melanogaster γ-TuRC (35.5 S Oegema et al., 1999). S1 A), which indicated that γ-TuRC-GFP sediments to 32–38 S, a range that is larger than individual γ-TuSCs (9.8 S) but comparable to the native D. First, the sucrose density gradient was calibrated with standards of known S values ( Fig. We characterized the biochemical composition of γ-TuRC-GFP in three ways. 1, B and C), and (3) sucrose density gradient centrifugation ( Fig. 1 A): (1) IgG affinity capture and tobacco etch virus (TEV) protease release, (2) gel filtration ( Fig. We collectively refer to this set of 10 recombinant proteins as “γ-TuRC-GFP.” A complex containing all γ-TuRC-GFP proteins could be isolated from infected insect cell lysates using three purification steps ( Fig. Based on a previous report that the structured portion of MZT2 (aa 35–80) binds to the N-terminus of GCP2 (GCP2-NHD Wieczorek et al., 2020a), monomeric enhanced green fluorescent protein (mEGFP) was also fused to MZT2’s unstructured C-terminus (aa 81–158). 1 A Huang et al., 2006 see Materials and methods). An IgG binding purification tag (ZZ) was fused to the N-terminus of MZT2 ( Fig. Encouraging yields were only obtained when a stoichiometric excess of γ-tubulin, GCP2, and GCP3 were co-overexpressed in insect cells with GCP4, GCP5, GCP6, NEDD1, MZT1, β-actin, and MZT2A (hereafter, MZT2 Wieczorek et al., 2020a ). We tested multiple strategies, including expressing various combinations of γ-TuRC components either in human cells under cytomegalovirus promoters or in insect cells using the baculovirus expression system. To address the need for a biochemically tractable system with which to study the function and assembly of a vertebrate γ-TuRC, we asked whether the human complex could be reconstituted with recombinant proteins. However, the role of the LB in the assembly and microtubule-nucleating activity of the γ-TuRC is currently unclear. The LB is composed of two copies of MZT1 that associate with the N-terminal α-helical domains (NHDs) of GCP3 and GCP6 in structurally mimetic “modules,” along with an actin-like protein ( Wieczorek et al., 2020a). Unexpectedly, the γ-TuRC also contains a prominent feature inside its conical structure, termed the “lumenal bridge” (LB). The γ-TuRC is an asymmetric, cone-shaped structure, in which GCP2-6 orient 14 γ-tubulin molecules in a helical arrangement that is poised to nucleate microtubules. The stoichiometry, location, and structures of these proteins in the context of the native human γ-TuRC were recently identified using high-resolution cryo-EM ( Wieczorek et al., 2020a, b Consolati et al., 2020). In vertebrates, the γ-TuRC contains at least 8 proteins, including γ-tubulin, the γ-tubulin complex proteins (GCPs) 2–6, and the mitotic spindle organizing proteins associated with a ring of γ-tubulin-1 and -2 (MZT1 and MZT2 Murphy et al., 2001 Teixidó-Travesa et al., 2010 Hutchins et al., 2010). The γ-tubulin ring complex (γ-TuRC) is an ∼2.3 MD assembly required for proper microtubule network formation in eukaryotes ( Knop et al., 1997 Raff et al., 1993 Stearns and Kirschner, 1994 Zheng et al., 1995). 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. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRC ΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. We show that γ-TuRC ΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. In addition, we generate a subcomplex, γ-TuRC ΔLB-GFP, which lacks MZT1 and actin. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. 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 formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC).
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