A stable core of GCPs 4, 5 and 6 promotes the assembly of γ-tubulin ring complexes

Abstract

{gamma}-tubulin is a major 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), containing {gamma}-tubulin and GCPs 2 and 3, and {gamma}-tubulin ring complexes ({gamma}TuRCs), containing 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 about 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-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 full-sized {gamma}TuRCs that are competent to nucleate microtubules. In addition, we investigate sequence extensions and insertions that are specifically found at the amino-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs, and we demonstrate that these are involved in the assembly or stabilization of the {gamma}TuRC. Summary statement{gamma}-tubulin ring complexes are templates for microtubule nucleation, composed of {gamma}-tubulin and GCP proteins. GCPs 4, 5, 6 form a stable sub-complex, driving the assembly of the full complex.