Assembly of the asymmetric human γ-tubulin ring complex by RUVBL1-RUVBL2 AAA ATPase

Abstract

The microtubule nucleator γ-tubulin ring complex (γTuRC) is essential for the function of microtubule organizing centers such as the centrosome. Since its discovery over two decades ago, γTuRC has evaded in vitro reconstitution and thus detailed structure-function studies. Here, we show that a complex of RuvB-like protein 1 (RUVBL1) and RUVBL2 “RUVBL” controls assembly and composition of γTuRC in human cells. Likewise, RUVBL assembles γTuRC from a minimal set of core subunits in a heterologous coexpression system. RUVBL interacts with γTuRC subcomplexes but is not part of fully assembled γTuRC. Purified, reconstituted γTuRC has nucleation activity and resembles native γTuRC as revealed by its cryo–electron microscopy (cryo-EM) structure at ~4.0-Å resolution. We further use cryo-EM to identify features that determine the intricate, higher-order γTuRC architecture. Our work finds RUVBL as an assembly factor that regulates γTuRC in cells and allows production of recombinant γTuRC for future in-depth mechanistic studies.

The gamma‐tubulin ring complex: Deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator

“…In the most extensive study, Zimmermann et al characterized the cryo-EM structure and assembly mechanism of the recombinant γ-TuRC and showed that the recombinant γ-TuRC recapitulates the structural organization of the native γ-TuRC isolated from frog extracts or human tissue culture cells. [60] Remarkably, even though Zimmermann et al did not co-express actin in their recombinant system, the purified γ-TuRC contained actin as part of the luminal bridge, indicating that insect cell actin can replace human actin in the recombinant human γ-TuRC. Moreover, the authors showed that co-expression of human GCP2, GCP3 and γ-tubulin together with both MZT1 and MZT2 allows assembly of a stable human γ-TuSC unit that had a tendency to selfinteract and form small γ-TuSC oligomers.…”

“…The central aspect of the study by Zimmermann et al is the discovery of the RUVBL1-RUVBL2 AAA+ ATPase (Figure 4A) as a cofactor for efficient assembly of the γ-TuRC, both for recombinant expression systems as well as native biogenesis of the γ-TuRC in mammalian cells. [60] The authors showed that RUVBL1-RUVBL2 associates with the human γ-TuSC in vitro and when co-expressed with recombinant γ-TuSC in insect cells. However, co-expression was not required for formation of stable γ-TuSC units, suggesting that the AAA+ ATPase is rather involved in follow-up steps of γ-TuRC assembly.…”

“…This is consistent with negative stain EM analysis, which indicates binding of the heterohexameric RUVBL1-RUVBL2 ATPase to the open ends of self-assembled γ-TuSC oligomers. [60] While Zimmermann et al identified RUVBL1-RUVBL2 activity to be important for efficient assembly of human γ-TuRC, the detailed mechanism remains unclear. RUVBL1 and RUVBL2 are highly conserved AAA+ ATPases with diverse functions ranging from chromatin remodeling and nonsense-mediated mRNA decay to the assembly of kinases (for example mTORC1, ATM, ATR), RNA polymerases and small nucleolar ribonucleoprotein complexes.…”

“…A likely scenario is that all GCP3 copies (and potentially also GCP5) [34] are associated with MZT1, but the remaining modules are not stably bound to the γ-TuRC core and therefore too flexible to be visualized in an averaging-based approach like cryo-EM. [40,41,43,60] Alternatively, only specific copies of GCP3 might be stably associated with MZT1, depending on their position and molecular surrounding in the γ-TuRC, while MZT1 may dissociate from other GCP3 copies after incorporation into the γ-TuRC.…”

Autoinhibition of Cnn binding to γ-TuRCs prevents ectopic microtubule nucleation and cell division defects

“…Recent structural studies have shown that γ-TuRCs purified from the cytosol of HeLa cells and Xenopus eggs are in a semi-open conformation in which γ-tubulin molecules do not perfectly match the geometry of a 13-protofilament microtubule (Consolati et al, 2020;Liu et al, 2020;Wieczorek et al, 2020). This is also observed in recombinantly generated human γ-TuRCs (Zimmermann et al, 2020;Wieczorek et al, 2021;Würtz et al, 2021). A conformational change into a fully closed ring that matches microtubule geometry is expected to increase the nucleation capacity of the γ-TuRC.…”