Mechanisms of Disease Programme, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Baldiri Reixac 10, 08028 Barcelona, Spain.
Structural Biology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029 Madrid, Spain.
Sci Adv. 2020 Dec 18;6(51). doi: 10.1126/sciadv.abe0894. Print 2020 Dec.
The microtubule nucleator γ-tubulin ring complex (γTuRC) is essential for the function of microtubule organizing centers such as the centrosome. Since its discovery over two decades ago, γTuRC has evaded in vitro reconstitution and thus detailed structure-function studies. Here, we show that a complex of RuvB-like protein 1 (RUVBL1) and RUVBL2 "RUVBL" controls assembly and composition of γTuRC in human cells. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous coexpression system. RUVBL interacts with γTuRC subcomplexes but is not part of fully assembled γTuRC. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC as revealed by its cryo-electron microscopy (cryo-EM) structure at ~4.0-Å resolution. We further use cryo-EM to identify features that determine the intricate, higher-order γTuRC architecture. Our work finds RUVBL as an assembly factor that regulates γTuRC in cells and allows production of recombinant γTuRC for future in-depth mechanistic studies.
微管核化因子 γ-微管蛋白环复合物（γTuRC）对于微管组织中心（如中心体）的功能至关重要。自二十多年前发现以来，γTuRC 一直难以在体外进行重组，因此无法进行详细的结构-功能研究。在这里，我们表明 RuvB 样蛋白 1（RUVBL1）和 RuvB 样蛋白 2（RUVBL2）“RUVBL”复合物控制着人类细胞中 γTuRC 的组装和组成。同样，RUVBL 在异源共表达系统中从一组最小的核心亚基组装 γTuRC。RUVBL 与 γTuRC 亚复合物相互作用，但不是完全组装的 γTuRC 的一部分。纯化的重组 γTuRC 具有成核活性，并通过其约 4.0-Å 的低温电子显微镜（cryo-EM）结构显示出类似于天然 γTuRC 的特性。我们进一步使用 cryo-EM 来确定决定复杂的、更高阶的 γTuRC 结构的特征。我们的工作发现 RUVBL 作为一种组装因子，调节细胞中的 γTuRC，并允许生产重组 γTuRC，以进行未来更深入的机制研究。
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