Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY.
Laboratory of Cell Biology, The Rockefeller University, New York, NY.
J Cell Biol. 2021 Mar 1;220(3). doi: 10.1083/jcb.202009146.
The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge" (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide-dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8-10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the holocomplex.
细胞微管网络的形成受到γ-微管蛋白环复合物（γ-TuRC）的调节。这个由>31 种蛋白质组成的∼2.3 MD 组装体包括γ-微管蛋白和 GCP2-6，以及 MZT1 和一个在“腔桥”（LB）中的肌动蛋白样蛋白。由于重新构建γ-TuRC 的挑战限制了对其组装和功能的分析。在这里，我们报告了一种人γ-TuRC（γ-TuRC-GFP）的生化重构，它是一种∼35 S 的复合物，可在体外核微管。此外，我们生成了一个缺少 MZT1 和肌动蛋白的亚复合物γ-TuRCΔLB-GFP。我们表明，γ-TuRCΔLB-GFP 以鸟嘌呤核苷酸依赖性方式核微管，并具有与全复合物相似的效率。电子显微镜显示，γ-TuRC-GFP 类似于天然的γ-TuRC 结构，而γ-TuRCΔLB-GFP 采用部分锥形形状，仅呈现 8-10 个γ-微管蛋白亚基，并且缺乏有序的腔桥。我们的结果表明，γ-TuRC 可以使用有限数量的蛋白质进行重构，并表明 LB 有助于围绕全复合物中的微管核“核心”自组装调节界面。
