub-complexes were mixed and applied to size-exclusion chromatography. The reconstituted Ndc80 holo complex is composed of four subunits with equal stoichiometry. The complex possesses microtubule-binding activity, and Total Internal Reflection Fluorescence (TIRF)-microscopy assays show that the complex binds the microtubule lattice. Interestingly, unlike the human complex, the fission yeast complex does not track depolymerising microtubule ends. Further analysis shows that under physiological ionic conditions, the Ndc80 holo complex does not detectably bind Dis1, but instead it interacts with Mal3/EB1, by which the Ndc80 complex tracks the growing microtubule plus end. This result substantiates the notion that the Ndc80 complex plays a crucial role in establishment of the dynamic kinetochore-microtubule interface by cooperating with chTOG and EB1.
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| 著者キーワード |
Dis1
Fission yeast
Kinetochore
Ndc80 complex
Microtubule
TIRF microscopy
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| 内容記述 |
This work was supported by Cancer Research UK and the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001163, FC001184), the UK Medical Research Council (FC001163, FC001184), and the Wellcome Trust (FC001163, FC001184) (T.S., T.T.), the Japan Society for the Promotion of Science KAKENHI Scientific Research (A) (16H02503) (T.T.), Challenging Exploratory Research (16K14672) (T.T.), the Naito Foundation (T.T.), the Uehara Memorial Foundation (T.T) and a Marie Curie fellowship (PIEF-GA-2009-253043) (S.P.M.).
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| 言語 |
英語
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| 資源タイプ | 学術雑誌論文 |
| 出版者 |
Elsevier
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| 発行日 | 2017-07 |
| 権利情報 |
© 2017 The Francis Crick Institute. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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| 出版タイプ | Version of Record（出版社版。早期公開を含む） |
| アクセス権 | オープンアクセス |
| 収録物識別子 |
[ISSN] 1046-5928
[DOI] 10.1016/j.pep.2017.05.002
[PMID] 28502666
[DOI] https://doi.org/10.1016/j.pep.2017.05.002
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