Reconstitution and characterization of budding yeast γ-tubulin complex
Reconstitution and characterization of budding yeast γ-tubulin complex
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DOI：
10.1091/mbc.02-01-0607
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发表时间：
2002-04-01
影响因子：
3.3
通讯作者：
Davis, TN
中科院分区：
文献类型：
--
作者：
Vinh, DBN;Kern, JW;Davis, TN
微管的成核是有丝分裂纺锤体组装的中心，这是每次细胞分裂所必需的。 γ-微管蛋白是中心体微管成核所必需的通用成分。为了阐明芽殖酵母中微管成核的机制，我们重建并表征了昆虫细胞中产生的酵母 T-微管蛋白复合物（Tub4p 复合物）。重组复合物与酵母细胞提取物中的天然复合物具有相同的沉降系数(11.6S)，并含有1分子Spc97p、1分子Spc98p和2分子Tub4p。重建的 Tub4p 复合物与预先形成的微管结合，并具有较低的成核活性，使我们能够开始详细分析增强这种成核活性的条件。我们测试了重组 Tub4p 复合物与纺锤体极体对接蛋白 Spc110p 的结合是否影响其成核活性。通过与酵母钙调蛋白 (Cmd1p) 共表达，重组 Spc110p 在昆虫细胞中的溶解度得到提高。 Spc110p/Cmd1p 复合物具有较小的沉降系数 (4.2 S) 和较大的斯托克斯半径 (14.3 nm)，表明其结构细长。 Tub4p 复合物通过 Spc98p 结合 Spc110p/Cmd1p，结合的 K-d 为 150 nM。当 Tub4p 复合物与 Spc110p/Cmd1p 结合时，其低成核活性并未增强，这表明它需要额外的成分或修饰才能实现强大的活性。最后，我们报告了酵母提取物中大型 22 S Tub4p 复合物的鉴定，其中含有 Spc97p 多聚体，类似于高等真核细胞中发现的 γ-微管蛋白环复合物。
Nucleation of microtubules is centra-1 to assembly of the mitotic spindle, which is required for each cell division. gamma-Tubulin is a universal component essential for microtubule nucleation from centrosomes. To elucidate the mechanism of microtubule nucleation in budding yeast we reconstituted and characterized the yeast T-tubulin complex (Tub4p complex) produced in insect cells. The recombinant complex has the same sedimentation coefficient (11.6 S) as the native complex in yeast cell extracts and contains one molecule of Spc97p, one molecule of Spc98p, and two molecules of Tub4p. The reconstituted Tub4p complex binds preformed microtubules and has a low nucleating activity, allowing us to begin a detailed analysis of conditions that enhance this nucleating activity. We tested whether binding of the recombinant Tub4p complex to the spindle pole body docking protein Spc110p affects its nucleating activity. The Solubility of recombinant Spc110p in insect cells is improved by coexpression with yeast calmodulin (Cmd1p). The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S) and a large Stokes radius (14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via Spc98p and the K-d for binding is 150 nM. The low nucleation activity of the Tub4p complex is not enhanced when it is bound to Spc110p/Cmd1p, suggesting that it requires additional components or modifications to achieve robust activity. Finally, we report the identification of a large 22 S Tub4p complex in yeast extract that contains multimers of Spc97p similar to gamma-tubulin ring complexes found in higher eukaryotic cells.