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bioRxiv · 10.1101/2020.09.21.306845

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

Wieczorek, M.·Ti, S.-C.·Urnavicius, L.·Molloy, K. R.·Aher, A.·Chait, B. T.·Kapoor, T. M.
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Abstract

The formation of cellular microtubule networks is regulated by the{gamma} -tubulin ring complex ({gamma}-TuRC). This [~]2.3 MDa assembly of >31 proteins includes{gamma} -tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge". The challenge of reconstituting the{gamma} -TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human{gamma} -TuRC ({gamma}-TuRC-GFP), a [~]35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex,{gamma} -TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that{gamma} -TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native{gamma} -TuRCs. Electron microscopy reveals that{gamma} -TuRC-GFP resembles the native{gamma} -TuRC architecture, while{gamma} -TuRCmini-GFP adopts a partial cone shape presenting only 8-10{gamma} -tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating "core" in the{gamma} -TuRC.

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Wieczorek, M., Ti, S.-C., Urnavicius, L., Molloy, K. R., Aher, A., Chait, B. T., Kapoor, T. M.. 2020-09-21. Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function. https://doi.org/10.1101/2020.09.21.306845

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