Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex
Misato Controls Mitotic Microtubule Generation by Stabilizing the TCP-1 Tubulin Chaperone Complex
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DOI：
10.1016/j.cub.2015.07.035
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发表时间：
2015-08-03
期刊：
影响因子：
9.2
通讯作者：
Wakefield JG
中科院分区：
文献类型：
--
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而成[1，2]。微管蛋白经历了一系列的蛋白质折叠和翻译后修饰以完成它们的功能[3，4]。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MST基因产物在调节果蝇有丝分裂MT生成中的作用[5]，但MST的分子功能仍不清楚。在这里，我们使用亲和纯化质谱仪(AP-MS)来鉴定果蝇胚胎中MST的相互作用伙伴。我们证明了MST与微管蛋白伴侣复合体TCP-1/CCT、异源六聚体微管蛋白前折叠蛋白复合体以及在产生MT活性微管蛋白中具有保守作用的蛋白质之间的化学计量学联系。我们发现，在体内，RNAi介导的任何TCP-1亚单位的耗尽都会复制前折叠蛋白编码基因旋转(MGR)突变的影响，导致含有少量MT的单极和无序的有丝分裂纺锤体。至关重要的是，我们证明了TCP1复合体的稳定性需要MST，而不是MGR，并且在突变株中，微管蛋白聚合的效率和微管蛋白的稳定性都受到了极大的影响。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，因此可能占据了TCP-1复杂的中央空腔。综上所述，我们的结果表明，MST作为TCP-1复合体的辅助因子，在正确组装纺锤体MT所需的微管蛋白折叠过程中发挥重要作用。
Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conservedmisato(mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in theDrosophilaembryo. We demonstrate that Mst associates stoichiometrically with the Tubulin chaperone complex, TCP-1/CCT, 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 inmstor the Prefoldin-encoding genemerry-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 inmstmutants. 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.