Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].
Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex [corrected].
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DOI：
10.1016/j.cub.2015.05.033
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发表时间：
2015-06-29
期刊：
影响因子：
--
通讯作者：
Wakefield JG
中科院分区：
文献类型：
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作者：
Palumbo V;Pellacani C;Heesom KJ;Rogala KB;Deane CM;Mottier-Pavie V;Gatti M;Bonaccorsi S;Wakefield JG
有丝分裂纺锤体主要由微管 (MT) 组成，微管由 α- 和 β- 微管蛋白异二聚体聚合产生。微管蛋白经历一系列蛋白质折叠和翻译后修饰以实现其功能。微管蛋白聚合的缺陷极大地影响纺锤体的形成并破坏染色体分离。我们最近描述了保守的 misato (mst) 基因产物在调节果蝇有丝分裂 MT 生成中的作用，但 Mst 的分子功能仍然未知。在这里，我们使用亲和纯化质谱 (AP-MS) 来鉴定果蝇胚胎中 Mst 的相互作用伙伴。我们证明 Mst 以化学计量与异八聚微管蛋白伴侣蛋白 1 (TCP-1) 复合物、异六聚微管蛋白预折叠蛋白复合物以及在生成 MT 活性微管蛋白中具有保守作用的蛋白质相关联。我们发现，RNAi 介导的任何 TCP-1 亚基的体内耗竭都会影响 mst 或 Prefoldin 编码基因旋转木马 (mgr) 突变的影响，导致单极且杂乱的有丝分裂纺锤体含有很少的 MT。至关重要的是，我们证明 TCP-1 复合体稳定性需要 Mst，而不是 Mgr，并且微管蛋白聚合效率和微管蛋白稳定性在 mst 突变体中都受到严重损害。此外，我们的结构生物信息学分析表明，Mst 类似于微管蛋白单体的三维结构，因此可能占据 TCP-1 复合体的中央空腔。总的来说，我们的结果表明 Mst 作为 TCP-1 复合物的辅助因子，在正确组装纺锤体 MT 所需的微管蛋白折叠过程中发挥着重要作用。 Misato 与微管蛋白伴侣蛋白-1 (TCP-1) 复合物发生生化相互作用 Misato 可能通过填充其微管蛋白折叠腔来稳定 TCP-1 复合物 Misato 或 TCP-1 复合物亚基的丢失会导致类似的有丝分裂表型 在没有 Misato 的情况下，微管蛋白不稳定并且无法有效聚合 Palumbo 等人。显示保守蛋白 Misato 与果蝇中的微管蛋白伴侣蛋白 1 (TCP-1) 复合物相互作用。在 Misato 缺失的情况下，TCP-1 亚基水平会降低，功能性微管蛋白不稳定，有丝分裂微管的生成也会受到影响。 Misato 的预测结构表明，它在微管蛋白缺失的情况下充当分子占位符，稳定 TCP-1 复合物。
Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs. Misato interacts biochemically with the Tubulin Chaperone Protein-1 (TCP-1) complex Misato stabilizes the TCP-1 complex, possibly by filling its Tubulin-folding cavity Loss of Misato or TCP-1 complex subunits leads to similar mitotic phenotypes In the absence of Misato, Tubulin is unstable and unable to efficiently polymerize Palumbo et al. show the conserved protein Misato interacts with the Tubulin Chaperone Protein-1 (TCP-1) complex in Drosophila. In the absence of Misato, TCP-1 subunit levels are reduced, functional Tubulin is unstable, and mitotic microtubule generation is compromised. The predicted structure of Misato suggests it acts as a molecular placeholder in the absence of Tubulin, stabilizing the TCP-1 complex.