Centre de Biologie du Développement, Centre de Biologie Intégrative, CNRS-Université Toulouse III, 31062 Toulouse, France.
Centre de Biologie du Développement, Centre de Biologie Intégrative, CNRS-Université Toulouse III, 31062 Toulouse, France
J Cell Sci. 2020 Jun 3;133(11):jcs244368. doi: 10.1242/jcs.244368.
γ-Tubulin is the main protein involved in the nucleation of microtubules in all eukaryotes. It forms two different complexes with proteins of the GCP family (γ-tubulin complex proteins): γ-tubulin small complexes (γTuSCs) that contain γ-tubulin, and GCPs 2 and 3; and γ-tubulin ring complexes (γTuRCs) that contain multiple γTuSCs in addition to GCPs 4, 5 and 6. Whereas the structure and assembly properties of γTuSCs have been intensively studied, little is known about the assembly of γTuRCs and the specific roles of GCPs 4, 5 and 6. Here, we demonstrate that two copies of GCP4 and one copy each of GCP5 and GCP6 form a salt (KCl)-resistant sub-complex within the γTuRC that assembles independently of the presence of γTuSCs. Incubation of this sub-complex with cytoplasmic extracts containing γTuSCs leads to the reconstitution of γTuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the N-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs. We also demonstrate that these are involved in the assembly or stabilization of the γTuRC.
γ-微管蛋白是所有真核生物中微管成核的主要蛋白。它与 GCP 家族（γ-微管蛋白复合物蛋白）的蛋白质形成两种不同的复合物：γ-微管蛋白小复合物（γTuSCs），包含 γ-微管蛋白和 GCPs2 和 3；以及 γ-微管蛋白环复合物（γTuRCs），除了 GCPs4、5 和 6 之外，还包含多个 γTuSCs。虽然 γTuSCs 的结构和组装特性已经得到了深入研究，但关于 γTuRC 的组装以及 GCPs4、5 和 6 的特定作用知之甚少。在这里，我们证明了两个 GCP4 拷贝和一个 GCP5 和 GCP6 拷贝形成一个盐（KCl）抗性亚复合物，该亚复合物在 γTuSCs 存在的情况下独立组装。将该亚复合物与含有 γTuSCs 的细胞质提取物孵育会导致能够起始微管的 γTuRC 的重建。此外，我们研究了在 GCP6 的 N 末端以及 GCP6 的 grip1 和 grip2 基序之间特异性发现的序列延伸和插入。我们还证明了这些序列延伸和插入参与了 γTuRC 的组装或稳定。
