© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs244368. doi:10.1242/jcs.244368 RESEARCH ARTICLE A stable sub-complex between GCP4, GCP5 and GCP6 promotes the assembly of γ-tubulin ring complexes Laurence Haren, Dorian Farache*, Laurent Emorine and Andreas Merdes‡ ABSTRACT grip1 and grip2 motifs, corresponding to the N-terminal and γ-Tubulin is the main protein involved in the nucleation of C-terminal halves of GCP4, the smallest GCP (Gunawardane et al., microtubules in all eukaryotes. It forms two different complexes with 2000; Guillet et al., 2011). The crystallographic structure of GCP4 α proteins of the GCP family (γ-tubulin complex proteins): γ-tubulin shows that these domains correspond to bundles of -helices. The small complexes (γTuSCs) that contain γ-tubulin, and GCPs 2 and 3; other GCPs contain additional specific sequences, mainly at the and γ-tubulin ring complexes (γTuRCs) that contain multiple γTuSCs extreme N-terminus or in the region that links the grip1 and grip2 in addition to GCPs 4, 5 and 6. Whereas the structure and assembly motifs, as in GCPs 5 and 6 (Guillet et al., 2011; Farache et al., 2016). properties of γTuSCs have been intensively studied, little is known Depletion of GCP2 or GCP3 leads to severe spindle about the assembly of γTuRCs and the specific roles of GCPs 4, 5 abnormalities, and depleted cells are not viable. Depletion of and 6. Here, we demonstrate that two copies of GCP4 and one copy GCP4, 5 or 6 can be tolerated in fission yeast or in somatic cells of Drosophila each of GCP5 and GCP6 form a salt (KCl)-resistant sub-complex but not in vertebrates, where removal of either of these γ within the γTuRC that assembles independently of the presence of GCPs prevents the formation of the TuRC and provokes spindle γTuSCs. Incubation of this sub-complex with cytoplasmic extracts defects (Anders et al., 2006; Vérollet et al., 2006; Farache et al., containing γTuSCs leads to the reconstitution of γTuRCs that are 2016; Cota et al., 2017). Rescue experiments with chimeric proteins competent to nucleate microtubules. In addition, we investigate containing N-terminal domains fused to C-terminal domains of a sequence extensions and insertions that are specifically found at the different GCP showed that the chimeras rescued the defects as long N-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs. as they carried the N-terminal domain of the depleted GCP (Farache We also demonstrate that these are involved in the assembly or et al., 2016). Thus, the GCPs are not functionally redundant, despite stabilization of the γTuRC. their structural similarities, and the function of individual GCPs are specified by their N-terminal domains. GCP2 and GCP3 interact KEY WORDS: Microtubule nucleation, γ-Tubulin ring complex, laterally through their N-terminal domains in γTuSC helices, Centrosome whereas γ-tubulin molecules are bound by the C-terminal domains (Kollman et al., 2010). FRET experiments also demonstrated a INTRODUCTION direct lateral interaction between GCP4 and GCP5 through their γ-Tubulin is a protein involved in the nucleation of microtubules. N-terminal domains (Farache et al., 2016), suggesting that the It assembles with so-called ‘gamma-tubulin complex proteins’ N-terminal domains specify lateral binding partners and, thereby, (GCPs) into multiprotein complexes of two different sizes. position the GCPs within the γTuRC helix. A ‘γ-tubulin small complex’ (γTuSC) comprises two molecules of In this context, the specific functions of GCPs 4, 5 and 6 in γ-tubulin that are bound by GCPs 2 and 3. A much larger ‘γ-tubulin γTuRC assembly need to be investigated. Because these proteins are ring complex’ (γTuRC) is formed by multiple γTuSCs that associate present only in one or two copies per complex (Murphy et al., 2001; with additional GCPs 4, 5 and 6, and several smaller accessory Choi et al., 2010), and because the rescue experiments with proteins into a helical structure of 2 MDa (Kollman et al., 2011; chimeras suggest that GCPs 4, 5 and 6 occupy non-random Farache et al., 2018). A few eukaryotes, such as Saccharomyces positions, their localization within the complex is of particular cerevisiae or Candida albicans contain only GCPs 2 and 3. In these interest. During the course of this work, three studies have described organisms, multiple γTuSCs are assembled that form a helix with the structure of native γTuRCs by cryo-electron microscopy, and the help of additional proteins, such as Spc110 or Mzt1 (Kollman have found a lateral association of four γTuSCs, bound to a lateral et al., 2010; Erlemann et al., 2012; Lyon et al., 2016; Lin et al., array of GCP4/GCP5/GCP4/GCP6, to which an additional γTuSC 2014, 2016). Most eukaryotes, however, express the full was associated (Consolati et al., 2019 preprint; Liu et al., 2020; complement of GCPs 2, 3, 4, 5 and 6, and form γTuRCs. It is Wieczorek et al., 2020). Altogether, this has raised the question believed that all these GCPs have similar structures. They are whether GCPs 4, 5 and 6 form assembly intermediates equivalent to characterized by sequence homology of two conserved regions, the γTuSCs. In this study, we demonstrate biochemically that GCPs 5 and 6, and two copies of GCP4 together form a stable, KCl-resistant γ Centre de Biologie du Développement, Centre de Biologie Intégrative, CNRS- core within the TuRC, which can be purified and drives the UniversitéToulouse III, 31062 Toulouse, France. assembly of free γTuSCs into a γTuRC that is competent to nucleate *Present address: Rosenstiel Basic Medical Sciences Research Center, Brandeis microtubules. University, Waltham, MA 02453, USA. ‡Author for correspondence ([email protected]) RESULTS γTuRC-specific GCPs 4, 5 and 6 form a core complex resistant D.F., 0000-0003-3715-9739; A.M., 0000-0002-3739-2728 to treatment with high concentrations of KCl γ Handling Editor: David Glover To determine how GCPs 4, 5 and 6 assemble within the TuRC, and Received 21 January 2020; Accepted 26 March 2020 to examine whether γTuSC-like intermediates are formed by these Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs244368. doi:10.1242/jcs.244368 proteins, we destabilized the γTuRC by treating HeLa cytoplasmic The core of GCP4/5/6 forms independently of γTuRC-assembly extracts with increasing concentrations of KCl. GCPs 4, 5 or 6 were To determine whether GCPs 4, 5 and 6 associate independently of immunoprecipitated and all interacting GCPs were identified by γTuRCs, we used RNA interference (RNAi) to deplete GCP2 western blotting (Fig. 1A, Fig. S1A). Whereas the full set of GCPs (Fig. 2A). Consistent with previously published findings on the co- was immunoprecipitated at 100 mM KCl, we observed an regulation of GCPs (Vérollet et al., 2006), we noticed that siRNAs increasing loss of γTuSCs from the immunoprecipitate at higher against individual GCPs also affected the protein levels of others concentrations of KCl. At 500 mM KCl, GCPs 4, 5 and 6 remained (GCP2 siRNA also decreased levels of GCP3, and levels of GCP6 the major constituents of the immunoprecipitate, irrespective of the were slightly affected by several siRNAs). Most importantly, loss of antibody used for precipitation. This indicated that the binding GCP2 caused the disappearance of γTuRCs in sucrose gradients affinities between GCPs 4, 5 and 6 are stronger than their affinities (Fig. 2B, Fig. S2) and GCPs 4, 5 and 6 immunoprecipitated together to γTuSCs. Consistently, γTuRCs have previously been found to with γ-tubulin in intermediate fractions. Here, the sub-complex dissociate and to release γTuSCs in response to treatment with KCl peaked at a sucrose density of ∼21% (fraction 5), whereas at high concentrations (Moritz et al., 1998; Oegema et al., 1999). disassembly of γTuRCs at 500 mM KCl yielded the strongest To investigate whether GCPs 4, 5 and 6 are associated in a single peak at 17% sucrose in fraction 4 (compare Fig. 1C with Fig. 2C). sub-complex within the γTuRC, we fractionated cytoplasmic This difference might be due to the loss of interactors at high extracts on gradients of 5–40% sucrose containing KCl (100 mM concentrations of KCl. or 500 mM; Fig. 1B, Fig. S1B). We noticed that the cell line used in Since Wieczorek et al. (2020) and Liu et al. (2020) suggested the these experiments (HeLa Flp-In T-REx) contains high levels of existence of γTuSC-like structures, containing complexes GCP4/5 GCP4 protein, part of which sedimented independently of γTuRCs or GCP4/6, we tested how the depletion of individual GCPs 4, 5 or 6 in low-density-fractions (Fig. S1B; Farache et al., 2016). To test affects the composition of the GCP4/5/6 sub-complex, and whether for mutual binding of GCPs 4, 5 and 6, we performed the proposed γTuSC-like structures do, indeed, exist. We depleted immunoprecipitation from each individual fraction in the gradient each of these three GCPs individually by using RNAi, which, in by using antibodies against GCP4 (Fig. 1C) or GCP5 (Fig. 1E, left each case, resulted in the loss of γTuRCs and the accumulation of panel). At 100 mM KCl, γTuSCs and γTuRCs sedimented mainly γTuSCs, as seen in sucrose gradients (Fig. 2A,B, Fig. S2). Next, we in fractions 3 and 7, at a mean density of 13% and 28% sucrose, immunoprecipitated the proteins from the gradient fractions, using respectively. GCP2 to GCP6 co-immunoprecipitated efficiently antibodies against GCP4, GCP5 or GCP6 (Fig. 2D). GCPs 4, 5 and from fraction 7 (Fig. 1C, first lane). By contrast, at 500 mM KCl, we 6 were systematically co-precipitated in the absence of GCP2.