Reconstitution of the recombinant human γ-tubulin ring complex

Abstract

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.

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

“…expressed the γ‐TuRC subunits γ‐tubulin, actin, GCP2‐6 and MZT1 and isolated structurally intact and microtubule nucleation‐competent γ‐TuRCs using a gentle and fast single step affinity purification against a double FLAG tag on GCP5, constituting the minimal system yielding functional γ‐TuRC to date. [ 58 ] Successful reconstitution of the γ‐TuRC in absence of MZT2 indicates that the function of the latter is not required for assembly of the γ‐TuRC, consistent with its peripheral position in the complex (Figure 2D). [ 43 ] In addition to the core γ‐TuRC subunits used by Würtz et al., Wieczorek et al.…”

“…Wieczorek et al, 2021 [59] Recombinant, insect cells (Sf9) [58] Recombinant, insect cells (Sf21/Hi5)…”

“…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 self‐interact and form small γ‐TuSC oligomers. Although MZT2 is not essential for the assembly of the γ‐TuRC [ 58 ] , its co‐expression enhanced the yield of soluble γ‐TuSC units in this recombinant system, suggesting that MZT2 stabilizes the N‐terminal region of GCP2, which it forms stable structural modules with. Whether MZT2 retains such a stabilizing function during and after the assembly of the γ‐TuRC in human cells remains to be investigated.…”

Modular assembly of the principal microtubule nucleator γ-TuRC

“…Based on this structural molecular census of γ-TuRC components, recombinant expression systems for the human γ-TuRC were established in several independent studies 9 , 13 , 14 , enabling direct genetic manipulation and thus detailed functional dissection of individual γ-TuRC components. Using recombinant systems, actin and MZT1 co-expression were observed to be essential for the structural integrity of the complex 14 , and the AAA+ ATPase RUVBL1/RUVBL2 was identified as an important factor for efficient γ-TuRC reconstitution 9 .…”

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.…”