The gammaTuRC revisited: a comparative analysis of interphase and mitotic human gammaTuRC redefines the set of core components and identifies the novel subunit GCP8

• Published in: Molecular biology of the cell 2010-11, Vol.21 (22), p.3963-3972
• Main Authors: Teixidó-Travesa, Neus, Villén, Judit, Lacasa, Cristina, Bertran, Maria Teresa, Archinti, Marco, Gygi, Steven P, Caelles, Carme, Roig, Joan, Lüders, Jens
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Biologia del desenvolupament; Blotting, Western; Cell Line, Tumor; Centrosome - metabolism; Citologia; Cytology; Developmental biology; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Interphase; Mass Spectrometry; Microscopy, Fluorescence; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; Microtubules - metabolism; Mitosis; Molecular Sequence Data; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Protein Binding; Protein Subunits - genetics; Protein Subunits - metabolism; RNA Interference; Sequence Homology, Amino Acid; Tubulin - genetics; Tubulin - metabolism
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E10-05-0408

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.