The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.
Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function
S.-C. Ti’s present address is School of Biomedical Sciences, Faculty of Medicine, University of Hong Kong, Hong Kong
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Michal Wieczorek, Shih-Chieh Ti, Linas Urnavicius, Kelly R. Molloy, Amol Aher, Brian T. Chait, Tarun M. Kapoor; Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function. J Cell Biol 1 March 2021; 220 (3): e202009146. doi: https://doi.org/10.1083/jcb.202009146
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