Asymmetric Molecular Architecture of the Human γ-Tubulin Ring Complex

Abstract

Highlightsd High resolution cryo-EM structure of the native human gtubulin ring complex (g-TuRC)d Identification and structural models for g-tubulin, GCP2, GCP3, GCP4, GCP5, and GCP6d Identification of an actin-like protein in the g-TuRC lumen d Insights into the regulation of microtubule nucleation by the g-TuRC

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

“…We demonstrate that GCPs 4, 5, and 6 form a stable sub-complex that permits the association with γTuSCs into a functional γTuRC. Within the sub-complex, the stoichiometric ratio of these GCPs matches the values found in recently reported structures of native γTuRCs (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). We find that GCPs 2 and 3 are also present in our preparations of the sub-complex, but at a molar ratio equivalent to half-a-γTuSC.…”

“…Since Wieczorek et al (2020) and Liu et al (2019) suggested the existence of γTuSC-like structures, containing pairs of GCPs 4/5 or GCPs 4/6, we tested how the depletion of individual GCPs 4, 5, or 6 affected the composition of the GCP4/5/6 sub-complex, and whether the proposed γTuSC-like structures did indeed exist. We depleted each of these three GCPs individually by RNAi, which resulted in each case in the loss of γTuRCs and in the accumulation of γTuSCs, as seen in sucrose gradients (Fig.…”

“…GCPs 4, 5, and 6 are essential for the assembly and/or for the stabilization of γTuRCs, since depletion of either component causes a reduction of γTuRCs both at the centrosome and in the cytoplasm (Izumi et al, 2008; Bahtz et al, 2012; Scheidecker et al, 2015; Farache et al, 2016; Cota et al, 2017). Besides GCPs 4, 5, 6, additional factors may still be needed, such as Mozart1, actin, or other proteins corresponding to unassigned densities in cryo-electron microscopy structures of native γTuRCs (Lin et al, 2016; Cota et al, 2017; Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). It is now clear that GCPs 4, 5, and 6 integrate into the helical wall of the γTuRC, and that they are laterally bound to γTuSCs, but their specific role within the γTuRC still remains to be determined (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019).…”

“…Besides GCPs 4, 5, 6, additional factors may still be needed, such as Mozart1, actin, or other proteins corresponding to unassigned densities in cryo-electron microscopy structures of native γTuRCs (Lin et al, 2016; Cota et al, 2017; Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). It is now clear that GCPs 4, 5, and 6 integrate into the helical wall of the γTuRC, and that they are laterally bound to γTuSCs, but their specific role within the γTuRC still remains to be determined (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). Our results show that they form a nucleus promoting the stable assembly of the 2MDa complex.…”

“…Because these proteins are present only in one or two copies per complex (Murphy et al, 2001; Choi et al, 2010), and because the rescue experiments with chimeras suggest that GCPs 4, 5, 6 occupy non-random positions, their localisation within the complex is of particular interest. During the course of this work, three studies have described the structure of native γTuRCs by cry-electron microscopy, and have found a lateral association of four γTuSCs, bound to a lateral array of GCP4/GCP5/GCP4/GCP6, to which an additional γTuSC was associated (Wieczorek et al, 2020; Consolati et al, 2019; Liu et al, 2019). Altogether, this has raised the question whether GCPs 4, 5, 6 form assembly intermediates equivalent to γTuSCs.…”