A stable sub-complex between GCP4, GCP5 and GCP6 promotes the assembly of γ-tubulin ring complexes

gamma-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 (gamma-tubulin complex proteins): gamma-tubulin small complexes (gamma TuSCs) that contain gamma-tubulin, and GCPs 2 and 3; and gamma-tubulin ring complexes (gamma TuRCs) that contain multiple gamma TuSCs in addition to GCPs 4, 5 and 6. Whereas the structure and assembly properties of gamma TuSCs have been intensively studied, little is known about the assembly of gamma TuRCs 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 gamma TuRC that assembles independently of the presence of gamma TuSCs. Incubation of this sub-complex with cytoplasmic extracts containing gamma TuSCs leads to the reconstitution of gamma TuRCs 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. gamma TuRC.