title: Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains.
abstract: Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex.
authors.affiliation: From the Centre de Biologie du Développement, CNRS-Université Toulouse III, 31062 Toulouse, France and.
authors.forename: Dorian
authors.initials: D
authors.lastname: Farache
authors.affiliation: Plateforme Imagerie-Microscopie, FR 3450 Pôle de Biotechnologie Végétale, 31326 Castanet-Tolosan, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Jauneau
authors.affiliation: From the Centre de Biologie du Développement, CNRS-Université Toulouse III, 31062 Toulouse, France and.
authors.forename: Cécile
authors.initials: C
authors.lastname: Chemin
authors.affiliation: From the Centre de Biologie du Développement, CNRS-Université Toulouse III, 31062 Toulouse, France and.
authors.forename: Marine
authors.initials: M
authors.lastname: Chartrain
authors.affiliation: From the Centre de Biologie du Développement, CNRS-Université Toulouse III, 31062 Toulouse, France and.
authors.forename: Marie-Hélène
authors.initials: MH
authors.lastname: Rémy
authors.affiliation: From the Centre de Biologie du Développement, CNRS-Université Toulouse III, 31062 Toulouse, France and andreas.merdes@univ-tlse3.fr.
authors.forename: Andreas
authors.initials: A
authors.lastname: Merdes
authors.affiliation: From the Centre de Biologie du Développement, CNRS-Université Toulouse III, 31062 Toulouse, France and laurence.haren@univ-tlse3.fr.
authors.forename: Laurence
authors.initials: L
authors.lastname: Haren
mesh_terms: D018360:Crystallography, X-Ray; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D014404:Tubulin
keywords: fluorescence resonance energy transfer (FRET); microtubule; microtubule nucleation; mitotic spindle; protein chimera; tubulin; γ-tubulin complex proteins; γ-tubulin ring complex