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

Abstract

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